Vaccines used for the treatment and prevention of infectious diseases in invertebrates

Abstract

This disclosure provides compositions and methods for treating invertebrates and populations of invertebrates and for vaccinating them against disease.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 616,346, filed December 29, 2023; U.S. Provisional Application No. 63 / 643,343, filed May 6, 2024; U.S. Provisional Application No. 63 / 661,827, filed June 19, 2024; and U.S. Provisional Application No. 63 / 716,070, filed November 4, 2024, the entire contents of each of which are incorporated herein by reference. Technical Field

[0002] This disclosure provides one or more methods for vaccinating, inducing immune responses, treating and / or preventing infections in invertebrates or groups of invertebrates (e.g., insects, bees, crustaceans and shrimp). Background Technology

[0003] Driven by climate change and globalization, the spread of infectious diseases has become an increasingly serious global problem, posing a growing public health concern as it threatens food security, access to nutritious food, animal health, human health, and plant health. Infectious diseases are caused by pathogenic microorganisms (such as bacteria, viruses, parasites, or fungi) and can spread rapidly from one infected host—human, animal, or plant—to another. Once an individual is infected, transmission can occur directly between individuals or through an intermediate host.

[0004] Infectious diseases are generally classified into three categories, which usually depend on the level of infection and the susceptibility of the host or patient. Diseases that can spread rapidly over long distances can lead to high mortality and disability and may cause global pandemics.

[0005] Since the root cause of infectious diseases lies outside the infected host, individual human, animal, or plant, human and animal health and agriculture employ three key strategies to reduce the risk of infection: enhancing the host or animal's resistance to infection through immunization; and reducing the presence of the pathogen itself or its carriers through the prophylactic use of pesticides, antibiotics, or the release of vectors with reduced reproductive capacity (such as fruit flies or mosquitoes).

[0006] Invertebrates, such as bees and other pollinating insects, general insects, and crustaceans like shrimp, are vital components of our ecosystems, contributing to biodiversity, food security, and climate change, but are also threatened by or carry out diseases. Therefore, new, non-chemical, and sustainable methods are needed to protect these animals, rather than eradicate them. Consequently, there is a need in the art for effective vaccines to prevent invertebrate and insect diseases. This disclosure addresses this need and provides relevant advantages. Summary of the Invention

[0007] In this disclosure, the applicant provides novel compositions and / or methods that reduce the infection burden of these important but underserved invertebrates by activating their innate immune systems (in the maternal and offspring generations), thereby reducing their mortality and their ability to spread infection among them and across species.

[0008] Treatment and prevention of bacterial and viral infections in invertebrates

[0009] The applicant provides a method for vaccinating, inducing an immune response, treating, and / or preventing one or more of a disease or infection caused by a microorganism (e.g., bacteria and / or viruses) or a microbial or viral disease to invertebrates or populations, the method comprising, substantially consisting of, or consisting of: administering an effective amount of a dead and / or inactivated non-pathogenic bacterial species or fragment thereof to the invertebrate, optionally in combination with a vector, thereby vaccinating, treating, preventing, or immunizing the invertebrate against the microbial disease or infection. In one aspect, the composition is administered to a maternal invertebrate, and the immune response, treatment, and / or prevention of the disease or infection is passed on to offspring. In one aspect, the invertebrates or populations thereof are identified in Table 1, or alternatively in Tables 1-4 and Experiment 4, such as insects (bees, e.g., honeybees or bumblebees), crustaceans, or shrimp, and exemplary dead and / or inactivated non-pathogenic bacterial species or fragments thereof are listed in the tables. Non-limiting examples of invertebrates include bees, honeybees, bumblebees, crustaceans, shrimp, fleas, ticks, flies, mites, mosquitoes, and their colonies.

[0010] In one aspect, the dead and / or inactivated non-pathogenic bacterial species comprises one or more Gram-positive bacteria and / or one or more Gram-negative bacteria. In another aspect, the dead and / or inactivated non-pathogenic bacterial species comprises one or more Gram-positive bacteria, is substantially composed of, or is composed of.

[0011] To the best of the applicant's knowledge, this is the first vaccine to provide protection against any viral disease or non-pathogenic bacterial or viral infection in invertebrates, crustaceans (such as shrimp), and insects (such as bees) and their offspring and populations, and is the first instance of transgenerational immunoprovocation (TGIP) using bacterial pathogens to provide protection against viruses and non-pathogenic bacteria in invertebrates (such as insects, bees, crustaceans, and shrimp). In another respect, the method is applied to specific invertebrates and diseases identified in Table 1, or alternatively in Tables 1-4, or as identified in Experiment 4.

[0012] In one aspect, this disclosure provides methods for one or more of the following: vaccination, inducing an immune response, treatment, and / or prevention of diseases caused by viral and / or bacterial pathogens in invertebrates or populations of invertebrates (such as insects, bees, crustaceans, and shrimp). The methods comprise, substantially comprise, or consist of: administering and / or immunizing the invertebrates or populations of invertebrates by applying, feeding, or administering an stimulant containing dead and / or inactivated non-pathogenic Gram-positive bacterial species to the invertebrates or populations, thereby immunizing the invertebrates or their offspring.

[0013] In one aspect of the method disclosed herein, the applied dead and / or inactivated non-pathogenic Gram-positive bacteria or cell wall fragments thereof comprise, or are substantially composed of, the dead and / or inactivated bacterial species or species of the genus *Paenibacillus* disclosed in Table 1, or alternatively Tables 1-4, such as those selected from *P. agarexedens*, *P. agaridevorans*, *P. alginolyticus*, *P. alkaliterrae*, *P. alvei*, *P. amylolyticus*, *P. anaericanus*, *P. antarcticus*, *P. apiarius*, *P. assamensis*, and *P. reductive azo*. azoreducens), nitrogen-fixing bacilli (P. azotofixans), Barcelona bacilli (P. barcinonensis), northern bacilli (P. borealis), Brazilian bacilli (P. brasilensis), P. brassicae[1], Campinasensis bacilli (P. campinasensis), Jinju bacilli (P. chinjuensis), chitinolyticus bacilli (P. chitinolyticus), chondroitinus bacilli (P. chondroitinus), cineris bacilli (P. cineris), cookii bacilli (P. cookii), curdlanolyticus bacilli (P. curdlanolyticus), daejeonensis bacilli (P. daejeonensis), dendritiformis bacilli (P. dendritiformis), durum bacilli (P. durum), ehimensis bacilli (P. ehimensis), and Egi bacilli (P. *Pseudomonas elgii*, *P. favisporus*, *P. glucanolyticus*, *P. glycanilyticus*, *P. gordonae*, *P. graminis*, *P. granivorans*, *P. hodogayensis*, *P. ilinosinica*.*P. jamilacea*, *P. kobensis*, *P. koleovorans*, *P. koreensis*, *P. kribbensis*, *P. lactis*, *P. larvae*, *P. lautus*, *P. lentimorbus*, *P. macerans*, *P. macquariensis*, *P. massiliensis*, *P. mendelii*, *P. motobuensis*, *P. naphthalenovorans*, *P. nematophilus*, *P. odorifer*, *P. fodder*. *P. pabuli*, *P. peoriae*, *P. phoenicis*, *P. phyllosphaerae*, *P. polymyxa*, *P. popilliae*, *P. pulvifaciens*, *P. rhizosphaerae*, *P. sanguinis*, *P. stellifer*, *Paenibacillusstellife*, *P. terrae*, *P. thiaminolyticus*, *P. timonensis*, *P. tundrae*, *P. turicensis*, *P. tylopili*, *P. validus*, *P. vortex*, *P. wound-forming bacteria*. *P. vulneris*, *P. wynnii*, and / or *P. xylanilyticus*, or combinations of two or more thereof; or selected from *Paenibacillus alvei*, *Paenibacillus dentritiformis*, *Paenibacillus amylolyticus*, *Paenibacillus campinatus*, *Paenibacillus*, and *Paenibacillus campinatus*.*Paenibacillus campinasensis*, *Paenibacillus chondroitinus*, *Paenibacillus chungangensis*, *Paenibacillus doosanensis*, *Paenibacillus glucanolyticus*, *Paenibacillus humicus*, *Paenibacillus lactis*, *Paenibacillus larvae*, *Paenibacillus lautus*, *Paenibacillus lentimorbus*, *Paenibacillus maceran*, *Paenibacillus macerans-like*, *Paenibacillus macquariensis*, *Paenibacillus* The bacteria include *Paenibacillus motobuensis*, *Paenibacillus phoenicis*, *Paenibacillus polymyxa*, *Paenibacillus popilliae*, *Paenibacillus puldeungensis*, *Paenibacillus residui*, *Paenibacillus stellife*, *Paenibacillus sthiaminolyticus*, *Paenibacillus validus*, and *Paenibacillus xylanisolvens*, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise, or are substantially composed of, or consist of, dead and / or inactivated larval *Paenibacillus larvae* (PL) or cell wall fragments thereof.

[0014] In another aspect, the composition applied in the method further comprises, or is substantially composed of, Gram-negative, dead and / or inactivated non-pathogenic bacteria or fragments of their cell walls, said bacteria being Vibrio sp., such as those selected from V. adaptatus, V. aerogenes, V. aestivus, V. aestuarianus, V. agarivorans, V. albensis, V. alfacsensis, V. alginolyticus, V. anguillarum, V. areeninigrae, V. artabrorum, V. atlanticus, V. atypicus, V. azureus, V. brasiliensis, V. bubulus, V. calviensis, V. campbellii, V. Vibrio casei, Vibrio chagasii, Vibrio cholerae, Vibrio cincinnatiensis, Vibrio coralliilyticus, Vibrio crassostreae, Vibrio cyclitrophicus, Vibrio diabolicus, Vibrio diazotrophicus, Vibrio ezurae, Vibrio fluvialis, Vibrio fortis, Vibrio furnissii, Vibrio gallicus, Vibrio gazogenes, Vibrio gigantis, Vibrio halioticoli, Vibrio harveyi, Vibrio hepatarius, Vibrio hippocampi, Vibrio hispanicus, Vibrio ichthyoenteri, V. Vibrio indicus, Vibrio kanaloae, Vibrio lentus, Vibrio litoralis, Vibrio logei, Vibrio mediterranei, Vibrio metschnikovii, Vibrio mimicus, Vibrio mytili, Vibrio natriegens, Vibrio navara (V.Vibrio navarrensis), Vibrio neonatus, Vibrio neptunius, Vibrio nereis, Vibrio nigripulchritudo, Vibrio ordalii, Vibrio orientalis, Vibrio pacinii, Vibrio parahaemolyticus, Vibrio pectenicida, Vibrio pelagius, Vibrio penaeicida, Vibrio pomeroyi, Vibrio ponticus, Vibrio proteolyticus, Vibrio rotiferianus, Vibrio ruber, Vibrio rumoiensis, Vibrio salmonicida, Vibrio scophthalmi, Vibrio splendidus, Vibrio superstes, Vibrio tapetis, Vibrio tasmani (V. Vibrio tasmaniensis), Vibrio tubiashii, Vibrio vulnificus, Vibrio wodanis and / or Vibrio xuii, or a combination of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio campbelli, Vibrio damsela, Vibrio harvestyi, Vibrio parahaemolyticus, Vibrio penaeicida, Vibrio vulnificus, Vibrio nereis, Vibrio tubiashi, Vibrio fluvialis, Vibrio splendidus, Vibrio nigripulchritudo, Hepatobacter Vibrio penaeiis, Vibrio alginolyticus, Vibrio anguillarum, Vibrio campbelli, Vibrio damsela, Vibrio harveyi, Vibrio parahaemolyticus.Parahaemolyticus. On the other hand, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise, or are substantially composed of, dead and / or inactivated larval spore-forming bacteria (PL) or cell wall fragments thereof, and are administered in combination with dead and / or inactivated non-pathogenic Vibrio bacteria or cell wall fragments thereof.

[0015] Treatment and prevention of viral infections in bees and bee colonies

[0016] Contrary to what is known in the art, and by way of example only, the applicant describes a method in one embodiment comprising, substantially composed of, or consisting of: administering, feeding, and / or immunizing invertebrates with an stimulant containing dead and / or inactivated non-pathogenic Gram-positive bacteria or bacterial species (e.g., Bacillus larvae (PL)) or fragments of their cell wall, to protect the invertebrates or their offspring from infection by a viral pathogen. Non-limiting examples of such invertebrates include shrimp, insects, crustaceans, and bees. For example, the applicant has demonstrated that administering a vaccine to bees containing the non-pathogenic bacterial pathogen Bacillus larvae (PL) reduces the amount of Deformity Wing Virus B (DWV-B) in bee colonies (i.e., queen bees and offspring of vaccinated bees). Thus, this vaccine provides beekeepers with a novel method to reduce the viral infection impact of Varroa destructor mite infestations on their bee colonies and also improves colony health by reducing the amount of DWV-B in the colony. Therefore, this disclosure provides these preventative and therapeutic methods.

[0017] Therefore, in one aspect, this disclosure provides a method for vaccinating, treating, preventing, or immunizing bees or bee colonies against one or more viral infections or viral diseases, the method comprising, substantially consisting of, or consisting of: administering to the bees or bee colony an effective amount of dead and / or inactivated Gram-positive bacteria (e.g., Bacillus larvae (PL)) or fragments thereof (e.g., cell wall fragments), optionally in combination with a carrier, thereby vaccinating, treating, or immunizing the bees or bee colony against the viral disease or infection. In one aspect, the queen bee is vaccinated, resulting in the queen bee and her offspring being vaccinated, treated, or immunized against the viral infection or viral disease.

[0018] In one aspect, the bee is a honeybee or the bee colony is a honeybee colony or community, and the virus causing the viral disease or infection is selected from Deformed Wing Virus A, Deformed Wing Virus B, Deformed Wing Virus C, Acute Bee Paralysis Virus, Israel Acute Bee Paralysis Virus, Kashmir Bee Virus, Slow Bee Paralysis Virus, Lake Sinai Virus 1, Lake Sinai Virus 2, Chronic Bee Paralysis Virus, Sack brood Virus, and Black Queen Cell Virus. On the other hand, the bees are bumblebees or bumblebee colonies or communities, and the viral disease or infection is caused by Devionevirus A, Devionevirus B, Devionevirus C, Acute Bee Paralysis Virus, Israel Acute Bee Paralysis Virus, Kashmir Bee Virus, Slow Bee Paralysis Virus, Lake Sinai Virus 1, Lake Sinai Virus 2, Chronic Bee Paralysis Virus, Sacbryovirus, or Black Queen Cell Virus. On the other hand, the bees are honeybees and the colony is a honeybee colony or community, and the virus causing the disease or infection is Devionevirus B (DWV-B).

[0019] In one embodiment disclosed above, the carrier contains bee food or bee feed.

[0020] In one aspect of the above method, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise, or are substantially composed of, the dead and / or inactivated bacterial species listed in Table 1, or, for example, bacterial species of the genus *Bacillus*. In another aspect, the dead and / or inactivated *Bacillus* species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus apis*, *Bacillus apis*, *Bacillus assamum*, *Bacillus azotobacter*, *Bacillus nitrogen-fixing*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brassicus*, and *P. brassicus*. ae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, Particle Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextrin, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus Stellife, thiamine-derived Bacillus, Paenibacillus validus, and xylolytic Bacillus, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0021] In another aspect, the composition applied in the method further comprises, or is substantially composed of, Gram-negative dead and / or inactivated non-pathogenic bacteria or fragments of their cell walls, said bacteria being Vibrio genus, such as selected from V. adaptatus, V. aerogenes, Vibrio summerensis, Vibrio estuarineis, Vibrio alginolyticus, V. albensis, Vibrio alfacoidus, Vibrio alginolyticus, Vibrio anguillarum, V. areninigrae, V. artabrorum, Vibrio atlantos, Vibrio atypical, Vibrio cyanobacterium, Vibrio brasiliensis, V. bubulus, V. calviensis, Vibrio cannii, V. casei, Vibrio chagusi, Vibrio cholerae, V. Vibrio cincinnatiensis, Vibrio lysozyme, Vibrio oysterii, Vibrio cyclohexanephaga, Vibrio demigodius, Vibrio diazotrophus, Vibrio smotherium, Vibrio fluvibrio, Vibrio viridissus, Vibrio fernsi, Vibrio gallus, Vibrio aerogenes, Vibrio giantis, Vibrio abaloneensis, Vibrio harveyi, Vibrio hepatis, Vibrio hippocampi, Vibrio schistosomiasis, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolois, Vibrio slow-moving, Vibrio shoreeri, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonate, Vibrio tsoriasis, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio parahaemolyticus, Vibrio parahaemolyticus, Vibrio prawnis, V. Vibrio pelagius, V. penaeicida, Vibrio pelagius ... On the other hand, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise, or consist substantially of, or are composed of, dead and / or inactivated larval spore-forming bacteria or their cell wall fragments, and are applied in combination with dead and / or inactivated non-pathogenic Vibrio bacteria or their cell wall fragments.

[0022] Treatment and prevention of bacterial and viral infections in crustaceans

[0023] A method is also provided for one or more of the following: vaccination, inducing an immune response, treatment, and / or prevention of diseases caused by viral and / or bacterial pathogens in crustaceans and crustacean populations. The method comprises, substantially comprises, or consists of: administering and / or immunizing the crustaceans or populations by applying, feeding, or administering an stimulant to the crustaceans or populations, said stimulant comprising a dead and / or inactivated non-pathogenic Gram-negative bacterial species or cell wall fragments thereof. In one aspect, the crustaceans or populations thereof are shrimp. In another aspect, the crustaceans are shrimp, provided that when *P. polymyxa* is the sole active agent in the vaccine, the shrimp or populations to be vaccinated do not include *P. polymyxa* used for vaccinating shrimp and their offspring against *V. parahaemolyticus* infection or disease.

[0024] On the other hand, the composition applied in the method comprises, or is substantially composed of, Gram-negative dead and / or inactivated non-pathogenic bacteria or fragments of their cell walls, said bacteria being Vibrio genus, such as selected from V. adaptatus, V. aerogenes, Vibrio summerensis, Vibrio estuarineis, Vibrio alginolyticus, V. albensis, Vibrio alfacoidus, Vibrio alginolyticus, Vibrio anguillarum, V. areninigrae, V. artabrorum, Vibrio atlantos, Vibrio atypical, Vibrio cyanobacterium, Vibrio brasiliensis, V. bubulus, V. calviensis, Vibrio cannii, V. casei, Vibrio chagusi, Vibrio cholerae, V. Vibrio cincinnatiensis, Vibrio lysozyme, Vibrio oysteriformis, Vibrio cyclohexanephaga, Vibrio demigodius, Vibrio diazotrophus, Vibrio smotherium, Vibrio fluvibrio, Vibrio viridissus, Vibrio fernsi, Vibrio gallus, Vibrio aerogenes, Vibrio giantis, Vibrio abaloneniformis, Vibrio harveyi, Vibrio hepatis, Vibrio hippocampi, Vibrio schistosomiasis, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolois, Vibrio slow-moving, Vibrio riparianis, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonate, Vibrio tsoriasis, Vibrio nervosa, Vibrio nigra, Vibrio ordalii, Vibrio orientalis, Vibrio pantherinae, Vibrio parahaemolyticus, Vibrio prawni, V. Vibrio pelagius, V. penaeicida, Vibrio pelagius ...

[0025] On the other hand, the composition applied in the method comprises, or is substantially composed of, Gram-negative dead and / or inactivated non-pathogenic bacteria or fragments of their cell walls, said bacteria being Vibrio genus, such as selected from V. adaptatus, V. aerogenes, Vibrio summerensis, Vibrio estuarineis, Vibrio alginolyticus, V. albensis, Vibrio alfacoidus, Vibrio alginolyticus, Vibrio anguillarum, V. areninigrae, V. artabrorum, Vibrio atlantos, Vibrio atypical, Vibrio cyanobacterium, Vibrio brasiliensis, V. bubulus, V. calviensis, Vibrio cannii, V. casei, Vibrio chagusi, Vibrio cholerae, V. Vibrio cincinnatiensis, Vibrio lysozyme, Vibrio oysteriformis, Vibrio cyclohexanephaga, Vibrio demigodius, Vibrio diazotrophus, Vibrio smotherium, Vibrio fluvibrio, Vibrio viridissus, Vibrio fernsi, Vibrio gallus, Vibrio aerogenes, Vibrio giantis, Vibrio abaloneniformis, Vibrio harveyi, Vibrio hepatis, Vibrio hippocampi, Vibrio schistosomiasis, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolois, Vibrio slow-moving, Vibrio riparianis, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonate, Vibrio tsoriasis, Vibrio nervosa, Vibrio nigra, Vibrio ordalii, Vibrio orientalis, Vibrio pantherinae, Vibrio parahaemolyticus, Vibrio prawni, V. Vibrio pelagius, V. penaeicida, Vibrio pelagius ... On the other hand, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise, or consist substantially of, or are composed of, dead and / or inactivated larval spore-forming bacteria or their cell wall fragments, and are applied in combination with dead and / or inactivated non-pathogenic Vibrio bacteria or their cell wall fragments.

[0026] On the other hand, the crustacean or group is a shrimp or a shrimp colony, and the infection or disease being treated is white spot syndrome virus (WSSV) in shrimp, and an effective amount of PL is administered alone or in combination with Vibrio parahaemolyticus (VP). The non-pathogenic bacteria can be administered according to methods known in the art, such as in feed or by injection.

[0027] Treatment and prevention of bacterial, viral, and fungal infections in the Culex and Anopheles subfamilies.

[0028] This document also provides compositions and methods for novel approaches to immunization and treatment of mosquito species and / or their hosts against non-pathogenic bacterial or viral infections or diseases, wherein the mosquito family includes, for example, Culexinae and Anophelesinae, using effective amounts of one or more dead and / or heat-inactivated Gram-positive bacteria or cell wall fragments thereof. See, for example, the lists of diseases and hosts in Tables 2 and 3. Non-limiting examples of mosquito species include Aedes albopictus, Aedes aegypti, and Aedes polynesiensis. Therefore, this document provides compositions and methods for vaccinating, treating, preventing, or immunizing mosquitoes and / or their offspring and / or mosquito hosts against one or more of viral infections, bacterial infections, bacterial diseases, or viral diseases, and respectively, the transmission of diseases. The methods comprise, substantially composed of, or consisting of: administering an effective amount of dead and / or inactivated Gram-positive bacteria (e.g., Bacillus larvae (PL)) or fragments thereof (e.g., cell wall fragments) to the mosquito or mosquito host, optionally in combination with a vector, thereby vaccinating, treating, or immunizing the mosquito, offspring, and / or host against the viral or bacterial disease or infection. The dead and / or inactivated Gram-positive bacteria are described herein, and a complete list thereof is incorporated herein by reference.Non-limiting examples of the group include one or more of the following: agar-solubilizing Bacillus, agar-eating Bacillus, alginate-solubilizing Bacillus, alkaliphilic Bacillus, honeycomb Bacillus, amylolytic Bacillus, anaerobic Bacillus, Antarctic Bacillus, bee Bacillus, Assam Bacillus, reducing azo Bacillus, nitrogen-fixing Bacillus, Barcelona Bacillus, Northern Bacillus, Brazilian Bacillus, and P. brassicae[1]. *Bacillus campinatus*, *Bacillus jinjuensis*, *Bacillus chitinosa*, *Bacillus chondroitinus*, *Bacillus ash*, *Bacillus cookerii*, *Bacillus coagulans*, *Bacillus daetianensis*, *Bacillus dendriticus*, *Bacillus sclerotiorum*, *Bacillus ehimeensis*, *Bacillus egigera*, *Bacillus nectarines*, *Bacillus pyriformis*, *Bacillus dextran*, *Bacillus dextran*, *Bacillus Gordonii*, *Bacillus graminearum*, *Bacillus granulatus*, *P.* *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, terrestrial Bacillus, thiamine Bacillus, timon Bacillus, P. tundrae, Zurich Bacillus, P. tylopili, P. validus, vortex Bacillus, wound Bacillus, Winnipeg Bacillus and / or xylolytic Bacillus, or combinations of two or more thereof.

[0029] On the other hand, the dead and / or inactivated Bacillus species are selected from *Bacillus apis*, *Bacillus dendriticus*, *Bacillus amyloliquefaciens*, *Bacillus campinatus*, *Bacillus chondroitinus*, *Bacillus hanjung*, *Paenibacillus doosanensis*, *Bacillus dextran*, *Bacillus humicus*, *Bacillus lactis*, *Bacillus larvae*, *Bacillus splenicus*, *Bacillus sclerotiorum*, *Bacillus sclerotiorum*, *Bacillus sclerotiorum*, *Bacillus sclerotiorum*, *Bacillus motobuensis*, *Bacillus foetida*, *Paenibacillus phoenicis*, *Bacillus polymyxa*, *Bacillus puldeungensis*, *Paenibacillus residui*, *Paenibacillus stellife*, *Bacillus thiamine*, and *Paenibacillus*. Validus and xylolytic Bacillus, or a combination of two or more of them.

[0030] In one aspect, the dead and / or inactivated bacteria are Paenibacillus sp., such as PL. The dead and / or inactivated Paenibacillus bacteria or cell wall fragments thereof can be fed to mosquitoes.

[0031] Composition

[0032] The composition comprises, or is substantially composed of, dead and / or inactivated Gram-positive or Gram-negative non-pathogenic bacteria or fragments of their cell walls. In one aspect, the non-pathogenic bacteria are Gram-positive bacteria of the genus *Bacillus*. In another aspect, the dead and / or inactivated *Bacillus* species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus vesicularus*, *Bacillus assamum*, *Bacillus reductiveis*, *Bacillus nitrogenophilus*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticulatus, Bacillus woundae, Bacillus venereum, and / or Bacillus xylolyticus, or a combination of two or more thereof; or alternatively selected from Bacillus apiaceus, Bacillus apiaceus, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus stellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0033] Alternatively, the composition comprises, or is substantially composed of, non-pathogenic bacteria selected from, *V. adaptatus*, *V. aerogenes*, *Vibrio summerensis*, *Vibrio estuarineis*, *Vibrio alginolyticus*, *V. albensis*, *Vibrio alphaca*, *Vibrio alginolyticus*, *Vibrio anguillarum*, *V. areninigrae*, *V. artabrorum*, *Vibrio atypical*, *Vibrio cyanobacterium*, *Vibrio brasiliensis*, *V. bubulus*, *V. calviensis*, *Vibrio cannii*, *V. casei*, *Vibrio chagusi*, *Vibrio cholerae*, *V. cincinnatiensis*, *Vibrio coralloides*, *Vibrio oysteri*, *Vibrio cyclohexane*, *Vibrio devilii*, *Vibrio diazotrophus*, *Vibrio zebufo*, *Vibrio fluvii*, *Vibrio virulentii*, *Vibrio fernigina*, *Vibrio gallophylla*, *Vibrio abaloneniformis*, *Vibrio harveyi*, *Vibrio hepatis*, and *V. Vibrio hippocampi, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolo, Vibrio slow-moving, Vibrio riparia, Vibrio logei, Vibrio mediterranei, Vibrio metili, Vibrio mytili, Vibrio navara, Vibrio neonatal, Vibrio tsoriopteris, Vibrio nervosa, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio pannioides, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio brevicornuate, Vibrio rotiferus, Vibrio rubrumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio superstes, Vibrio rubrumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio supreme, Vibrio rubrumoiensis, Vibrio rubrumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio superstes, Vibrio rubrumoiensis, Vibrio supreme, Vibrio scophthalmi, Vibrio supreme ... Vibrio tapetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vortanense, and / or Vibrio xuensis, or combinations of two or more thereof; or alternatively selected from the genus Vibrio, such as Hepatobacter penaeiis, Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio meranti, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawni, Vibrio vulnificus, Vibrio nerei, Vibrio tasmani, Vibrio fluvibrio, Vibrio splenium, Vibrio niger, alone or in combination with non-pathogenic dead and / or inactivated Gram-positive bacteria. In one embodiment, the dead or inactivated Gram-positive bacteria is a combination of PL or its cell wall fragments with Vibrio genus. These compositions may further comprise a carrier, such as invertebrate food, like shrimp feed, and be used for the treatment of shrimp.

[0034] This document also provides a composition comprising, or substantially comprising, whole cells or cell wall fragments of dead and / or inactivated Gram-negative bacteria selected from V. adaptatus, V. aerogenes, Vibrio summerensis, Vibrio estuarineis, Vibrio alginolyticus, V. albensis, Vibrio alphafascis, Vibrio alginolyticus, Vibrio anguillarum, V. areninigrae, V. artabrorum, Vibrio atlantos, Vibrio atypical, Vibrio cyanobacterium, Vibrio brasiliensis, V. bubulus, V. calviensis, Vibrio cannii, and V. Vibrio casei, Vibrio chagusi, Vibrio cholerae, Vibrio cincinnatiensis, Vibrio lysozyme, Vibrio oysteri, Vibrio cyclohexane, Vibrio demigodius, Vibrio diazotrophus, Vibrio smotherium, Vibrio fluvibrio, Vibrio virulentii, Vibrio furnis, Vibrio gallobryo, Vibrio aerogenes, Vibrio giantis, Vibrio abaloneniformis, Vibrio harveyi, Vibrio hepatis, Vibrio hippocampi, Vibrio myasthenia gravis, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolo, Vibrio slow-moving, Vibrio shoreeri, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonate, Vibrio tsoriata, Vibrio nervosa, Vibrio nigra, Vibrio nigra, Vibrio ordalii, Vibrio orientalis, Vibrio pantherinae, Vibrio parahaemolyticus, Vibrio prawni, V. Vibrio pelagius, V. penaeicida, Vibrio brevis, Vibrio nigra, Vibrio rotiferus, Vibrio rubrum, Vibrio rumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio superstes, Vibrio tapetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vortanensis and / or Vibrio xuei, or combinations of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio mermaidii, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawnic, Vibrio vulnificus, Vibrio tasmani, Vibrio tasmani, Vibrio rivubilis, Vibrio splenium, Vibrio nigra, or combinations of two or more thereof, and Gram-positive bacterial vaccines and vectors listed in Table 1. In one aspect, the vector comprises feed for invertebrates such as shrimp. In another aspect, the composition is formulated for oral administration, injection administration and / or submersion administration of the invertebrates, or combinations thereof. On the other hand, the invertebrates were identified in Tables 1-4 and Experiment 4. Attached Figure Description

[0035] Figure 1The figure illustrates an exemplary method for obtaining bacterial fragments that can be used as antigens. This figure is reproduced from Lodish, H.(ed) Molecular Cell Biology, Sixth Edition, 2008, WH Freeman and Company.

[0036] Figure 2 Box plots show the DWV-B load as measured by PCR analysis. Samples were collected from care bee colonies one week before vaccination and four months after vaccination. One sample was submitted from a colony with a vaccinated queen and one sample from a colony with an unvaccinated queen in each of our eight farms (N=8 controls, N=8 vaccinated). In each sample, 50 care bees were equally mixed from 10 hives in the same treatment group. Before vaccination, there was no difference in DWV-B levels between treatment groups (Wilcoxon rank-sum test, W=31, P=0.96). After vaccination, the DWV-B load was significantly lower in the vaccinated population compared to the control population (Wilcoxon rank-sum test, W=54.5, P=0.021). The (*) between bars indicates significance of the Wilcoxon rank-sum test (P<0.05).

[0037] Figure 3 Box plots show the Varroa destructor mite counts in vaccinated and unvaccinated bee populations one week before vaccination (N=35 unvaccinated, N=38 vaccinated) and six months after vaccination (N=44 vaccinated and N=44 unvaccinated). Counts were performed using standard alcohol washing. A two-sample Welch t-test showed no difference in mite counts between groups before vaccination (T=0.38, df=61, P=0.71) or after vaccination (T=0.38, df=61, P=0.71).

[0038] Figures 4A-4B The image shows shrimp that were vaccinated (with P. larvae (PL) and / or Vibrio parahaemolyticus (VP)) and unvaccinated, respectively, after being treated with AHPND (…). Figure 4A ) or WSSV ( Figure 4B Comparison of survival rates after an attack. Figure 4A Following vaccination / treatment of female broodstock shrimp with PL and PL / PV combination products, the survival rate of the next generation of shrimp at 10g size on day 6 after EMS challenge. Figure 4BSurvival rate of the next generation of shrimp at 2g size on day 12 after vaccination / treatment of female parent shrimp with PL and PL / PV combination products.

[0039] Figures 5A-5B : Showing the injection of vaccines into shrimp. The vaccine is delivered to the female parent shrimp via injection. Figure 5A In short, the shrimp are removed from their rearing tanks and injected. During each ovulation cycle, the treatment is administered intramuscularly (IM) at a rate of 100 µL per shrimp, along the ventral axis between the thoracic and pleural legs. Figure 5B A 0.3 mL insulin syringe (30G) with an 8 mm (5 / 6 inch) needle can be used. After injection, return the shrimp to the rearing tank.

[0040] Figures 6A-6C Artificial feeding systems for mosquitoes. (Fig. 6A) Aedes aegypti mosquitoes satiated on an artificial blood meal substitute diet. Food coloring was added to different SkitoSnacks; (Fig. 6B) A glass membrane feeder for mosquitoes. Warm water was used in this device to keep the food at body temperature. Mosquitoes ingested the food through a Parafilm® membrane (Sigma Aldrich, St. Louis, MO, USA); (Fig. 6C) Hemotek feeding system (Hemotek Ltd., Great Harwood, UK). Reprinted from https: / / www.mdpi.com / 1660-4601 / 13 / 12 / 1267. Detailed Implementation

[0041] definition

[0042] As used herein and in the appended claims, singular forms such as “a,” “an,” and “the,” and similar designations, in the context of describing an element, shall be construed as encompassing both the singular and the plural, unless otherwise stated herein or the context clearly contradicts it.

[0043] As used herein, “about” is understood by those skilled in the art and may vary to some extent depending on the context in which it is used. Where the use of the term “about” is unclear to those skilled in the art based on the context in which it is used, “about” refers to plus or minus 10% of a particular term.

[0044] Those skilled in the art will understand that, for any and all purposes, all scopes disclosed herein also cover any and all possible subscopes and combinations thereof. Furthermore, those skilled in the art will understand that the scope includes each individual member.

[0045] The term “exemplary” as used herein means “as an example, instance, or illustration”, and not “preferred” or “superior to other implementations”.

[0046] As used in this article, the terms "non-pathogenic" or "non-disease species" refer to bacterial species that may or may not be pathogenic but do not cause the targeted, treated, and / or preventable infectious diseases (such as viral diseases).

[0047] "Invertebrates" refers to animals that do not develop or retain a vertebral column, which evolved from the notochord. It is a paraphyletic group that includes all animals except for vertebrates (i.e., vertebrates) in the subphylum Chordata.

[0048] As used in this article, the term "host" refers to an animal infected with a virus or bacteria, regardless of whether the animal is sick from carrying the virus or bacteria.

[0049] "Insects" are hexapod invertebrates belonging to the class Insecta. They are the largest group in the phylum Arthropoda. Insects have a chitinous exoskeleton, a three-part body, three pairs of articulated legs, compound eyes, and a pair of antennae. Such non-limiting examples include arachnids, bees, mites, ticks, mosquitoes, flies, and those identified in Table 1, or alternatively in Tables 1-4 and Experiment 4.

[0050] "Mites" are tiny arachnids with four pairs of legs in adults, and are associated with ticks. Many species live in the soil, while some parasitize plants or animals.

[0051] Flies are insects belonging to the order Diptera, which use a pair of wings to fly. Diptera is a large order containing more than 150,000 species, including horseflies, fruit flies, houseflies, tsetse flies, spiral flies, giant mosquitoes, hoverflies, and mosquitoes.

[0052] "Mosquitoes," or the "Family of Culicoides," refers to a small family of flies comprising about 3,600 species. They have slender, segmented bodies, a pair of wings, three pairs of long, hairy legs, and specialized, highly elongated piercing-sucking mouthparts. Mosquitoes transmit saliva to their hosts during their bites, causing itchy rashes. Furthermore, blood-sucking species can ingest pathogens during their bites and transmit them to other hosts. These species include vectors for parasitic diseases such as malaria and filariasis, as well as vectors for arbovirus diseases such as yellow fever and dengue fever. Through disease transmission, mosquitoes cause more than 725,000 deaths annually.

[0053] "Crustaceans" refers to the group of invertebrates that make up the arthropods. It belongs to the subphylum Crustacea and is a large, diverse, and primarily aquatic group of arthropods, including the order Decapoda (shrimp, prawn, crab, lobster, and crayfish), ostracods, branchiopods, fish lice, krill, paddlefoot, isopods, barnacles, copepods, mysids, amphipods, and mantis shrimp.

[0054] "Shrimp" refers to crustaceans with slender bodies that primarily move by swimming. Shrimp generally belong to the infraorder Caridea or suborder Dendrobranchiata within the order Decapoda, although some crustaceans outside this order are also called "shrimp." Non-limiting examples of shrimp include rock shrimp, pink shrimp, tiger shrimp, Chinese white shrimp, brown shrimp, white shrimp, Atlantic northern shrimp, and tiger prawn.

[0055] As used in this article, the term "bee" refers to any bee belonging to the genus *Apis*, characterized primarily by the production and storage of honey and the construction of perennial social nests using wax. For example, two bee species, *A. mellifera* or *A. ceranaindica*, are commonly kept by beekeepers. Bees include, but are not limited to, *Apis andreniformis* and *Apis florea* of the subgenus *Micrapis*, *Apis dorsata* of the subgenus *Megapis*, and *Apis cerana*, *Apis koschevnikovi*, *Apis mellifera*, and *Apis nigrocincta* of the subgenus *Apis*.

[0056] As used herein, the terms "swarm" or "bee colony" or "bumblebee colony" refer to a social unit of bees (e.g., honeybees or bumblebees), encompassing groups also referred to herein as communities. The social unit can be any form of systematic organization used by bees to promote the survival of the colony or community. Typically, a "swarm" consists of thousands of bees that cooperate in nest building, food gathering, and larval rearing. Each member of a "swarm" has a specific task to perform, requiring the collective effort of the entire colony for survival and reproduction. A swarm typically contains a queen bee, thousands of worker bees, and hundreds of drones in late spring and summer. Generally, a "swarm" consists of closely related bees, with colony size depending on the species (e.g., thousands of honeybee individuals, hundreds of bumblebee individuals), who cooperate in nest building. Therefore, a swarm is a "honeybee colony."

[0057] Typically, a bee colony reaches its peak in late spring and summer, and its lowest point in winter. The social structure of the colony is maintained by the queen and worker bees and relies on an effective communication system. Domesticated bees are raised in "beehives" or "honeybee hives." Therefore, the term "beehive" or "honeybee hive" refers to a structure used as habitat for a bee colony (such as a honeybee colony).

[0058] As used herein, the term "carrier" encompasses any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents. A carrier can also be a suitable feed for the invertebrates being treated, such as bees, shrimp, mites, mosquitoes, ticks, etc. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0059] The term "effective amount" or "effective amount of..." or any grammatically equivalent term or expression refers to the amount sufficient to achieve treatment or prevention of a viral disease or symptom in an individual invertebrate or population (e.g., a bee colony) when applied by any means, for example, to adults or larvae, for the treatment or prevention of a viral disease or symptom in an individual invertebrate or population (e.g., a bee colony). Typically, the effective dose of antigen used for the treatment and / or immunization of adults and their respective offspring is approximately 1.5 x 10^6 doses per gram of food. 7 Or 1.5 x 10 4 – 1.5 x 10 11 Individual antigen units and the range thereof. In one embodiment, an "effective amount" means an amount sufficient to inoculate the adult worm and its larvae when fed to the adult worm, such that larvae from inoculated adult worms are at least 50%, or at least 45%, or at least 40%, or at least 35%, or at least 30%, or at least 25%, or at least 20%, or at least 15%, or alternatively at least 10%, or at least 5%, higher than larvae from uninoculated adult worms.

[0060] The term "caregiver bee" as used in this article refers to the bee that feeds worker bee larvae worker bee royal jelly, which is secreted by glands that secrete royal jelly.

[0061] The term "worker bee" as used in this article refers to any female (eusocial) bee that lacks the full reproductive capacity of a queen bee colony.

[0062] The term "larva" refers to the three developmental stages of a bee, collectively known as the larva. A bee begins as an egg, which hatches into larvae (in plural), who then undergo metamorphosis as pupae. Larvae are legless and exclusively feed.

[0063] The term "preventive" refers to a medicine that serves to prevent disease (such as viral diseases).

[0064] As used in this article, “vaccination” refers to means of generating immunity against viral diseases (such as DWV-B and those listed in Table 1 below, or alternatively in Tables 1-4 and Experiment 4) to treat or prevent the occurrence of disease or symptoms (preventive treatment) or to suppress the spread of disease in larvae, offspring, colonies or bee swarms (slowing down or stopping its development).

[0065] As used herein, the term “genus” has its conventional meaning as known in the art. Generally, a genus is defined as a rank in biological taxonomy used for biological classification, above species and below family. For example, the genus *Paenibacillus* is a facultative anaerobic, endospore-producing bacterium, classified by Ash et al. in 1994 (see, for example, Ash, C., Priest, FG & Collins, MD (1994). *Paenibacillus gen. nov.* and *Paenibacillus polymyxa comb. nov.* In Validation of the Publication of NewNames and New Combinations Previously Effectively Published Outside the IJSB, List no. 51. Int J Syst Bacteriol 44, 852). A disease pathogen is an infectious biological agent that causes disease in a host, characterized by certain features such as interference with the organism's functions, and severely damages the host, including its death. Broad-spectrum protection is achieved when immunization with bacteria of a specific genus or multiple species of that genus protects the host from subsequent infection by pathogens not contained in the vaccine preparation.

[0066] The term "treatment" aims to elicit an immune response in the adult worm and then pass it on to its offspring.

[0067] The term "inducing an immune response" refers to the process by which a vaccine or treatment presents non-pathogenic antigens to developing eggs in the ovaries of adult worms.

[0068] The term "vaccination bacteria" refers to pathogenic or non-pathogenic bacteria that serve as active immunizing or therapeutic bacteria in vaccine preparations. Vaccination bacteria will vary depending on the invertebrate being treated and the viral disease being treated or prevented. In one instance, vaccination bacteria are PL, used for the treatment and / or prevention of Deformity Wing Virus B (DWV-B) in bees or bumblebees and / or bee or bumblebee colonies, and White Spot Syndrome Virus (WSSV) in shrimp.

[0069] The terms “vaccine,” “vaccine formulation,” and / or “vaccination bacteria” refer to a dead and / or inactivated whole cell or cell wall fragment (antigen fragment) that has been shown to treat and / or prevent infection by microorganisms (e.g., bacteria, viruses, or fungi) when administered to invertebrates. The bacteria and / or its fragments may be the sole active agent in the vaccine or may be used in combination with other active agents. It should be understood that, unless explicitly stated otherwise, cell wall fragments may be used in conjunction with or as a substitute for whole bacterial cells.

[0070] "Antigen / unit" refers to the number of cells or antigen fragments of non-pathogenic pathogens.

[0071] The term “application” or “administration” refers to any suitable manner in which a bacterial vaccine is provided to an invertebrate that is a target of treatment. Non-limiting examples of such methods include providing the vaccine in the invertebrate’s food, administering the vaccine by injection, or dissolving the bacterial vaccine in the surrounding environment, such as dissolving or suspending it in a tank or enclosure containing invertebrates (e.g., shrimp) and immersing the invertebrates in the dissolved or suspended bacterial vaccine.

[0072] The term "dosage" refers to the amount of bacterial vaccine administered to an invertebrate (such as an insect or shrimp) in a single injection, immersion, or feeding or per unit of food.

[0073] Paenibacillus larvae is a bacterial species found worldwide that causes American foulbrood, a deadly disease of bee larvae. It is a Gram-positive, rod-shaped bacterium that forms spores that can remain viable for at least thirty-five years. Strains and genotypes are known (see, for example, Appl. Environ. Microbiol. (2005) Nov. 71(11):7551-7555) and are commercially available from the American Type Culture Collection (ATCC). When used in the methods of this disclosure, non-limiting examples of the group include one or more Bacillus subtilis, Bacillus alginate-eating Bacillus, Bacillus alginate-eating Bacillus, alkaliphilic Bacillus, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobic Bacillus, Bacillus antarcticis, Bacillus vesicularis, Bacillus assamica, Bacillus reductive azotozobacteria, Bacillus nitrogen-fixing Bacillus, Bacillus barbarus, Bacillus northernis, Bacillus brassica, and P. brassica. e[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, Particle Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timon-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P. validus, Vorticella-derived Bacillus, Wound-derived Bacillus, Winnipeg-derived Bacillus and / or Xylolytic Bacillus.

[0074] As used in this article, the term Vibrio refers to a genus of Gram-negative bacteria with curved rod-shaped (comma-shaped) stems, several of which can cause foodborne or soft tissue infections known as vibriosis. Infections are usually associated with consuming undercooked seafood. Such non-limiting examples include one or more of the following: *V. adaptatus*, *V. aerogenes*, *Vibrio estuarineis*, *Vibrio alginolyticus*, *V. albensis*, *Vibrio alphaca*, *Vibrio alginolyticus*, *Vibrio anguillarum*, *V. areninigrae*, *V. artabrorum*, *Vibrio atypical*, *Vibrio cyanobacterium*, *Vibrio brasiliensis*, *V. bubulus*, *V. calviensis*, *Vibrio cannii*, *V. casei*, *Vibrio chagusi*, *Vibrio cholerae*, *V. cincinnatiensis*, *Vibrio lysomorphus*, *Vibrio oysterii*, *Vibrio cyclohexane*, *Vibrio demigodius*, *Vibrio diazotrophus*, *Vibrio smotherium*, *Vibrio fluvii*, *Vibrio virgaurea*, *Vibrio fernsi*, *Vibrio gallophylla*, *Vibrio abaloneniformis*, *Vibrio harveyi*, *Vibrio hepatis*, *V. hippocampi*, *Vibrio esculenti*, *V. ichthyoenteri*, *V. Vibrio indicus, Vibrio canarano, Vibrio slow-moving, Vibrio shoal, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonatal, Vibrio tsinae, Vibrio nervosa, Vibrio nigra, Vibrio nigra, Vibrio ordalii, Vibrio orientalis, Vibrio panniae, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio nigra, Vibrio rotiferus, Vibrio rubrumoiensis, Vibrio scoramii, Vibrio scophthalmi, Vibrio splenium, Vibrio superstes, Vibrio tapepetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vulnificus, and / or Vibrio xuei, or combinations of two or more thereof.

[0075] Method of implementing this disclosure

[0076] Preparation of bacterial vaccine compositions – general methods

[0077] Preparation of bacterial vaccine compositions

[0078] This disclosure provides an invertebrate vaccine composition comprising, or at least two, or at least three, or at least four or more dead and / or inactivated bacterial whole-cell or cell wall fragments, or substantially composed of, or composed of, such bacteria. In one aspect, an invertebrate vaccine composition is provided comprising, or at least two, or at least three, or at least four or more dead and / or inactivated Gram-positive bacteria and / or at least one, or at least two, or at least three, or at least four or more dead and / or inactivated Gram-negative bacteria, or substantially composed of, or composed of, such bacteria. In one embodiment, the invertebrate vaccine composition comprises, or substantially composed of, whole-cell or cell wall fragments of dead and / or inactivated Gram-positive and / or Gram-negative bacteria. In another embodiment, the Gram-positive and / or Gram-negative bacteria are selected from any one or more of the vaccine bacteria listed in Tables 1-4 and / or Experiment 4. By way of example only, the vaccine composition may comprise, or consist substantially of, whole cells or cell wall fragments of dead and / or inactivated P. larvae and P. thiaminolyticus, for vaccinating black soldier flies against Hermetiaillucens totivirus. The ratio of the vaccine components may vary, for example, 1:1; 1:2; 1:3; 1:4; 1:1:4; 1:2:4; 1:3:4. In one aspect, the ratio is 1:1.

[0079] The bacterial pathogen selected for the vaccine composition will vary depending on the infectious disease (e.g., viral disease) and the invertebrate being treated (see, for example, Table 1, or alternatively Tables 1-4 and / or Experiment 4).

[0080] Inoculate the seed culture of the selected bacteria, culture it, and amplify it if necessary. For example, the bacteria can be amplified from a frozen glycerol stock of bacteria, and the inoculated plates are grown at suitable temperatures and conditions for the bacteria, such as in a dark growth chamber at 35°C for 4-12 days.

[0081] Harvest bacterial colonies, for example by washing a plate with 5 mL of ice-cold H2O and scraping it into a Falcon tube (or glass bottle). Measure the optical density (OD600) at 600 nm using standard methods. Mix equal volumes of the harvested bacterial culture and H2O in two replicates into cuvettes. Based on the OD600 reading, prepare the antigen solution to the desired concentration, for example, at least 1.5 x 10⁻⁶. 9Bacterial cells / mL. Inactivated bacterial solution. Non-limiting examples of inactivating the collected bacteria include using formalin, diethyleneimine (BEI), or heat and pressure (autoclaving). Any method known in the art for killing bacteria can be used. After inactivation, the cells are lysed and large fragments of the bacterial cell wall are separated by centrifugation, for example, at a speed of about 10,000 g to about 60,000 g for 1–4 hours (or longer, depending on volume, size, and concentration). The supernatant is recovered and can be further fractionated as described below. Alternatively, whole-cell preparations can be used. The supernatant can be freeze-dried, lyophilized, or formulated into a solution and administered to selected invertebrates in various forms, such as feed, spray, injection, or immersion. One method of administration includes, but is not limited to, preparing a feed selected for the selected invertebrates, which can be purchased pre-packaged at a supplier or prepared by methods known in the art. By way of example only, invertebrate feed can be coated with a bacterial vaccine for administration to invertebrates.

[0082] The dosage can be determined using standard methods, such as pre-inactivation cell counting or ELISA-based bacterial potency testing. See, for example, pdf.sciencedirectassets.com / 278679 / 1-s2.0-S1877282X11X00036 / 1-s2.0-S1877282X11000348 / main.pdf?X-Amz-Security-Token=IQoJb3JpZ2luX2VjEHsaCXVzLWVhc3QtMSJGMEQCIFHI0kqWKpEoBlN9Mhcg0MVIOXd2ncxY5xEL6FKYb%2FfOAiBTCDnHlABBoPZKjzbYY4yLT4i6kuMTt63YCYqFPzCjS, last accessed on December 18, 2024.

[0083] The composition can be formulated to provide a vaccine composition for delivering a total dose of dead and / or inactivated bacteria or fragments of their cell wall, containing approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 7 Or 1.5 x 10 4 1 antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species or fragment thereof, consisting of one antigen unit.

[0084] Preparation of bacterial fragments for use as vaccine antigens

[0085] Figure 1 An exemplary method for obtaining bacterial fragments to be used as antigens is illustrated. BugBuster® is used for fresh or frozen cell pellets. Cells are harvested from liquid cultures by centrifugation at 10,000 × g for 10 minutes using a weighed centrifuge tube. For small-scale extractions (1.5 ml or less), centrifugation can be performed in 1.5-ml tubes at 14,000–16,000 × g. Decant the liquid, allowing the pellet to drain and removing as much liquid as possible. The pellet can be weighed. Once the cells are harvested but not inactivated (e.g., by autoclaving or the method described above), pellet them and store at +4°C. Resuspend the cell pellet at room temperature with BugBuster® Master Mix by pipetting or gentle vortexing, using 5 ml of reagent per gram of wet cell paste. This typically corresponds to approximately 2.5 ml per 50-ml culture. Incubate the cell suspension slowly for 10–20 minutes on a shaking platform or rotary mixer at room temperature, then incubate for 1 hour at +25°C, shaking, and remove insoluble cell debris by centrifugation at 16,000 × g for 20 minutes at 4°C. Transfer the supernatant to a new tube. Store the clarified extract on ice for a short period (2–3 hours) or freeze at -20°C until needed. Transfer the cell homogenate to centrifuge tubes. Centrifuge according to the following protocol and transfer the supernatant to a new centrifuge tube.

[0086] Centrifugation steps used (at 4°C): 1) 800×g in 10 minutes 2) 10 minutes, 1500g 3) 10000×g over 60 minutes 4) 3 hours 20000×g

[0087] Add 500 µl of PBS to each precipitate and vortex.

[0088] The collected fragments can be used as antigens in food to vaccinate or treat invertebrates such as shrimp or queen bees and their offspring.

[0089] The composition can be formulated to provide a vaccine composition for delivering a total dose of dead and / or inactivated bacteria or fragments of their cell wall, containing approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 7 Or 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species or fragment thereof, consisting of one antigen unit.

[0090] Using the methods described above, Tables 1-4 and / or Experiment 4 identified bacterial antigens (pathogens) for use alone or in combination in vaccine compositions for the treatment of diseases caused by pathogenic pathogens in invertebrate populations and vaccine formulations for the treatment or prevention of such diseases.

[0091] Table 1

[0092] Table 2 – Vector-borne Diseases

[0093] Table 2 provides additional examples of vector-borne diseases, where the provided bacterial vaccines are used to reduce pathogen load in insects (i.e., vectors), which further serves as a method for the prevention, treatment, or immunization against infectious diseases transmitted to plants, animals, and / or humans. For the diseases listed in Table 1, the method involves applying an effective amount of a non-pathogenic, dead, and / or inactivated Gram-positive bacterial pathogen or its cell wall fragments, alone or in combination with a Gram-negative bacterial pathogen or its cell wall fragments. The method of application is described herein.

[0094] Table 2 – Vector-borne Diseases

[0095] Table 3 – Viral Pathogens in Shrimp – DNA Viruses (Reprinted from https: / / www.biomin.net / species / aquaculture / shrimp-diseases / , last accessed on December 28, 2023)

[0096] Table 4 – Viral Pathogens in Shrimp – RNA Viruses (Reprinted from https: / / www.biomin.net / species / aquaculture / shrimp-diseases / , last accessed on December 28, 2023)

[0097] Preparation of larval Bacillus vaccine and larval Bacillus parahaemolyticus (VP) combination product composition

[0098] The following describes an exemplary method for preparing a composition comprising an effective amount of one or more dead and / or inactivated whole or fragmented larval Bacillus and Vibrio species.

[0099] Larval Bacillus (PL) can be prepared to provide vaccines or treatments for shrimp.

[0100] For this purpose, seed cultures were inoculated from frozen glycerol stock in PL. The inoculated plates were then grown in a dark growth chamber at 35°C for 4–12 days.

[0101] Harvest larval Bacillus colonies by washing the plate with 5 mL of ice-cold H2O and scraping the colonies into Falcon tubes (or glass vials). Measure the optical density (OD600) at 600 nm using standard methods. Mix equal volumes of the harvested bacterial culture and H2O in two replicates into cuvettes. Based on the OD600 reading, prepare the antigen solution to the desired concentration—at least 1.5 x 10⁻⁶. 9 Bacterial cells / mL. Inactivated bacterial solution. Non-limiting examples of inactivating the collected bacteria are formalin, diethyleneimine (BEI), or heat and pressure (autoclaving). Any method of killing bacteria known in the art can be used. After inactivation, cells can be lysed and large fragments separated by centrifugation, for example at a speed of about 10,000 g to about 60,000 g for 1–4 hours (or longer, depending on volume, size, and concentration). The supernatant is recovered and can be further fractionated as described below. Alternatively, whole-cell preparations can be used.

[0102] The supernatant can be freeze-dried, lyophilized, or formulated into a solution and applied to bees in various forms, such as feed, spray, or injection. One method of application includes, but is not limited to, the preparation of insect feed, such as queen bee candy, which can be purchased pre-packaged in beekeeping supply stores or prepared by methods known in the art. The supernatant can be used in combination vaccine compositions with one or more, two or more, three or more, or four or more whole or fragmented bacterial species that are dead and / or inactivated.

[0103] For the production of VP used in Experiment 3, VP strain ATCC-27519 was used. The bacteria were cultured in Marine 2216 broth at 37°C ± 2°C on a shaker at 250 ± 50 rpm for one (1) day. Cultures in the growth phase of the exponential growth phase were inactivated by autoclaving (121°C, 15 min). Inactivation was confirmed by spreading the culture on Marine 2216 agar plates (10 plates) and incubating it at 37°C for 14 days, observing for visible colony growth. Additionally, the culture was spread on TSA blood agar plates (2 plates) and incubated at 37°C for 24–48 hours, observing for visible colony growth. No growth was observed on any of the inoculated plates, confirming sterility. The heat-inactivated vaccine was stored at 2–8°C until use for vaccination.

[0104] The composition can be formulated to provide a vaccine composition for delivering a total dose of dead and / or inactivated bacteria or fragments of their cell wall, containing approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 7 Or 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species or fragment thereof, consisting of one antigen unit.

[0105] Preparation for parameter studies and live bacteria challenge tests. Vibrio and WSSV challenge materials were prepared by the ShrimpVet Laboratory in Vietnam.

[0106] Premixed feed with vaccine or placebo

[0107] Commercial shrimp pellet feed is coated with vaccine-treated material or placebo. (>10) 9 The vaccine treatment is coated onto saturated feed pellets at a dose of 10 cells / mL. Placebo (sterile culture medium) treatment is similarly coated onto pelleted feed. The premixed feed treatment is stored at 2–8°C until used for vaccination.

[0108] Preparation of PL and VP bacterial fragments for use as vaccine antigens

[0109] Figure 1 An exemplary method for obtaining PL and / or VP bacterial fragments for use as antigens is illustrated. BugBuster® can be used for fresh or frozen cell pellets. Harvest cells from liquid cultures by centrifuging at 10,000 × g for 10 minutes using a weighed centrifuge tube. For small-scale extractions (1.5 ml or less), centrifugation can be performed in 1.5-ml tubes at 14,000–16,000 × g. Decant the liquid, allowing the pellet to drain and removing as much liquid as possible. Weigh the pellet. Once the cells are harvested but not autoclaved, they can be pelleted and stored at +4°C. Resuspend the cell pellet at room temperature with BugBuster® Master Mix by pipetting or gentle vortexing, using 5 ml of reagent per gram of wet cell paste. This typically corresponds to approximately 2.5 ml per 50-ml culture. Incubate the cell suspension slowly on a shaking platform or rotary mixer at room temperature for 10–20 minutes. Incubate at +25°C for 1 hour, shaking. Remove insoluble cell debris by centrifugation at 16,000 × g for 20 min at 4°C. Transfer the supernatant to a new tube. Store the clarified extract on ice for a short period (2–3 hours) or freeze at –20°C until needed. Transfer the cell homogenate to a centrifuge tube. Centrifuge according to the following protocol and transfer the supernatant to a new centrifuge tube.

[0110] Centrifugation steps used (at 4°C): 1) 800×g in 10 minutes 2) 10 minutes, 1500g 3) 10000×g over 60 minutes 4) 3 hours 20000×g

[0111] Add 500 µl of PBS to each precipitate and vortex.

[0112] The collected PL fragments, alone or in combination with VP, can be used as antigens in the composition for vaccinating or treating bees, shrimp, and their offspring.

[0113] The composition can be formulated to provide a vaccine composition for delivering a total dose of dead and / or inactivated bacteria or fragments of their cell wall, containing approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 6 To approximately 1.5 x 1011 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 7 Or 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species or fragment thereof, consisting of one antigen unit.

[0114] In one exemplary embodiment, the composition and / or vaccine preparation is given to worker bees, who will mix the vaccine with royal jelly in their mandibular glands and feed it to the queen bee, or give it directly to the queen bee, to effectively treat or immunize the queen bee and her offspring larvae against disease-causing viruses, such as Deformity Wing Virus B (DWV-B).

[0115] In another exemplary embodiment, the composition and / or vaccine preparation is administered to broodstock shrimp to effectively treat and immunize the broodstock shrimp and their larval offspring against disease-causing bacteria and viruses, such as vibriosis and white spot syndrome virus (WSSV). The composition and / or vaccine preparation can be administered to broodstock shrimp as a shrimp feed coated with the composition and / or vaccine preparation, as an injectable agent, or by immersing or soaking the broodstock shrimp in a solution or suspension of the composition and / or vaccine preparation.

[0116] For shrimp feed, commercially available feeds can be used, and vaccines can be combined with feeds through simple mixing or by coating the feed with a vaccine. A non-limiting example of a commercially available feed is Skretting SAPPHIRE feed (see here www.skretting.com / en-in / feed-and-services-for-aquaculture / sapphire-4853 / ), which is also described below as an example of a commercially available feed.

[0117] Vaccination methods

[0118] Treatment or prevention of invertebrate populations

[0119] This disclosure provides a method for preventing or treating infections (e.g., viral or microbial infections) and / or diseases in invertebrates or, for example, those invertebrate populations identified in Tables 1 or alternatively Tables 1-4 and Experiment 4 (e.g., shrimp, crustaceans, and insects such as bees, e.g., honeybees). In one aspect, the method comprises, or is substantially composed of, or consists of: administering or feeding a bacterial vaccine to an invertebrate population that produces or supports invertebrate offspring or the offspring themselves, said bacterial vaccine containing dead and / or inactivated bacteria or fragments thereof specifically selected for treatment against said invertebrate population and viruses causing viral diseases. In one aspect, said administration comprises, or is substantially composed of, oral administration, injection, immersion, or any combination of two or more thereof.

[0120] In another aspect, the method comprises, substantially, or consists of: feeding an effective amount of a bacterial vaccine composition containing dead and / or inactivated bacteria or fragments thereof to adult invertebrates in a population, and then the adult invertebrates passing on immunity and / or treatment to their offspring in the population. In another aspect, the vaccine is administered to the offspring in the population. In one aspect, the vaccine preparation is contained in or mixed with the typical food of the invertebrate acting as a carrier. By way of example only, the invertebrate food may be coated with the vaccine preparation. In one aspect, approximately 1.5 x 10⁻⁶ ppm is provided or fed to the invertebrates (e.g., shrimp, insects such as queen bees, worker bees, caretaker bees, and / or larvae). 7 Or 1.5 x 10 4 To approximately 1.5 x 10 8 Antigen units per gram of food (see Table 1 for a list of paired invertebrates, bacterial vaccines and viruses causing viral infections, Table 2 for a list of vector-borne diseases that can be transmitted from vectors (e.g., insects) to animals, plants and / or humans, and Tables 3 and 4 for a list of shrimp virus pathogens causing shrimp diseases, and mosquito-borne transmission in Experiment 4).

[0121] In one aspect of the method, the administration comprises feeding the invertebrate a suitable amount of food, wherein each dose of vaccine, composition, or formulation administered contains dead and / or inactivated bacteria or fragments thereof, comprising approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species or fragment thereof, consisting of one antigen unit.

[0122] In another aspect, the method further includes detecting microbial infections in the invertebrate population prior to administration of the vaccine (such methods are known in the art). In yet another aspect, the bacterial vaccine is selected to specifically treat and / or prevent the viral infection and the invertebrate population, and then an effective amount of the vaccine is administered to the invertebrate population (see Table 1 for a list of paired invertebrates, bacterial vaccines, and viral infections treated with bacterial vaccines; Table 2 for a list of vector-borne diseases that can be transmitted from vectors (e.g., insects) to animals, plants, and / or humans; and Tables 3 and 4 for a list of shrimp viral pathogens and viral diseases).

[0123] On the other hand, for the treatment of infected populations, the vaccine preparation is administered to infected or uninfected adult worms that produce offspring and introduced into the population, and then the vaccine preparation is passed on to the offspring.

[0124] Vaccination methods: Bacterial or microbial treatment or prevention in invertebrates and invertebrate populations.

[0125] This disclosure provides a method for preventing or treating microbial infections and / or diseases in invertebrate populations or invertebrates (e.g., those identified in Table 1, or alternatively Tables 1-4 and Experiment 4, such as shrimp, crustaceans, and insects like bees, e.g., honeybees). In one aspect, the method comprises, or is substantially composed of, or consists of: administering or feeding a bacterial vaccine to an invertebrate population that produces or supports invertebrate offspring or the offspring themselves, said bacterial vaccine containing dead and / or inactivated bacteria or fragments thereof specifically selected for treatment against said invertebrate population and viruses causing viral diseases. In one aspect, said administration comprises, or is substantially composed of, oral administration, injection, immersion, or any combination of two or more thereof.

[0126] In another aspect, the method comprises, substantially, or consists of: feeding an effective amount of a bacterial vaccine composition containing dead and / or inactivated bacteria or fragments thereof to adult invertebrates in a population, and then the adult invertebrates passing on immunity and / or treatment to their offspring in the population. In another aspect, the vaccine is administered to the offspring in the population. In one aspect, the vaccine preparation is contained in or mixed with the typical food of the invertebrate acting as a carrier. By way of example only, the invertebrate food may be coated with the vaccine preparation. In one aspect, approximately 1.5 x 10⁻⁶ ppm is provided or fed to the invertebrates (e.g., shrimp, insects such as queen bees, worker bees, caretaker bees, and / or larvae). 7 Or 1.5 x 10 4 To approximately 1.5 x 10 8 Antigen units per gram of food (see Table 1 for a list of paired invertebrates, bacterial vaccines and viruses causing viral infections, Table 2 for a list of vector-borne diseases that can be transmitted from vectors (e.g., insects) to animals, plants and / or humans, and Tables 3 and 4 for a list of shrimp virus pathogens causing shrimp diseases) and mosquito-borne diseases in Experiment 4.

[0127] In one aspect of the method, the administration comprises feeding the invertebrate a suitable amount of food, wherein each dose of vaccine, composition, or formulation administered contains dead and / or inactivated bacteria or fragments thereof, comprising approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species or fragment thereof, consisting of one antigen unit.

[0128] In another aspect, the method further includes detecting microbial infections in the invertebrate population prior to administration of the vaccine (such methods are known in the art). In yet another aspect, the bacterial vaccine is selected to specifically treat and / or prevent the viral infection and the invertebrate population, and then an effective amount of the vaccine is administered to the invertebrate population (see Table 1 for a list of paired invertebrates, bacterial vaccines, and viral infections treated with bacterial vaccines; Table 2 for a list of vector-borne diseases that can be transmitted from vectors (e.g., insects) to animals, plants, and / or humans; and Tables 3 and 4 for a list of shrimp viral pathogens and viral diseases and Experiment 4 for mosquito-borne diseases and transmission).

[0129] On the other hand, for the treatment of infected populations, the vaccine preparation is administered to infected or uninfected adult worms that produce offspring and introduced into the population, and then the vaccine preparation is passed on to the offspring.

[0130] Treatment or prevention of microbial and viral infections in bees and shrimp

[0131] bee

[0132] This disclosure provides a method for preventing or treating microbial infections and / or diseases in bees, such as honeybees, and wherein the virus causing the viral infection is selected from Aberrant wingvirus A, Aberrant wingvirus B, Aberrant wingvirus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, and Black queen cell virus. The method comprises, or is substantially composed of, or consists of: administering or feeding a bacterial vaccine to bees that produce or support bee offspring or the offspring themselves, the bacterial vaccine comprising dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof as described herein. In one aspect, the method comprises, or is substantially composed of, or consists of: feeding or injecting an effective amount of a Gram-positive bacterial vaccine composition to the queen bee, who then transmits immunity and / or treatment to her offspring. In another aspect, administering the Gram-positive bacterial vaccine to the offspring. In one aspect, the Gram-positive bacterial vaccine preparation is contained in or mixed in the typical food of queen bees, worker bees, caretaker bees, or bee larvae, said food acting as a carrier. In another aspect, approximately 1.5 x 10⁻⁶ ppm is provided or fed to queen bees, worker bees, caretaker bees, or bee larvae. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 8 One antigen unit per gram of food.

[0133] In one aspect of the method, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise dead and / or inactivated bacterial species of the genus *Bacillus*. In another aspect, the dead and / or inactivated bacterial species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus vesicularus*, *Bacillus assamum*, *Bacillus reductiveazobia*, *Bacillus nitrogen-fixing*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus stellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0134] In one aspect of the method, the application comprises feeding the bees a suitable amount of food, wherein each dose of vaccine, composition, or formulation administered contains dead and / or inactivated Gram-positive bacteria, such as PL, containing approximately 1.5 x 10⁻⁶. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 5 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 7 Or 1.5 x 10 4 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 5 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 6 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 7 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ units. 8 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 9 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 10 At least one dead and / or inactivated Gram-positive bacterium or its cell wall fragment per gram of food containing one antigen unit per gram of food.

[0135] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee husbandry practices. The queen bee, along with 8-10 worker bees, is placed in a "queen cage" and provided with sufficient feed (queen candy) to sustain her for one to two weeks. The oral vaccine is added to the queen candy, on which the queen and worker bees will feed for 3-8 days, after which she is transferred to a new hive to prepare for egg-laying and to establish a new protected colony.

[0136] In another implementation, the vaccine is placed in a queen candy in a queen transport box, or in a mating box or queen rearing box. The care bees will consume the vaccine along with the queen candy and transfer it to their royal jelly glands, where it is mixed with royal jelly fed to the queen larvae or the queen bee.

[0137] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee hive.

[0138] This disclosure provides a method for preventing or treating viral infections and / or diseases in bees, such as honeybees, and wherein the virus causing the viral infection is Deformed Wing Virus B (DWV-B). The method comprises, or is substantially composed of, or consists of: administering or feeding a bacterial vaccine to bees that produce or support bee offspring or the offspring themselves, the bacterial vaccine comprising dead and / or inactivated P. larvae as described herein. In one aspect, the method comprises, or is substantially composed of, feeding an effective amount of a dead and / or inactivated P. larvae bacterial vaccine composition to a queen bee, who then passes on immunity and / or treatment to her offspring. In another aspect, administering the dead and / or inactivated P. larvae vaccine to the offspring. In one aspect, the dead and / or inactivated P. larvae vaccine preparation is contained in or mixed in a typical diet of a queen bee, worker bee, caretaker bee, or bee larvae, the diet acting as a carrier. In one aspect, approximately 1.5 x 10 liters of honey are provided or fed to the queen bee, worker bees, caretaker bees, or bee larvae. 4 To approximately 1.5 x 10 8 One antigen unit per gram of food.

[0139] In one aspect of the method for treating or preventing DWV-B, the administration comprises feeding bees an appropriate amount of food, wherein each dose of vaccine, composition, or formulation administered contains dead and / or inactivated PL or cell wall fragments thereof, comprising approximately 1.5 x 10⁻⁶ ppm. 4 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ units. 7 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 4 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 5One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 6 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 7 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 8 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 9 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ units. 10 At least one dead and / or inactivated PL or its cell wall fragment per gram of food containing one antigen unit per gram of food.

[0140] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee husbandry practices. The queen bee, along with 8-10 worker bees, is placed in a "queen cage" and provided with sufficient feed (queen candy) to sustain her for one to two weeks. The oral vaccine is added to the queen candy, on which the queen and worker bees will feed for 3-8 days, after which she is transferred to a new hive to prepare for egg-laying and to establish a new protected colony.

[0141] In another implementation, the vaccine is placed in a queen candy in a queen transport box, or in a mating box or queen rearing box. The care bees will consume the vaccine along with the queen candy and transfer it to their royal jelly glands, where it is mixed with royal jelly fed to the queen larvae or the queen bee.

[0142] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee hive.

[0143] bumblebee

[0144] This disclosure also provides a method for preventing or treating microbial infections and / or diseases in bees, such as bumblebees, and wherein the virus causing the viral infection is selected from Aberrant wingvirus A, Aberrant wingvirus B, Aberrant wingvirus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, and Black queen cell virus. The method comprises, or is substantially composed of, or consists of: administering or feeding a bacterial vaccine to a bumblebee that produces or supports bumblebee offspring or the offspring themselves, the bacterial vaccine comprising dead and / or inactivated Gram-positive bacteria or fragments thereof as described herein. In one aspect, the method comprises, or is substantially composed of, or consists of: feeding an effective amount of a dead and / or inactivated Gram-positive bacterial vaccine composition to a bumblebee queen, who then transmits immunity and / or treatment to her offspring.

[0145] In one aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated bacterial species of the genus *Bacillus*. In another aspect, the dead and / or inactivated bacterial species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus vesicularus*, *Bacillus assamum*, *Bacillus reductiveazobia*, *Bacillus nitrogen-fixing*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus stellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0146] On the other hand, the deceased and / or inactivated Gram-positive bacterial vaccine is administered to the offspring. In one aspect, the deceased and / or inactivated Gram-positive bacterial vaccine preparation is contained in or mixed with typical food of bumblebee queens, bumblebee workers, bumblebee caretakers, or bumblebee larvae, said food serving as a carrier. In another aspect, approximately 1.5 x 10⁻⁶ liters of the vaccine are provided or fed to bumblebee queens, bumblebee workers, bumblebee caretakers, or bumblebee larvae. 4 To approximately 1.5 x 10 8 One antigen unit per gram of food.

[0147] In one aspect of the method, administration includes feeding bumblebees an appropriate amount of food, wherein each dose of vaccine, composition, or formulation administered contains dead and / or inactivated Gram-positive bacteria or fragments of their cell walls, comprising approximately 1.5 x 10⁻⁶ cells. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 5 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶7 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ units. 8 To approximately 1.5 x 10 10 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 4 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 5 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ units. 6 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 7 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 8 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 9 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic PL or fragment thereof per gram of food containing one antigen unit.

[0148] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee husbandry practices. The queen bee, along with 8-10 worker bees, is placed in a "queen cage" and provided with sufficient feed (queen candy) to sustain her for one to two weeks. The oral vaccine is added to the queen candy, on which the queen and worker bees will feed for 3-8 days, after which she is transferred to a new hive to prepare for egg-laying and to establish a new protected colony.

[0149] In another implementation, the vaccine is placed in a queen candy in a queen transport box, or in a mating box or queen rearing box. The care bees will consume the vaccine along with the queen candy and transfer it to their royal jelly glands, where it is mixed with royal jelly fed to the queen larvae or the queen bee.

[0150] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee hive.

[0151] On the other hand, the above method further includes detecting (such methods are known in the art) the virus or viral infection of the bee or bumblebee before administering the dead and / or inactivated PL vaccine, and administering the PL vaccine to the bee or bumblebee, such as queen bee, worker bee, caretaker bee, or bee larvae.

[0152] shrimp

[0153] WSSV (Vitiligo Syndrome Virus)

[0154] White spot syndrome virus (WSSV) is by far the most destructive pathogen in farmed shrimp. It infects all farmed shrimp and is responsible for many of the economic impacts of disease in shrimp production worldwide.

[0155] White spot syndrome virus (WSSV) is a highly virulent pathogen affecting farmed shrimp. It can cause mortality rates of up to 100% within 3–10 days. WSSV is a large, enveloped double-stranded DNA virus belonging to the genus Whispovirus in the family Nimaviridae. It has a broad host range in crustaceans such as shrimp, crabs, lobsters, and crayfish, and primarily affects commercially farmed marine shrimp species. The virus infects all age groups, leading to significant mortality. Ectodermal and mesodermal tissues, such as gills, lymphoid organs, and epidermal epithelium, are the primary sites of infection. White spot disease caused by WSSV remains a major obstacle to sustainable shrimp farming.

[0156] White spot syndrome virus (WSSV) has a wide host range, affecting farmed and wild marine shrimp, crabs, lobsters, crayfish, mantis shrimp, copepods, and freshwater species such as the giant freshwater prawn (Macrobrachium rosenbergii). It poses a potential threat to most commercially important shrimp species, including *Penaeus monodon*, *Penaeus vannamei*, *Penaeus indicus*, *Penaeus japonicus*, *Penaeus chinensis*, *Penaeus penicillatus*, *Penaeus azteus*, *Penaeus merguiensis*, *F. duorarum*, and *P. stylirostris*.

[0157] Acute hepatopancreatic necrosis disease (AHPND)

[0158] Vibrio infection is a significant bacterial disease caused by opportunistic Vibrio species and remains one of the most serious threats to shrimp farmers. Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, Vibrio splendidus, Vibrio salmonicida, Vibrio vulnificus, and Vibrio parahaemolyticus strains have been identified as the main pathogens of vibrio infection. Acute hepatopancreatic necrosis disease (AHPND), originally called Early Death Syndrome (EMS), is a relatively new bacterial disease affecting farmed shrimp. Acute hepatopancreatic necrosis disease (AHPND) is caused by infection with a strain of Vibrio parahaemolyticus (VpAHPND) carrying a plasmid of approximately 70 kbp. This plasmid contains genes encoding Pir toxins from Photorhabdus insects, specifically PirA and PirB. Although other Vibrio species have been isolated from clinical AHPND cases, only VpAHPND has been identified as the causative agent.

[0159] AHPND affects a variety of shrimp species, including commercially important species such as *Penaeus monodon*, *L. vannamei*, and *Macrobrachium rosenbergii*, as well as *Artemia franciscana*. Early-life shrimp are particularly susceptible to AHPND infection. AHPND is characterized by high mortality, typically occurring when shrimp are one month old or larvae are approximately 20-30 days old. The disease is characterized by severe hepatopancreatic atrophy, distinctive histopathological changes in the acute phase, and subsequent extensive shedding of hepatopancreatic or digestive tract epithelial cells within the first 30 days after stocking. Notably, the bacteria causing AHPND primarily target the digestive gland (hepatopancreas), damaging the R (absorption), B (vacuole), F (fibrous), and E (embryonic) cells of the hepatopancreas, leading to dysfunction and significant shrimp mortality. Affected shrimp exhibit lethargy, anorexia, slow growth, an empty digestive tract, and a pale to white hepatopancreas.

[0160] Traditional methods, such as antibiotics and disinfectants, have shown limited effectiveness in mitigating or curing AHPND. Furthermore, their use is associated with alterations in the host's gut microbiota, immunity, and the development of antibiotic resistance in bacterial pathogens. For example, the Mexican strains of Vibrio parahaemolyticus causing AHPND (13-306D / 4 and 13-511 / A1) carry the tetB gene responsible for tetracycline resistance, while Vibrio campbellii from China carries multiple antibiotic resistance genes. Therefore, there is an urgent need to develop innovative health management strategies, such as vaccination, to prevent or control AHPND in shrimp farming. This effort is crucial for ensuring future food security and providing economic stability for farmers. Crustacean and shrimp vaccine compositions and administration methods

[0161] This disclosure provides a method for preventing or treating microbial infections and / or diseases in crustaceans such as shrimp, wherein the shrimp are selected from rock shrimp, pink shrimp, tiger shrimp, Chinese white shrimp, brown shrimp, white shrimp, Atlantic northern shrimp, tiger prawn, Aesop shrimp, banana shrimp, and blue shrimp. In one aspect of the method, the pathogen causing the microbial infection is selected from white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), baculovirus penaenii (BP), shrimp iridovirus (IRIDO), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), or covert mortality Noda virus (CMNV), or those identified in Tables 3 and 4 below. On the other hand, the bacterial vaccine comprises whole cells or cell wall fragments of dead and / or inactivated Gram-positive and / or Gram-negative bacteria. On the other hand, the shrimp or population to be vaccinated does not include *P. polymyxa* in the vaccine or feed used for vaccination against *V. parahaemolyticus* infection or disease, when *P. polymyxa* is the sole active agent in the vaccine. On the other hand, vaccines for treating shrimp, populations, or offspring do not include *V. anguillarum*, used for treating or preventing infections in shrimp caused by *Vibrio* species.

[0162] In one aspect of the method, the whole cell or cell wall fragment of the dead and / or inactivated Gram-negative bacteria comprises a dead and / or inactivated species of Vibrio. On the other hand, the Gram-negative bacteria are selected from *V. adaptatus*, *V. aerogenes*, *Vibrio summerensis*, *Vibrio estuarineis*, *Vibrio alginolyticus*, *V. albensis*, *Vibrio alphaca*, *Vibrio alginolyticus*, *Vibrio anguillarum*, *V. areninigrae*, *V. artabrorum*, *Vibrio atlantos*, *Vibrio atypical*, *Vibrio cyanobacterium*, *Vibrio brasiliensis*, *V. bubulus*, *V. calviensis*, *Vibrio cannii*, *V. casei*, *Vibrio chagusi*, *Vibrio cholerae*, *V. cincinnatiensis*, *Vibrio lysomorphus*, *Vibrio oysteri*, *Vibrio cyclohexane*, *Vibrio demigodius*, *Vibrio diazotrophus*, *Vibrio smotherium*, *Vibrio fluvii*, *Vibrio robustus*, *Vibrio fernsi*, *Vibrio gallophyllus*, *Vibrio aurorum*, *Vibrio gibberella*, *Vibrio abaloneniformis*, *Vibrio harveyi*, *Vibrio hepatis*, *V. hippocampi*, *Vibrio myasthenia gravis*, *V. ichthyoenteri*, and *V. Vibrio indicus, Vibrio canarolosum, Vibrio slow-moving, Vibrio shoal, Vibrio logei, Vibrio mediterranei, Vibrio mechnikhovella, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonate, Vibrio tsinae, Vibrio nervosa, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio panniae, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio nigra, Vibrio rotaeni, Vibrio rumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio superstes, Vibrio rumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio supreme, Vibrio rumoiensis, Vibrio supreme. Vibrio tapetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vodanensis and / or Vibrio xuensis, or a combination of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio mermaidii, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawni, Vibrio vulnificus, Vibrio nerei, Vibrio tasmani, Vibrio fluvialis, Vibrio splenium and / or Vibrio niger, or a combination of two or more thereof.

[0163] In another aspect of the method, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise dead and / or inactivated bacterial species of the genus *Bacillus*. In another aspect, the dead and / or inactivated bacterial species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus vesicularus*, *Bacillus assamum*, *Bacillus reductiveazobia*, *Bacillus nitrogen-fixing*, *Bacillus Barcelona*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Bacillus residui*, *Paenibacillus sttellife*, thiamine-derived *Bacillus*, *Paenibacillus validus*, and xylolytic *Bacillus*, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval *Bacillus* (PL) or cell wall fragments thereof. In yet another aspect, the bacterial vaccine comprises dead and / or inactivated bacterial species of the genus *Bacillus* and / or the genus *Vibrio*.

[0164] In one aspect of the method, the method comprises or is substantially composed of or consists of: administering a bacterial vaccine to a shrimp that produces or supports shrimp offspring or the offspring themselves, the bacterial vaccine comprising whole cells or cell wall fragments of dead and / or inactivated Gram-positive and / or Gram-negative bacteria. In another aspect of the method, the bacterial vaccine comprises *V. adaptatus*, *V. aerogenes*, *Vibrio estuarineis*, *Vibrio estuarineis*, *Vibrio alginolyticus*, *V. albensis*, *Vibrio alphaca*, *Vibrio alginolyticus*, *Vibrio anguillarum*, *V. areninigrae*, *V. artabrorum*, *Vibrio atypical*, *Vibrio cyanobacterium*, *Vibrio brasiliensis*, *V. bubulus*, *V. calviensis*, *Vibrio cannii*, *V. casei*, *Vibrio chagusi*, *Vibrio cholerae*, *V. cincinnatiensis*, *Vibrio lysomorphus*, *Vibrio oysteri*, *Vibrio cyclohexane*, *Vibrio demigodius*, *Vibrio diazotrophus*, *Vibrio smotherium*, *Vibrio fluvii*, *Vibrio virulentus*, *Vibrio fernigina*, *Vibrio gallophylla*, *Vibrio aerogenes*, *Vibrio megaterium*, *Vibrio abaloneniformis*, *Vibrio harveyi*, *Vibrio hepatis*, *V. hippocampi*, *Vibrio myasthenia gravis*, *V. ichthyoenteri*, and *V. Vibrio indicus, Vibrio canarolosum, Vibrio slow-moving, Vibrio shoal, Vibrio logei, Vibrio mediterranei, Vibrio mechnikhovella, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonate, Vibrio tsinae, Vibrio nervosa, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio panniae, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio nigra, Vibrio rotaeni, Vibrio rumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio superstes, Vibrio rumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio supreme, Vibrio rumoiensis, Vibrio supreme. Vibrio tapetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vortanense, and / or Vibrio xuensis, or combinations of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio mermaidii, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawni, Vibrio vulnificus, Vibrio nerei, Vibrio tasmani, Vibrio fluvialis, Vibrio splenium, and / or Vibrio niger, or combinations of two or more thereof, of dead and / or inactivated whole cells or cell wall fragments. In another aspect of the method, the bacterial vaccine comprises dead and / or inactivated PL whole cells or cell wall fragments.

[0165] In one aspect, administration includes oral administration (e.g., feeding), injection, and / or immersion, or is substantially composed of, or consists of, the bacterial vaccine. In another aspect, shrimp feed is coated with the bacterial vaccine. In yet another aspect, the shrimp, shrimp producing or supporting offspring, or the offspring themselves are immersed in a solution or suspension of the bacterial vaccine. In yet another aspect, the bacterial vaccine is prepared as a solution or suspension in which an effective amount of the dead and / or inactivated bacterial vaccine composition is added to water (e.g., sterile aged brackish water or seawater), and the shrimp, shrimp producing or supporting offspring, or the offspring themselves are immersed in the solution or suspension. For example, for oral (os) vaccination, it is administered for two to seven days, whether after injection or alone. Medicated feed pellets are top-coated with 9 mL / kg of vaccine and fed at a rate of up to 10% of body weight per day, divided into one meal per day.

[0166] Mix the vaccine with drinking water / distilled water until homogeneous. The mixture should be at the desired top-coating concentration and include 20% extra volume to compensate for volume loss due to adhesion / cohesion and to maintain spray pressure. Place the homogeneous solution into a spray bottle and weigh it. Place 1 kg of commercial shrimp feed into a container. For example, Skretting SAPPHIRE feed pellets (see here: https: / / www.skretting.com / en-in / feed-and-services-for-aquaculture / sapphire-4853 / , last accessed October 27, 2024), and gradually spray 100 g of the test product solution onto the surface of the commercial feed. Mix the feed continuously throughout the process.

[0167] In one aspect, the method comprises, substantially, or consists of: feeding female broodstock shrimp an effective amount of a bacterial vaccine composition containing dead and / or inactivated Gram-positive and / or Gram-negative whole cells or cell wall fragments, and then the female broodstock shrimp passing on immunity and / or treatment to their offspring. In another aspect, the dead and / or inactivated bacterial vaccine is administered to the offspring. In one aspect, the dead and / or inactivated bacterial vaccine preparation is contained in or mixed in a typical diet of the female broodstock shrimp or shrimp larvae, the diet acting as a carrier. In one aspect, approximately 1.5 x 10⁻⁶ ppm is provided or fed to the female broodstock shrimp or shrimp larvae. 4 To approximately 1.5 x 10 8 One antigen unit per gram of food.

[0168] In one aspect of the method, the application comprises feeding, injecting, and / or immersing female broodstock shrimp in a suitable amount of bacterial vaccine, wherein each dose of vaccine, composition, or formulation administered contains dead and / or inactivated Gram-positive and / or Gram-negative bacteria, comprising approximately 1.5 x 10⁻⁶ bacteria.7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 7 Or 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 A vaccine consisting of at least one dead, non-pathogenic bacterial antigen unit.

[0169] In one embodiment, the bacterial vaccine is injected into female broodstock shrimp.

[0170] In another embodiment of the method, the application includes feeding female broodstock shrimp an effective amount of the dead and / or inactivated bacterial vaccine composition, injecting female broodstock shrimp with an effective amount of the dead and / or inactivated bacterial vaccine composition, immersing female broodstock shrimp in an effective amount of the dead and / or inactivated bacterial vaccine composition, or any combination of two or more thereof. In one aspect, the bacterial vaccine composition comprises about 1.5 x 10⁻⁶ ppm. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 7 Or 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species with one antigen unit.

[0171] In one embodiment of the method, the administration comprises feeding offspring an effective amount of the dead and / or inactivated bacterial vaccine composition, injecting offspring with an effective amount of the dead and / or inactivated bacterial vaccine composition, immersing offspring in an effective amount of the dead and / or inactivated bacterial vaccine composition, or any combination of two or more thereof. In one aspect, the bacterial vaccine composition comprises about 1.5 x 10⁻⁶ ppm. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 104 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic bacterial species with one antigen unit.

[0172] In one aspect of the invention, the vaccine composition comprises, or is substantially composed of, a combination vaccine prepared by mixing effective amounts of inactivated P. larvae and V. parahaemolyticus to provide shrimp with protective benefits against WSSV and / or AHPND.

[0173] Group treatment or prevention of bees and shrimp

[0174] bee colony

[0175] This disclosure provides a method for preventing or treating microbial infections and / or diseases in bee colonies, wherein the pathogen causing the infection is selected from Aberrant wingvirus A, Aberrant wingvirus B, Aberrant wingvirus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, and Black queen cell virus. In one aspect, the method comprises, or is substantially composed of, or consists of: administering or feeding a bacterial vaccine preparation containing Gram-positive bacteria to a bee colony that produces or supports bee colony (community) offspring or bee offspring. In another aspect, the method comprises, or is substantially composed of, or consists of: feeding an effective amount of the bacterial vaccine composition to the queen bee or queen-supporting bee in the colony (community), whereby the queen bee or queen-supporting bee transmits immunity and / or treatment to its offspring in the colony or community. In another aspect, the PL vaccine is administered to the offspring in the colony. In one aspect, the vaccine preparation is contained in or mixed in a typical bee diet that serves as a carrier. In one aspect, approximately 1.5 x 10 liters of honey are provided or fed to bees in a colony or community, such as the queen bee, worker bees, caretaker bees, and / or bee larvae. 4 To approximately 1.5 x 10 8 One antigen unit per gram of food.

[0176] In one aspect of the method, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise dead and / or inactivated bacterial species of the genus *Bacillus*. In another aspect, the dead and / or inactivated bacterial species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus vesicularus*, *Bacillus assamum*, *Bacillus reductiveazobia*, *Bacillus nitrogen-fixing*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus stellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0177] In one aspect of the method, the application comprises feeding the bees in the colony a suitable amount of food containing an effective amount of dead and / or inactivated Gram-positive bacteria, wherein the amount of dead and / or inactivated Gram-positive bacteria applied per dose of vaccine, composition, or formulation contains approximately 1.5 x 10⁻⁶ bacteria. 4 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 4 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 5 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 6 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 7 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 8 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 9 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 10 One antigen unit per gram of food.

[0178] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee husbandry practices. The queen bee, along with 8-10 worker bees, is placed in a "queen cage" and provided with sufficient feed (queen candy) to sustain her for one to two weeks. The oral vaccine is added to the queen candy, on which the queen and worker bees will feed for 3-8 days, after which she is transferred to a new hive to prepare for egg-laying and to establish a new protected colony.

[0179] In another implementation, the vaccine is placed in a queen candy in a queen transport box, or in a mating box or queen rearing box. The care bees will consume the vaccine along with the queen candy and transfer it to their royal jelly glands, where it is mixed with royal jelly fed to the queen larvae or the queen bee.

[0180] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee hive.

[0181] This disclosure provides a method for preventing or treating viral infections and / or diseases in bee colonies, wherein the virus causing the viral infection is Deformed Wing Virus B (DWV-B). In one aspect, the method comprises, or is substantially composed of, or consists of: administering or feeding a bee colony that produces or supports bee colony (colony) offspring or bee offspring a dead and / or inactivated PL bacterial vaccine preparation comprising dead and inactivated whole cells or cell wall fragments. In another aspect, the method comprises, or is substantially composed of, feeding an effective amount of a bacterial vaccine composition comprising dead and / or inactivated PL or fragments thereof to the queen bee or queen-supporting bee in the colony (colony), whereby the queen bee or queen-supporting bee transmits immunity and / or treatment to its offspring in the colony or colony. In another aspect, the dead and / or inactivated PL vaccine is administered to the offspring in the colony. In one aspect, the vaccine preparation is contained in or mixed in a typical bee diet that serves as a carrier. In one aspect, approximately 1.5 x 10 liters of honey are provided or fed to bees in a colony or community, such as the queen bee, worker bees, caretaker bees, and / or bee larvae. 4 To approximately 1.5 x 10 8 One antigen unit per gram of food.

[0182] In one aspect of the method, the application comprises feeding the bees in the colony an appropriate amount of food, wherein each dose of vaccine, composition, or formulation administered contains dead and / or inactivated PL, comprising approximately 1.5 x 10⁻⁶ ppm. 4 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 4 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 5One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 6 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 7 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 8 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 9 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 10 At least one dead, non-pathogenic PL or fragment thereof per gram of food containing one antigen unit.

[0183] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee husbandry practices. The queen bee, along with 8-10 worker bees, is placed in a "queen cage" and provided with sufficient feed (queen candy) to sustain her for one to two weeks. The oral vaccine is added to the queen candy, on which the queen and worker bees will feed for 3-8 days, after which she is transferred to a new hive to prepare for egg-laying and to establish a new protected colony.

[0184] In another implementation, the vaccine is placed in a queen candy in a queen transport box, or in a mating box or queen rearing box. The care bees will consume the vaccine along with the queen candy and transfer it to their royal jelly glands, where it is mixed with royal jelly fed to the queen larvae or the queen bee.

[0185] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee hive.

[0186] In another aspect, the method further includes detecting (such methods are known in the art) the virus or viral infection in the bee colony or population prior to the administration of the PL vaccine (e.g., DWV-B). In another aspect, for the treatment of a virally infected colony, the vaccine preparation is administered to uninfected adults that produce offspring (uninfected queen bees) and introduced into the colony, and then the vaccine preparation is passed on to the offspring.

[0187] bumblebee colony

[0188] This disclosure provides a method for preventing or treating microbial infections and / or diseases in a bumblebee colony. In one aspect of the method, the microbial infection is a virus selected from Aberrant wingvirus A, Aberrant wingvirus B, Aberrant wingvirus C, Acute Bee Paralysis Virus, Israel Acute Bee Paralysis Virus, Kashmir Bee Virus, Slow Bee Paralysis Virus, Lake Sinai Virus 1, Lake Sinai Virus 2, Chronic Bee Paralysis Virus, Sacbryovirus, and Black Bee Queen Cell Virus. In one aspect, the method comprises, or is substantially composed of, or consists of: administering or feeding a bacterial vaccine preparation to a bumblebee colony that produces or supports offspring of a bumblebee colony (community) or bumblebee offspring, the bacterial vaccine preparation comprising dead and / or inactivated whole cells of Gram-positive bacteria or fragments of their cell walls. In another aspect, the method comprises, or is substantially composed of, or consists of: feeding an effective amount of a bacterial vaccine composition to a queen bumblebee or a queen bumblebee supporting a queen bumblebee in the colony (community), and then the queen bumblebee or queen bumblebee supporting a queen bumblebee transfers immunity and / or treatment to its offspring in the colony or community. On the other hand, the bacterial vaccine is administered to the offspring in the colony. In one aspect, the bacterial vaccine preparation is contained in or mixed with typical bumblebee food that serves as a carrier. In another aspect, approximately 1.5 x 10-1 liters are provided or fed to bumblebees in the colony or community, such as queen bees, worker bees, caretaker bees, and / or larvae. 4 To approximately 1.5 x 10 8 One antigen unit per gram of food.

[0189] In one aspect of the method, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise dead and / or inactivated bacterial species of the genus *Bacillus*. In another aspect, the dead and / or inactivated bacterial species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus vesicularus*, *Bacillus assamum*, *Bacillus reductiveazobia*, *Bacillus nitrogen-fixing*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus stellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0190] In one aspect of the method, the application comprises feeding the bumblebees in the colony an appropriate amount of food, wherein each dose contains approximately 1.5 x 10^6 dead and / or inactivated Gram-positive bacteria or fragments of their cell walls. 4 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit per gram of food, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶4 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 5 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 6 One antigen unit per gram of food, or at least 1.5 x 10⁻⁶ 7 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 8 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 9 One antigen unit per gram of food, or at least approximately 1.5 x 10⁻⁶ 10 One antigen unit per gram of food.

[0191] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee husbandry practices. The queen bee, along with 8-10 worker bees, is placed in a "queen cage" and provided with sufficient feed (queen candy) to sustain her for one to two weeks. The oral vaccine is added to the queen candy, on which the queen and worker bees will feed for 3-8 days, after which she is transferred to a new hive to prepare for egg-laying and to establish a new protected colony.

[0192] In another implementation, the vaccine is placed in a queen candy in a queen transport box, or in a mating box or queen rearing box. The care bees will consume the vaccine along with the queen candy and transfer it to their royal jelly glands, where it is mixed with royal jelly fed to the queen larvae or the queen bee.

[0193] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee hive.

[0194] In another aspect, the method further includes detecting a virus or viral infection in the bumblebee colony or population prior to the administration of the PL vaccine (such methods are known in the art). In another aspect, for the treatment of infected colonies, the vaccine is administered to uninfected adults that produce offspring (uninfected queen bees), which are then introduced into the colony, and the vaccine formulation is subsequently passed on to the offspring.

[0195] Shrimp Group

[0196] This disclosure provides a method for preventing or treating microbial infections and / or diseases in shrimp populations, wherein the shrimp are selected from rock shrimp, pink shrimp, tiger shrimp, Chinese white shrimp, brown shrimp, white shrimp, Atlantic northern shrimp, tiger prawn, Aesop shrimp, banana shrimp, and blue shrimp. In one aspect of the method, the pathogen causing the infection is selected from white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic tissue necrosis virus (IHHNV), shrimp baculovirus (BP), shrimp iridovirus (IRIDO), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), or latent death Nodamura virus (CMNV) or those identified in Tables 3 and 4 below; and wherein the bacterial vaccine comprises dead and / or inactivated whole cells of Gram-positive and / or Gram-negative bacteria or fragments of their cell walls. On the other hand, the Gram-negative bacteria are selected from *V. adaptatus*, *V. aerogenes*, *Vibrio summerensis*, *Vibrio estuarineis*, *Vibrio alginolyticus*, *V. albensis*, *Vibrio alphaca*, *Vibrio alginolyticus*, *Vibrio anguillarum*, *V. areninigrae*, *V. artabrorum*, *Vibrio lataniae*, *Vibrio atypical*, *Vibrio cyanobacterium*, *Vibrio brasiliensis*, *V. bubulus*, *V. calviensis*, *Vibrio cannii*, *V. casei*, *Vibrio chagusi*, *Vibrio cholerae*, *V. cincinnatiensis*, *Vibrio lysomorphus*, *Vibrio oysteri*, *Vibrio cyclohexane*, *Vibrio demigodius*, *Vibrio diazotrophus*, *Vibrio smotherium*, *Vibrio fluvii*, *Vibrio virulentus*, *Vibrio fernsi*, *Vibrio gallophylla*, *Vibrio abaloneniformis*, *Vibrio harveyi*, *Vibrio hepatis*, *V. hippocampi*, *Vibrio myasthenia gravis*, *V. ichthyoenteri*, and *V. Vibrio indicus, Vibrio canarolosum, Vibrio slow-moving, Vibrio shoal, Vibrio logei, Vibrio mediterranei, Vibrio mechnikhovella, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonate, Vibrio tsinae, Vibrio nervosa, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio panniae, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio nigra, Vibrio rotaeni, Vibrio rumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio superstes, Vibrio rumoiensis, Vibrio scophthalmi, Vibrio splenium, Vibrio supreme, Vibrio rumoiensis, Vibrio supreme. Vibrio tapetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vodanensis and / or Vibrio xuensis, or a combination of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio mermaidii, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawni, Vibrio vulnificus, Vibrio nerei, Vibrio tasmani, Vibrio fluvialis, Vibrio splenium and / or Vibrio niger, or a combination of two or more thereof.On the other hand, the shrimp or population to be vaccinated does not include *P. polymyxa* in the vaccine or feed used for vaccination against *V. parahaemolyticus* infection or disease, when *P. polymyxa* is the sole active agent in the vaccine. On the other hand, vaccines used to treat shrimp, populations, or offspring do not include *V. anguillarum*, used to treat or prevent infections in shrimp caused by *Vibrio* species.

[0197] In another aspect of the method, the dead and / or inactivated Gram-positive bacteria or their cell wall fragments comprise dead and / or inactivated bacterial species of the genus *Bacillus*. In another aspect, the dead and / or inactivated bacterial species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus vesicularus*, *Bacillus assamum*, *Bacillus azotobacter*, *Bacillus nitrogen-fixing*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Bacillus residui*, *Paenibacillus sttellife*, thiamine-derived *Bacillus*, *Paenibacillus validus*, and xylolytic *Bacillus*, or combinations of two or more thereof. In another aspect, the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval *Bacillus* (PL) or cell wall fragments thereof. In yet another aspect, the bacterial vaccine comprises dead and / or inactivated bacterial species of the genus *Bacillus* and / or the genus *Vibrio*.

[0198] In one aspect, the method comprises, or is substantially composed of, or consists of: administering a bacterial vaccine preparation to a population of female broodstock shrimp or their offspring. In one embodiment of the method, the administration comprises feeding the female broodstock shrimp or their offspring an effective amount of the bacterial vaccine composition, injecting the female broodstock shrimp or their offspring with an effective amount of the bacterial vaccine composition, immersing the female broodstock shrimp or their offspring in an effective amount of the bacterial vaccine composition, or any combination of two or more thereof.

[0199] In another aspect, the method comprises, substantially, or consists of: feeding, submerging, and / or injecting an effective amount of a bacterial vaccine composition to female broodstock shrimp, who then pass on immunity and / or treatment to their offspring in a colony or community. In another aspect, the bacterial vaccine is administered to the offspring in the colony. In one aspect, the vaccine preparation is contained in or mixed with a typical shrimp diet that serves as a carrier. In one aspect, approximately 10 [units of something] are provided or fed to shrimp in a colony or community, such as female broodstock shrimp and / or shrimp larvae. 4 To about 10 8 One antigen.

[0200] In some embodiments, the methods of administration include, but are not limited to, oral administration, injection, and submersion. In one embodiment, for injection administration, 10 mg / tail is used per tail. 4 -10 8 The vaccine composition of the present invention is injected into shrimp at a dose of CFU. In another embodiment, for administration by immersion, a vaccine stock solution is prepared by dissolving and / or suspending the vaccine composition of the present invention in water (e.g., sterile aged brackish water or seawater), and the shrimp are immersed in the suspension. As an example only, the bacterial vaccine composition is added to water and diluted in 20 L of water to achieve a 10:10 concentration. 2 - 10 5 For a concentration of CFU / ml, the soaking time is approximately 30 minutes or longer, such as 1 hour, 2 hours, 3 hours or longer.

[0201] Therefore, this disclosure also provides for using heat-inactivated Vibrio cells as adjuvants to improve the immunogenicity of vaccines made from formalin-inactivated Vibrio cells or BEI-inactivated Vibrio cells.

[0202] In one embodiment, the vaccine composition is delivered to female broodstock shrimp by injection. In yet another embodiment, the vaccine is delivered to female broodstock shrimp and / or shrimp offspring by feeding them feed coated with the vaccine composition.

[0203] Vaccine compositions for the treatment and prevention of viral and bacterial infections in invertebrates.

[0204] An invertebrate vaccine and / or composition and / or formulation is provided, comprising, or consisting substantially of, whole-cell or cell wall fragments of at least one, or at least two, or at least three, or at least four or more dead and / or inactivated non-pathogenic bacterial species. In one aspect, the invertebrate vaccine and / or composition and / or formulation comprises, or consists substantially of, whole-cell or cell wall fragments of at least one, or at least two, or at least three, or at least four or more dead and / or inactivated non-pathogenic Gram-positive and / or Gram-negative bacterial species.

[0205] A method for preparing invertebrate vaccines and / or compositions and / or formulations is also provided, comprising isolating whole cells or cell wall fragments from at least one, or at least two, or at least three, or at least four or more dead, non-pathogenic bacterial species as shown in Table 1, or alternatively identified in Tables 1-4 and Experiment 4.

[0206] In one embodiment, the method further includes mixing the isolated antigen units with insect food or a carrier, queen bee flakes, or a gel. In one aspect, the bacterial species is a species of the genus *Bacillus*, such as *Bacillus apis* or *Bacillus dendriticus*, or a combination thereof. In another aspect, the dead *Bacillus* species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus apis*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus apis*, *Bacillus assamum*, *Bacillus azotocinus*, *Bacillus nitrogenophilus*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brasiliensis*, and *P.* brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus stellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or combinations thereof. On the other hand, the dead *Bacillus* species are *Bacillus apis* and *Bacillus dendriticus*.

[0207] On the other hand, the dead and / or inactivated whole cells or cell wall fragments from at least one dead and / or inactivated non-pathogenic bacterial species are selected from V. adaptatus, V. aerogenes, Vibrio summerensis, Vibrio estuarineis, Vibrio alginolyticus, V. albensis, Vibrio alfacoidus, Vibrio alginolyticus, Vibrio anguillarum, V. areninigrae, V. artabrorum, Vibrio atlantos, Vibrio atypical, Vibrio cyanobacterium, Vibrio brasiliensis, V. bubulus, V. calviensis, Vibrio cannii, V. casei, Vibrio chagusi, Vibrio cholerae, V. cincinnatiensis, Vibrio coralloides, Vibrio oysteriformis, Vibrio cyclohexanephaga, Vibrio devilii, Vibrio diazotrophus, Vibrio well-faced, Vibrio fluvibrio, Vibrio virulentis, Vibrio furnis, Vibrio gallophylla, Vibrio abaloneniformis, Vibrio harveyi, Vibrio hepatis, V. Vibrio hippocampi, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolo, Vibrio slow-moving, Vibrio riparia, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonatal, Vibrio tsoriopteris, Vibrio nervosa, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio pannioides, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio brevicornuate, Vibrio rotiferus, Vibrio rubrumensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio brevicornuate, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae. Vibrio superstes, V. tapepetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vortanense, and / or Vibrio xuei, or combinations of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio mermaidii, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawnicella, Vibrio vulnificus, Vibrio nerei, Vibrio tasmani, Vibrio fluvialis, Vibrio splenium, and / or Vibrio niger, or combinations of two or more thereof. In another aspect, the invertebrate vaccine and / or composition and / or formulation comprises dead and / or inactivated bacterial species of the genus Bacillus and / or Vibrio.

[0208] In one respect, each dose provides at least one, or at least two, or at least three, or at least four or more dead, non-pathogenic bacterial species, in an amount of about 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5 One antigen unit, or at least 1.5 x 10⁻⁶ 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 One antigen unit.

[0209] A composition is also provided comprising about 1.5 x 10 4 To approximately 1.5 x 10 8 One antigen unit per gram of invertebrate food containing dead and / or inactivated bacteria, or alternatively approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 6 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 7 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 8 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 8 To approximately 1.5 x 10 10One antigen unit per gram of invertebrate food, or approximately 1.5 x 10-1 8 To approximately 1.5 x 10 9 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 4 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 5 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 6 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 7 One antigen unit per gram of invertebrate food, or at least approximately 1.5 x 10⁻⁶ units. 8 One antigen unit per gram of invertebrate food, or at least approximately 1.5 x 10⁻⁶ units. 9 One antigen unit per gram of invertebrate food, or at least approximately 1.5 x 10⁻⁶ units. 10 One antigen unit per gram of invertebrate food.

[0210] Non-limiting examples of the carrier may be solid or liquid carriers and may include preservatives, insect nutrients, invertebrate nutrients, or other colorants as necessary. In one specific embodiment, the carrier is invertebrate food (shrimp food) or insect food, such as queen bee flakes or sugar feed. In an example where the carrier is invertebrate food, the invertebrate food is coated with the bacterial vaccine and / or composition and / or formulation. In another embodiment, the carrier is adapted for injecting the bacterial vaccine and / or composition and / or formulation. In yet another embodiment, the carrier is water (e.g., sterile aged brackish water or seawater) for dissolving and / or suspending the bacterial vaccine and / or composition and / or formulation, such that the invertebrate can be immersed in the solution or suspension.

[0211] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee husbandry practices. The queen bee, along with 8-10 worker bees, is placed in a "queen cage" and provided with sufficient feed (queen candy) to sustain her for one to two weeks. The oral vaccine is added to the queen candy, on which the queen and worker bees will feed for 3-8 days, after which she is transferred to a new hive to prepare for egg-laying and to establish a new protected colony.

[0212] In another implementation, the vaccine is placed in a queen candy in a queen transport box, or in a mating box or queen rearing box. The care bees will consume the vaccine along with the queen candy and transfer it to their royal jelly glands, where it is mixed with royal jelly fed to the queen larvae or the queen bee.

[0213] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee hive.

[0214] In some embodiments, the methods of administration include, but are not limited to, oral administration, injection, and / or immersion (shrimp). In one embodiment, for injection administration, the dosage is 10 mg per individual. 4 -10 8 The vaccine composition of the present invention is administered to invertebrates, including bees and shrimp, at a dose of CFU. In another embodiment, for administration by immersion, a vaccine stock solution is prepared by immersion in the vaccine composition of the present invention and the vaccine is administered to shrimp, for example, prepared in a 1 L tank from sterile aged brackish water or seawater, and the solution can be diluted to 10 in 20 L of water to achieve a concentration of 10. 2 -10 5 The concentration is achieved by soaking for approximately 30 minutes or longer, such as 1 hour, 2 hours, 3 hours or longer.

[0215] Vaccines for treating vector-borne diseases

[0216] Vector-borne diseases (see, for example, Table 2), such as mosquito-borne diseases or mosquito-borne pests, are diseases caused by bacteria, viruses, or parasites transmitted by mosquitoes or other insects such as ticks. For example, nearly 700 million people are infected with mosquito-borne diseases each year, resulting in more than 1 million deaths. Mosquito-borne diseases include malaria, dengue fever, West Nile virus, chikungunya, yellow fever, filariasis, tularemia, canine filariasis, Japanese encephalitis, St. Louis encephalitis, western equine encephalitis, eastern equine encephalitis, Venezuelan equine encephalitis, Ross River fever, Bama forest fever, La Crosse encephalitis, and Zika fever, as well as the newly discovered Keystone virus and Rift Valley fever.

[0217] The applicant hereby describes a novel method for immunizing and treating insects or populations thereof carrying vector-borne diseases (e.g., ticks or mosquito species, including Culexinae and Anophelesinae) with an effective amount of one or more dead and / or heat-inactivated Gram-positive bacteria or cell wall fragments thereof. Examples of mosquito species include Aedes albopictus, Aedes aegypti, and Aedes polynesiana. The dead and / or inactivated Gram-positive bacteria are described herein (see, for example, Table 2), a complete list of which is incorporated herein by reference.

[0218] In one aspect, the insect is a mosquito, and the disease is selected from Chikunguna, dengue fever, Rift Valley fever, yellow fever, Zika, O'nyong'nyong virus, Japanese encephalitis, Zika, or West Nile fever. In another aspect, the disease is selected from dengue fever, Rift Valley fever, Zika, yellow fever, Japanese encephalitis, or West Nile fever. In another aspect, the insect is a mosquito, and the disease is selected from dengue fever or Zika. In one aspect, the insect is a mosquito, and the disease is Zika. In another aspect, the insect is a mosquito, and the disease is dengue fever.

[0219] In another aspect, the insect is a tick, and the disease is selected from Crimean-Conglo Haemorrhagic Fever, Lyme Disease, Relapsing Fever, Rickettsial Diseases, Spotted Fever, Q Fever, Tick-borne encephalitis, or Tularaemia. In another aspect, the insect is a tick, and the disease is selected from Lyme Disease, Relapsing Fever, Rickettsial Disease, Spotted Fever, Q Fever, Tick-borne encephalitis, or Tularaemia. In another aspect, the insect is a tick, and the disease is selected from Lyme Disease, Spotted Fever, Q Fever, Tick-borne encephalitis, or Tularaemia. In another aspect, the insect is a tick, and the disease is Lyme Disease.

[0220] In one aspect, the dead and / or inactivated bacteria are Paenibacillus sp., such as PL. The dead and / or inactivated Paenibacillus bacteria or cell wall fragments thereof can be microinjected into female mosquitoes, or mixed into human and animal blood and fed to the insects.

[0221] The applicant's method provides a means for generating insect species such as ticks or mosquitoes that are specifically immunized and / or treated, as needed. The generated immunized and treated insects, such as ticks or mosquitoes, can then be introduced into a population of insects such as ticks or mosquitoes by introducing individuals that have undergone specific species immunization or treatment with an effective amount of said dead and / or inactivated bacteria or cell wall fragments thereof to control infection pressure within the insect population, thereby suppressing the population's ability to become an effective disease vector and reducing the risk of infection transmission to another species such as animals, humans, or plants.

[0222] The applicant also provides a method for immunizing or treating insects such as ticks (e.g., Aedes mosquito species) with dead bacteria, comprising injecting or feeding female insects such as ticks or mosquitoes (e.g., Aedes mosquitoes) with dead and / or inactivated Bacillus bacteria or cell wall fragments thereof. In one aspect, the dead or inactivated bacteria are PL or cell wall fragments thereof. The Aedes mosquito species include those selected from Aedes albopictus, Aedes aegypti, and Aedes polynesiana.

[0223] This document also provides a method for reducing the ability of treated and immunized animals to contract infectious diseases (including bacterial and viral diseases such as Zika and dengue fever (see Tables 1 and 2)) and to become vectors of such bacteria and viruses, by administering to the animals dead and / or inactivated bacteria or cell wall fragments thereof, as described herein, such as those of the genus *Bacillus*. In one aspect, the dead or inactivated bacteria are *PL* or cell wall fragments thereof. The method disclosed herein can also prevent infection in the next generation by immunizing or treating the parent insect (e.g., ticks or mosquitoes).

[0224] This method uses dead and / or inactivated Gram-positive bacteria, such as Bacillus or fragments of their cell walls, to suppress / eliminate pathogenic viral and bacterial levels in insects carrying vector-borne diseases (e.g., ticks or mosquitoes) and as a means of infectious disease control, employing strategies to reduce or block the transmission of pathogens by insects carrying vector-borne diseases (e.g., ticks or mosquitoes). These strategies include immune-based vector suppression and replacement, treating female insects carrying vector-borne diseases (e.g., female ticks or mosquitoes) with dead and / or inactivated Bacillus bacteria or fragments of their cell walls. One mechanism is transgenerational immunization, through which immunization / treatment of the female insect transmits immunity to the next generation, thereby reducing or preventing infection with viruses and bacteria, and thus reducing the offspring's ability to become disease vectors and transmit infectious diseases.

[0225] Those skilled in the art will understand that this method can be used to protect populations of vector-borne insects (e.g., ticks or mosquitoes), including F1 offspring, from carrying effective levels of infectious disease pathogens, including viruses and bacteria, by treating and immunizing female mosquitoes with one or more dead and / or inactivated strains of *Bacillus* or cell wall fragments thereof, and by introducing these immunized and treated female insects carrying vector-borne diseases (e.g., ticks or mosquitoes). In one embodiment, the dead and / or inactivated *Bacillus* or cell wall fragments thereof are PL. When the female insects carrying vector-borne diseases (e.g., ticks or mosquitoes) mate and lay eggs, the resulting insects carrying vector-borne diseases (e.g., ticks or mosquitoes) will be effectively protected from infection upon ingestion of blood containing infectious disease pathogens.

[0226] This approach helps control the growing burden of vector-borne diseases by effectively immunizing and treating a population in which the natural vector population cannot be infected and become disease carriers.

[0227] An invertebrate vaccine and / or composition and / or formulation is provided, comprising, or consisting substantially of, whole cells or cell wall fragments of at least one, at least two, at least three, at least four or more dead and / or inactivated non-pathogenic bacterial species. In one aspect, the invertebrate vaccine and / or composition and / or formulation comprises, or consists substantially of, whole cells or cell wall fragments of at least one, at least two, at least three, at least four or more dead and / or inactivated non-pathogenic Gram-positive and / or Gram-negative bacterial species. In one aspect, the composition or formulation comprises *Bacillus* spp. or cell wall fragments thereof, such as those described above. In another aspect, the *Bacillus* spp. or cell wall fragments thereof are *PL* or cell wall fragments thereof.

[0228] A method for preparing invertebrate vaccines and / or compositions and / or formulations is also provided, comprising isolating whole cells or cell wall fragments from at least one, or at least two, or at least three, or at least four or more dead, non-pathogenic bacterial species as identified in Table 1.

[0229] In one aspect, the bacterial species is a species of the genus *Bacillus*, such as *Bacillus vesicularis* or *Bacillus dendriticis*, or a combination thereof. In another aspect, the dead *Bacillus* species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularis*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus apis*, *Bacillus assamum*, *Bacillus reductiveis*, *Bacillus nitrogen-fixingis*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brassicae*, *P. brassicae*, etc. [1], Campinas Bacillus, Jinju Bacillus, Chitinous Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough-Clearing Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Glucan-Degrading Bacillus, Polysaccharide-Degrading Bacillus, Gordon Bacillus, Gramineae Bacillus, Particle-Eating Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextrin, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus Stellife, thiamine-derived Bacillus, Paenibacillus validus, and xylolytic Bacillus, or combinations thereof. On the other hand, the dead Bacillus species are *Bacillus apis* and *Bacillus dendriticus*. On the other hand, the bacteria are *PL*.

[0230] In one respect, each dose provides at least one, or at least two, or at least three, or at least four or more dead, non-pathogenic bacterial species, in an amount of about 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 6 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 7 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10⁻⁶ 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 4 One antigen unit, or at least 1.5 x 10 5One antigen unit, or at least 1.5 x 10⁻⁶ 6 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10⁻⁶ 8 One antigen unit, or at least about 1.5 x 10⁻⁶ 9 One antigen unit, or at least about 1.5 x 10⁻⁶ 10 One antigen unit.

[0231] On the other hand, the dead and / or inactivated bacteria are approximately 1.5 x 10⁻⁶. 4 To approximately 1.5 x 10 8 Administer one antigen unit per gram of invertebrate food, or alternatively, approximately 1.5 x 10⁻⁶ units. 4 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 7 Or 1.5 x 10 5 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 6 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 7 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 8 To approximately 1.5 x 10 11 One antigen unit per gram of invertebrate food, or approximately 1.5 x 103 8 To approximately 1.5 x 10 10 One antigen unit per gram of invertebrate food, or approximately 1.5 x 10-1 8 To approximately 1.5 x 10 9 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 4 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 5 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 6 1 antigen unit / gram of invertebrate food, or at least 1.5 x 10 7 One antigen unit per gram of invertebrate food, or at least approximately 1.5 x 10⁻⁶ units. 8 One antigen unit per gram of invertebrate food, or at least approximately 1.5 x 10⁻⁶ units. 9 One antigen unit per gram of invertebrate food, or at least approximately 1.5 x 10⁻⁶ units. 10 One antigen unit per gram of invertebrate food.

[0232] Non-limiting examples of the carrier may be solid or liquid carriers and may include preservatives, insect nutrients, invertebrate nutrients, or other colorants as necessary. In another embodiment, the carrier is adapted for injection of the bacterial vaccine and / or composition and / or formulation. In yet another embodiment, the carrier is water (e.g., sterile aged brackish water or seawater) for dissolving and / or suspending the bacterial vaccine and / or composition and / or formulation, such that invertebrates can be immersed in the solution or suspension.

[0233] Experiment 1

[0234] Vaccine preparation and inoculation in bees

[0235] The vaccine preparation was derived from a strain of *P. larvae*, initially isolated from a bee colony in New York State in 2018. The vaccine is a proprietary aqueous suspension of inactivated *P. larvae* vegetative-stage bacilli, supplied by Diamond Animal Health. The vaccine has passed all purity regulatory tests and has been identified as ERIC I genotype. Bacterial counts were performed prior to inactivation using flow cytometry and OD600. The vaccine was mixed with queen bee feed (48 ml corn syrup per 500 g powdered sugar) at a ratio of 1 ml per 100 g (or 1 ml water per 100 g queen bee feed for the control).

[0236] Vaccination

[0237] A field experiment was conducted using 200 vaccinated and 200 unvaccinated populations. The applicant demonstrated that, shortly before vaccination, adult bees from both the control and experimental populations had statistically similar DWV-B numbers. Four months post-vaccination, the experimental population showed a significantly lower DWV-B number compared to the control population.

[0238] Young adult (caregiver) bees from eight study sites were used at two time points. At each site, caregivers from 10 control groups and 10 vaccinated groups were pooled. The caregivers were then sent to the National Agricultural Genotyping Center for quantifying the presence of DWV-B by PCR. Following vaccination (but not before), DWV-B levels in the vaccinated groups were lower than in the control groups at all eight sites. On average, DWV-B presence was reduced by 83% per farm, with a maximum reduction of 99.86% and a minimum reduction of 20%.

[0239] By way of example only, the applicant has shown that the viral load of DWV-B was significantly reduced in bee samples mixed from queen bee populations vaccinated with Bacillus larvae vaccine compared to unvaccinated queen bee populations. The treatment groups had the same DWV-B load before vaccination; the difference became apparent 4 months post-vaccination.

[0240] Experiment 2

[0241] The reduction in teratogenic virus B levels in bee colonies after queen bee vaccination with inactivated larval Bacillus subtilis

[0242] Deformed Wing Virus (DWV) is an RNA virus belonging to the family Iflaviridae. It is one of the most widespread and devastating diseases affecting bees (Chen & Siede, 2007; de Miranda & Genersch, 2010), and studies typically find DWV in over 90% of the populations tested (Natsopoulou et al., 2017; Kevil et al., 2019; Paxton et al., 2022). Pupae infected with DWV develop into abnormal adult bees with underdeveloped, nonfunctional wings, swollen abdomens, reduced adult size, and severely shortened lifespan (de Miranda & Genersch, 2010). There is evidence that even subclinical levels of DWV in bees can impair cognitive function, reduce foraging efficiency, and shorten lifespan (Benaets et al., 2017; Chen et al., 2021).

[0243] DWV is classified into three main strains: the common DWV-A (Lanzi et al., 2006) and DWV-B (Ongus et al., 2004), and the less common DWV-C (Mordecai et al., 2016). While DWV-A has historically been the most prevalent, DWV-B is rapidly replacing it globally due to the global spread of Varroa destructor (Paxton et al., 2022). DWV-B accumulates at higher levels in bees and larvae (Norton et al., 2020), likely because it is more easily transmitted by Varroa destructor than DWV-A (Ryabov et al., 2014, 2019). Due to their extensive pathology, both DWV-A and DWV-B are directly associated with overwintering failure (Kevill et al., 2019; Natsopoulou et al., 2017) and are of great concern to beekeepers.

[0244] DWV is highly associated with the obligate honeybee ectoparasite Varroa destructor (Wilfert et al., 2016; Barroso-Arévalo et al., 2019; Posada-Florez et al., 2019), making it the most strongly associated factor for beekeepers with colony loss (Engebretson et al., 2022). Varroa mites parasitically feed on the fat body tissue of developing pupae and adult bees (Ramsey et al., 2019; Warner et al., 2024), making them effective vectors for DWV transmission (Natsopoulou et al., 2017; Posada-Florez et al., 2019). Varroa mites also weaken the host immune system by downregulating the expression of host immune genes, thereby increasing the severity of DWV infection (Nazzi et al., 2012). In the absence of Varroa mites, DWV persists in the colony through vertical transmission from the infected queen to her offspring, or through direct horizontal transmission via cross-feeding or larval feeding (Yue & Genersch, 2005; Yue et al., 2007). Typically, mite-free transmission produces low viral loads and results in subclinical DWV (Locke et al., 2017).

[0245] Studies have shown that feeding virus-specific dsRNA to larvae or adult bees to activate the RNAi system before infection with DWV or Israel Acute Paralysis Virus (IAPV) can reduce viral load, mortality, and symptoms caused by specific viral infections (Hunter et al., 2010; Desai et al., 2012). Potassium channel manipulation has also proven to be an effective treatment for IAPV (Fellows et al., 2023). However, neither of these methods is currently approved for field use, and there are no licensed specific treatments or preventative measures for DWV (Smeele et al., 2023). Currently, the primary approach to reducing DWV levels in populations is vector control through reducing Varroa mite levels (Woodford et al., 2022). This is typically done using insecticides and acaricides such as amitraz (Warner et al., 2024), which are widely used in agricultural pest control. However, when used on bees, these chemicals can reduce viral immunity (O'Neal et al., 2017) and contaminate honey (Pohorecka et al., 2018).

[0246] Another mechanism for direct disease management in bee colonies is protection through vaccination. Currently, vaccination with Paenibacillus larvae vaccines is commercially approved for protecting bee larvae from American foulbrood via transgenerational immunoprovocation (TGIP), a process of transferring immunization experience from parent to offspring (Hernández López et al., 2014; Dickel et al., 2022). Inoculation of parent insects with inactivated pathogens has been shown to provide homologous protection via TGIP across a broad spectrum of invertebrate lineages (Tetreau et al., 2019), including honeybees (Freitak et al., 2014; Hernández López et al., 2014). Some have hypothesized that vaccination with inactivated DWV-A could provide transgenerational protection, but results have been mixed to date. Context-dependent TGIP benefits have been recorded after DWV-A was transmitted to queen bees via sexual transmission (Lang et al., 2022), but no benefit has been observed after oral administration (Leponiemi et al., 2021).

[0247] Here, the applicant provides, for the first time, evidence that oral vaccination of queen bees with inactivated Gram-positive bacteria, *Paenibacillus larvae*, directly reduces DWV-B levels in bee colonies in the field, independent of Varroa destructor load. To the best of our knowledge, these data represent the first use of inactivated bacteria in TGIP to provide heterologous protection against the virus and the first instance of controlling DWV in the field without relying on mite control.

[0248] method

[0249] Queen bee vaccination

[0250] On May 31, 2023, 400 Italian honeybee queens (Apis mellifera ligustica) were purchased from Vidalia Apicultural Services in Toms County, Georgia. 200 queens were vaccinated with a completely inactivated P. larvae vaccine (PCN 2915.00, manufactured according to Diamond Animal Health's proprietary production guidelines), while the remaining 200 queens remained unvaccinated. For every 50 bees to be vaccinated, 3 mL of the P. larvae vaccine solution was added to 300 g of queen bee candy (approximately 1:7.5 w / w corn syrup to powdered sugar), yielding 1.5 x 10g of queen bee candy per queen. 8One dead larval Bacillus cell line was used. Queen bees were isolated in queen cages and placed in battery boxes in groups of 50. 300g of candy containing the vaccine was placed in two strips between the queen cages in each battery box. Approximately 2100 care bees were then placed in the battery boxes to care for the queens. The queens and care bees were left in the battery boxes for 8 days to allow the candy to be fully consumed before being placed in the hives. Due to the difficulty of procuring 400 queens on the same day, 200 unvaccinated queens were not included in the vaccination protocol and thus served as a true control rather than a placebo control. Queen replacement in all hives was performed on June 8, 2023.

[0251] Experimental subjects

[0252] In May 2023, 400 established bee colonies from eight farms in Toms County, Georgia, were included in the study. Each farm contained between 39 and 78 colonies, roughly divided into vaccinated and unvaccinated groups. Before the study began, the colonies were inspected to ensure they were healthy and free from pests and diseases. At the start of the study, each colony consisted of one deep hive and one shallow hive. Most colonies maintained this size throughout the study, but farm staff made some expansions and reductions according to their standard colony management practices. Varroa mites were treated monthly with amitraz, and the colonies were fed sugar water to supplement their nectar intake during periods of nectar scarcity.

[0253] Sample collection

[0254] Ten caretaker bees were collected immediately before the experimental queen was introduced into the colonies in May 2023 from 100 vaccinated and 100 unvaccinated colonies, and then collected again four months after the queen was introduced in September 2023. Caretaker bees were collected from above the brood comb and visually identified by their color and intact dorsal hairs. The caretaker bees were placed in 15ml Falcon tubes and flash-frozen in the field with dry ice. The bees were then stored in the laboratory at -80°C until submission.

[0255] Sample Analysis

[0256] In all eight farms, a pooled sample of care bees was sent from each experimental group for analysis. Each pooled sample contained 50 bees collected in equal proportions from 10 colonies (N=8 samples / treatments / time points).

[0257] Blind samples were sent to the National Agricultural Genotyping Center (NAGC) in Fargo, North Dakota, for analysis. NAGC is an accredited testing facility used by beekeepers and scientists in North America. DWV-B levels were analyzed using qPCR. Sample controls, positive controls, reagent controls, and template-free controls were tested. The PCR cycling conditions are proprietary to NAGC and therefore cannot be reported here.

[0258] Similar methods were used to detect DWV-A, DWV-C, American foulbrood, European foulbrood, and cysticercus virus in the samples, but few or no instances of these diseases were observed, and therefore these results were not reported.

[0259] Mite quantification

[0260] Mites were collected from bee colonies in May and November 2023. Counting was performed using the Veto-pharma Varroa Easy Check kit. Approximately 300 bees were collected from above the brood comb in the sample hive and washed in 70% ethanol to separate the mites from the bees. The number of mites below the sieve was quantified and reported as mites / 100 bees. Washing was performed from the colonies in May (N=35 unvaccinated, N=38 vaccinated) and November (N=44 vaccinated and N=44 unvaccinated).

[0261] Statistical analysis

[0262] Since DWV-B levels follow an exponential distribution, the Wilcox rank-sum test was used for comparison. Mite counts were compared using the Welch two-sample t-test. All statistical analyses were performed using R version 4.3.0 (R Core Team, 2024).

[0263] result

[0264] DWV-B level

[0265] One week prior to vaccination, DWV-B levels were similar across groups (Wilcoxon rank-sum test, W=31, P=0.96), and four months post-vaccination, DWV-B levels in vaccinated hives were significantly lower than in control hives (Wilcoxon rank-sum test, W=54.5, P=0.021). Figure 1 In all eight facilities, the number of DWV-B cells in the vaccinated group was lower than that in the unvaccinated group, with an average reduction of 83%.

[0266] Mite count

[0267] One week before vaccination (Welch two-sample t-test, T=0.38, df=61, P=0.71) or six months after vaccination (Welch two-sample t-test, T=-1.30, df=48, P=0.20), there was no difference in mite count between the treatment groups. Figure 2 Following standard beekeeping practices, all bee colonies were regularly treated with amitraz for mites. These results showed no difference in mite load among the treatment groups.

[0268] discuss

[0269] This study provides the first evidence that vaccination of queen bees with Penibacillus larvae vaccine in field conditions provides heterologous, transgenerational protection against DWV-B. To our knowledge, these data are also the first to demonstrate that vaccination of organisms with only an innate immune system using inactivated bacteria provides protection against viral diseases. Although there was no difference in Varroa destructor numbers between vaccinated and unvaccinated populations, viral load was reduced, indicating that the effect was independent of mite load. This experiment was conducted on full-size bee colonies in a commercial apiary, without involving any special colony management practices other than the administration of vaccinated queen bees. These results suggest that queen bee vaccination with Penibacillus larvae vaccine may be an effective method for direct control of DWV-B in bee colonies in the field.

[0270] Most laboratory studies on DWV have been conducted in the absence of the Varroa destructor, which is present in bee populations worldwide, virtually impossible to eradicate, highly correlated with elevated DWV levels, and directly weakens and disrupts the bee's immune response (Annoscia et al., 2019; Kuster et al., 2014; Ramsey et al., 2019). Previous studies in the laboratory investigating homologous transgenerational protection against DWV have either failed to show protection (Leponiemi et al., 2021) or only showed protection under specific conditions (Lang et al., 2022). Therefore, the applicant's observation of heterologous protection against DWV-B following vaccination of queen bees with Bacillus larvae is surprising. Arthropods, including bees, lack an adaptive immune system (Baxter et al., 2017) and rely solely on innate immunity to defend against pathogens (Morfin et al., 2021). It has been noted that honeybees express only one-third of the immune regulatory genes found in solitary insects, relying instead on social immunity, such as grooming, task division, and temperature regulation. This reduction in genes may increase crosstalk between immune pathways and potentially lead to decreased specificity of immune responses, thereby increasing the likelihood that vaccination will induce generalized immune benefits.

[0271] While the applicant has not yet determined the mechanism by which bacterial inoculation helps reduce viral infections, they hypothesize that the vaccine may enhance one or more common pathways of the bee's innate immune system, leading to enhanced innate immune function in bee larvae (Nazzi & Le Conte, 2016; Annoscia et al., 2019). Innate immune responses typically function by increasing the expression of generalized defenses such as AMPs and siRNAs (Kingsolver et al., 2013; Mondotte et al., 2020). It has been demonstrated that queen bumblebees injected with heat-inactivated bacteria pass on transgenerational immune benefits to their daughters, even in the absence of infection, with daughters upregulating genes associated with AMPs and the Toll signaling pathway (Barribeau et al., 2016). However, simply altering immune function does not guarantee disease reduction. Upregulation of both RNAi gene and siRNA activity was observed in response to DWV infection, but this did not inhibit DWV accumulation to high levels (Norton et al., 2023). This may be partly due to the immunosuppressive syndrome associated with DWV infection, which includes a strong downregulation of NF-κB, a transcription factor that helps resist a wide range of environmental challenges (Nazzi et al., 2012). Future transcriptomic studies should look for changes in worker bee gene regulation after queen bee inoculation with Bacillus larvae to elucidate how these complex interactions affect bee immunity.

[0272] If the larval Bacillus vaccine does indeed improve general immune function, the applicant anticipates seeing similar heterologous protection against multiple diseases in the field. Although the applicant tested for protective effects against five other diseases, none were detected in three, and two were present only at subclinical levels, making efficacy assessment impossible.

[0273] Experiment 3

[0274] Vaccines for crustaceans and shrimp

[0275] vaccine preparation

[0276] A vaccine treatment prepared from heat-inactivated *P. larvae* and *V. parahaemolyticus* was used to vaccinate a population of Litopenaeus vannamei (L.) broodstock to evaluate whether vaccines prepared from major shrimp pathogens *V. parahaemolyticus* and *P. larvae* could provide nonspecific protection against WSSV and AHPND attacks in offspring. The *P. larvae* vaccine strain is a Gram-positive pathogen of bees. *V. parahaemolyticus* is a known Gram-negative pathogen of shrimp, and some strains of this bacterium are involved in the pathogenicity of AHPND in shrimp.

[0277] Heat-inactivated vaccines from Bacillus larvae and Vibrio parahaemolyticus were prepared. Bacillus larvae were cultured in MYPGP (Mueller-Hinton broth, yeast extract, potassium phosphate, glucose, and pyruvate) broth at 37°C ± 2°C and 250 ± 50 rpm for three (3) days, and then inactivated by autoclaving (121°C, 15 min). Inactivation was confirmed by spreading the vaccine on MYPGP agar plates (10 plates) and incubating at 37°C for 14 days for visible colony growth. Additionally, the vaccine was spread on TSA blood agar plates (2 plates) and incubated at 37°C for 24–48 hours for visible colony growth. No growth was observed on any of the inoculated plates, confirming sterility.

[0278] Similarly, Vibrio parahaemolyticus was cultured in Marine 2216 broth at 37°C ± 2°C on a shaker at 250 ± 50 rpm for one (1) day. Cultures in the growth phase of the inactivation index were inactivated by autoclaving (121°C, 15 min). Inactivation was confirmed by spreading the culture onto Marine 2216 agar plates (10 plates) and incubating at 37°C for 14 days, observing for visible colony growth. Additionally, the culture was spread onto TSA blood agar plates (2 plates) and incubated at 37°C for 24–48 hours, observing for visible colony growth. No growth was observed on any of the inoculated plates, confirming sterility. The heat-inactivated vaccine was stored at 2–8°C until use for vaccination.

[0279] Premixed feed with vaccine or placebo

[0280] Commercial shrimp pellet feed is coated with vaccine-treated material or placebo. (>10) 9 The vaccine treatment is coated onto saturated feed pellets at a dose of 10 cells / mL. Placebo (sterile culture medium) treatment is similarly coated onto pelleted feed. The premixed feed treatment is stored at 2–8°C until used for vaccination.

[0281] Shrimp

[0282] Litopenaeus vannamei broodstock were selected and vaccinated with different vaccine preparations as shown in Table 5. Healthy female broodstock were selected, tagged with eyestalk markers, and checked regularly to determine ovarian development stage. SPF status for major shrimp pathogens was checked using standard PCR methods. The included shrimp were weighed and counted, and distributed to 90 L broodstock rearing tanks (two replicates per treatment, 4–6 females per tank). All major water quality parameters were checked regularly, and routine husbandry practices were performed according to the standard procedures of the test facility. The shrimp were fed a combination of live broodstock feed and pelleted feed.

[0283] Table 5. Experimental Design

[0284] Vaccination or placebo treatment

[0285] A few days before the female shrimp prepares to lay eggs (determined by visual observation of ovarian development), on the 4th day, the shrimp are injected (intramuscularly into the dorsal muscle) with the appropriate treatment (Table 5).

[0286] In addition, as shown in Table 5, some groups were fed either the vaccine premixed feed treatment or the placebo feed for four consecutive days (days 0, 1, 2, and 3 of the trial).

[0287] Shrimp in each treatment tank were allowed to mate with males in appropriate proportions according to standard shrimp rearing procedures. The offspring larvae produced from each treatment group were reared separately, fed according to standard rearing practices, and continuously monitored until they reached a size suitable for attack by invasive species.

[0288] AHPND and WSSV attacks

[0289] Attack on shrimp weighing 1-2 grams

[0290] When the offspring shrimp reached 1-2 grams in size, an appropriate number of juvenile shrimp from each treatment group were divided into two attack groups and attacked with either AHPND or WSSV (Table 5). The clinical symptoms of disease and daily mortality were observed in each attack group. The observation period was 12 days for AHPND and 16 days for WWSV.

[0291] A second attack on shrimp weighing 10 grams

[0292] The remaining unattacked progeny shrimp in each treatment group were allowed to grow until they reached a size of approximately 10 grams. Then, an appropriate number of shrimp from each treatment group were divided into two attack groups and attacked again with AHPND and WSSV as described above.

[0293] Determining the cause of death in the AHPND attack group

[0294] Mortality was monitored daily for 12 days, and 20% of the daily mortality rate was sampled for re-isolation of the challenge strain of Vibrio parahaemolyticus. Hepatocellular and pancreatic samples were aseptically streaked onto Marine 2216E agar and incubated at 34-7°C for 24 hours. The re-isolated presumed Vibrio parahaemolyticus challenge strain was confirmed by AHPND-specific PCR.

[0295] Determining the cause of death in the WSSV attack group

[0296] Mortality was monitored daily for 16 days, and an attempt was made to re-isolate the WSSV strain used to attack AHPND by sampling 20% ​​of the daily mortality rate and testing the liver and pancreas samples using a WSSV-specific PCR method.

[0297] discuss

[0298] Calculate the cumulative mortality rate (CPM) for each vaccine-treated or placebo group at the post-attack days. Calculate the relative survival rate (RPS) for the vaccine-treated groups using the following formula:

[0299] (1 – [CPM of vaccinated group / CPM of unvaccinated placebo group]) × 100

[0300] After confirming that the residuals are normally distributed and that the datasets have equal variances, one-way ANOVA (α = 0.05) is used to determine differences in CPM. If the differences are significant (P < 0.05), a Tukey post-hoc test is performed to determine which groups have differences. Statistical analysis is performed using GraphPad® Prism (or other appropriate software).

[0301] Experiment 4

[0302] Vaccines used to treat vector-borne diseases such as mosquito-borne or tick-borne diseases.

[0303] This approach helps control the growing burden of vector-borne diseases by effectively immunizing and treating a population in which the natural vector population cannot be infected and become disease carriers.

[0304] In one aspect, the insect is a mosquito, and the disease is selected from chikungunya, dengue fever, Rift Valley fever, yellow fever, Zika, Onyonniyong virus, Japanese encephalitis, Zika, or West Nile fever. In another aspect, the disease is selected from dengue fever, Rift Valley fever, Zika, yellow fever, Japanese encephalitis, or West Nile fever. In another aspect, the insect is a mosquito, and the disease is selected from dengue fever or Zika. In one aspect, the insect is a mosquito, and the disease is Zika. In another aspect, the insect is a mosquito, and the disease is dengue fever.

[0305] In one aspect, the applied bacterial species is a species of the genus *Bacillus*, such as *Bacillus vesicularis* or *Bacillus denticulatus*, or a combination thereof. In another aspect, the dead *Bacillus* species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularis*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus apis*, *Bacillus assamum*, *Bacillus reductiveis*, *Bacillus nitrogen-fixingis*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brassicae*, *P. brassicae*, etc. [1], Campinas Bacillus, Jinju Bacillus, Chitinous Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough-Clearing Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Glucan-Degrading Bacillus, Polysaccharide-Degrading Bacillus, Gordon Bacillus, Gramineae Bacillus, Particle-Eating Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextrin, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus *Stellife*, *Thioamine-like* Bacillus, *Paenibacillus validus*, and *Xylolytic* Bacillus, or combinations thereof. On the other hand, the dead *Bacillus* species are *Bacillus apis* and *Bacillus dendriticus*.

[0306] On the other hand, the dead and / or inactivated whole cell or cell wall fragments from at least one dead and / or inactivated non-pathogenic bacterial species are selected from V. adaptatus, V. aerogenes, Vibrio summerensis, Vibrio estuarineis, Vibrio alginolyticus, V. albensis, Vibrio alfacoidus, Vibrio alginolyticus, Vibrio anguillarum, V. areninigrae, V. artabrorum, Vibrio atlantos, Vibrio atypical, Vibrio cyanobacterium, Vibrio brasiliensis, V. bubulus, V. calviensis, Vibrio cannii, V. casei, Vibrio chagusi, Vibrio cholerae, V. cincinnatiensis, Vibrio lysomorphus, Vibrio oysteri, Vibrio cyclohexane, Vibrio devilii, Vibrio diazotrophus, Vibrio davidii, Vibrio fluvibrio, Vibrio virulentii, Vibrio furnis, Vibrio gallophylla, Vibrio abaloneniformis, Vibrio harveyi, Vibrio hepatis, V. Vibrio hippocampi, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolo, Vibrio slow-moving, Vibrio riparia, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonatal, Vibrio tsoriopteris, Vibrio nervosa, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio pannioides, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio brevicornuate, Vibrio rotiferus, Vibrio rubrumensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio brevicornuate, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae. Vibrio superstes, V. tapepetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vortanense, and / or Vibrio xuei, or combinations of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio mermaidii, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawni, Vibrio vulnificus, Vibrio nerei, Vibrio tasmani, Vibrio fluvialis, Vibrio splenium, and / or Vibrio niger, or combinations of two or more thereof. In another aspect, the invertebrate vaccine and / or composition and / or formulation comprises dead and / or inactivated bacterial species of the genus Bacillus and / or Vibrio genus.

[0307] In another aspect, the insect is a tick, and the disease is selected from Crimean-Congo hemorrhagic fever, Lyme disease, relapsing fever, rickettsial disease, spotted fever, Q fever, tick-borne encephalitis, or tularemia. In another aspect, the insect is a tick, and the disease is selected from Lyme disease, relapsing fever, rickettsial disease, spotted fever, Q fever, tick-borne encephalitis, or tularemia. In another aspect, the insect is a tick, and the disease is selected from Lyme disease, spotted fever, Q fever, tick-borne encephalitis, or tularemia. In another aspect, the insect is a tick, and the disease is Lyme disease.

[0308] In one aspect, the applied bacterial species is a species of the genus *Bacillus*, such as *Bacillus vesicularis* or *Bacillus denticulatus*, or a combination thereof. In another aspect, the dead *Bacillus* species are selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularis*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarcticus*, *Bacillus apis*, *Bacillus assamum*, *Bacillus reductiveis*, *Bacillus nitrogen-fixingis*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brassicae*, *P. brassicae*, etc. [1], Campinas Bacillus, Jinju Bacillus, Chitinous Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough-Clearing Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Glucan-Degrading Bacillus, Polysaccharide-Degrading Bacillus, Gordon Bacillus, Gramineae Bacillus, Particle-Eating Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextrin, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus *Stellife*, *Thioamine-like* Bacillus, *Paenibacillus validus*, and *Xylolytic* Bacillus, or combinations thereof. On the other hand, the dead *Bacillus* species are *Bacillus apis* and *Bacillus dendriticus*.

[0309] On the other hand, the dead and / or inactivated whole cell or cell wall fragments from at least one dead and / or inactivated non-pathogenic bacterial species are selected from V. adaptatus, V. aerogenes, Vibrio summerensis, Vibrio estuarineis, Vibrio alginolyticus, V. albensis, Vibrio alfacoidus, Vibrio alginolyticus, Vibrio anguillarum, V. areninigrae, V. artabrorum, Vibrio atlantos, Vibrio atypical, Vibrio cyanobacterium, Vibrio brasiliensis, V. bubulus, V. calviensis, Vibrio cannii, V. casei, Vibrio chagusi, Vibrio cholerae, V. cincinnatiensis, Vibrio lysomorphus, Vibrio oysteri, Vibrio cyclohexane, Vibrio devilii, Vibrio diazotrophus, Vibrio davidii, Vibrio fluvibrio, Vibrio virulentii, Vibrio furnis, Vibrio gallophylla, Vibrio abaloneniformis, Vibrio harveyi, Vibrio hepatis, V. Vibrio hippocampi, Vibrio ichthyoenteri, Vibrio indicus, Vibrio canarolo, Vibrio slow-moving, Vibrio riparia, Vibrio logei, Vibrio mediterranei, Vibrio mimicus, Vibrio mytili, Vibrio sodium-dependent, Vibrio navara, Vibrio neonatal, Vibrio tsoriopteris, Vibrio nervosa, Vibrio nervosa, Vibrio ordalii, Vibrio orientalis, Vibrio pannioides, Vibrio parahaemolyticus, Vibrio pelagius, Vibrio penaeicida, Vibrio brevis, Vibrio brevicornuate, Vibrio rotiferus, Vibrio rubrumensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio brevicornuate, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae, Vibrio rumoiensis, Vibrio scoraniae, Vibrio scophthalmi, Vibrio scoraniae. Vibrio superstes, V. tapepetis, Vibrio tasmani, Vibrio tasmani, Vibrio vulnificus, Vibrio vortanense, and / or Vibrio xuei, or combinations of two or more thereof; or alternatively selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio cannii, Vibrio mermaidii, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio prawnicella, Vibrio vulnificus, Vibrio nerei, Vibrio tasmani, Vibrio fluvialis, Vibrio splenium, and / or Vibrio niger, or combinations of two or more thereof. In another aspect, the invertebrate vaccine and / or composition and / or formulation comprises dead and / or inactivated bacterial species of the genus Bacillus and / or Vibrio.

[0310] Laboratory tests can demonstrate that the ability to prevent or reduce the infection rate of the next generation of populations after ingesting blood infected with diseases such as Zika by using dead and / or inactivated Gram-positive bacteria or fragments of their cell walls, through application to insects such as mosquitoes or ticks (e.g., Aedes aegypti, Aedes albopictus, or Aedes polynesian), treatment of female insects such as mosquitoes or ticks, and immunization / vaccination.

[0311] Adult female mosquitoes (Higgs WT control and TZIKV-C) or ticks were divided into the following groups: a) vaccinated, b) positive control, c) placebo control, and d) negative control.

[0312] The method described by Masters SW et al. (2020) (Rearing Aedes aegypti Mosquitoesin a Laboratory Setting Centers for Disease Control and Prevention (.gov)https: / / stacks.cdc.gov › cdc › cdc_96121_DS1) and modified for the growth of tick populations were used to feed each group of the next generation of mosquitoes or ticks.

[0313] In short, F1 generation mosquitoes or ticks from groups a), b), and c) were infected with a pathogen, such as ZIKV (FSS13025, Cambodian strain 2010; GenBank accession number JN860885) via membranous blood meal (JT Ladner, et al., Complete genome sequences of five Zika virus isolates. Genome Announc 4, e00377-16(2016)). Control groups received uninfected blood, such as blood uninfected with Zika. On days 4 and 14 post-feeding, the midguts of blood-feeding mosquitoes or ticks were dissected, and ZIKV RNA copy numbers were measured using real-time RT-qPCR. The experiment was performed in triplicate.

[0314] To determine virus transmission, saliva was collected from individual mosquitoes or ticks on day 14 post-infection, and ZIKV titers were measured using the half-tissue culture infection dose assay.

[0315] Therefore, oral administration of inactivated Gram-positive bacteria to female insects such as mosquitoes or ticks via blood meal may be an effective way to reduce the replication capacity of pathogenic viruses or bacteria (such as Zika virus) in the next generation of disease-carrying insects. Implementation Plan

[0316] A method for vaccinating, treating, or immunizing an invertebrate, a population of invertebrates, its offspring, or an invertebrate host against one or more of a bacterial or viral infection or disease, the method comprising administering to the invertebrate or population an effective amount of a dead and / or inactivated nonpathogenic Gram-positive bacterium or a fragment of its cell wall thereof, thereby vaccinating, treating, or immunizing the invertebrate against the bacterial or viral disease or infection, optionally wherein the invertebrate is a crustacean or an insect.

[0317] The method according to embodiment 1 further includes administering an effective amount of dead and / or inactivated Gram-negative bacteria or cell wall fragments thereof to the invertebrate or the population of invertebrates, and optionally wherein the invertebrate is a crustacean or an insect.

[0318] 3. The method according to embodiment 1 or 2, wherein the bacterial or viral infection is caused by a single row of any of the tables 1-4 or by a pathogen as shown in Experiment 4, and the dead and / or inactivated non-pathogenic Gram-positive bacteria are identified in the corresponding row of the tables 1-4 or as shown in Experiment 4.

[0319] 4. The method according to any one of embodiments 1-3, wherein the invertebrate is an insect and / or the invertebrate population is an insect or an insect population or community, and the virus causing the viral disease or infection is selected from Aberrant wing virus A, Aberrant wing virus B, Aberrant wing virus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, Queen cell virus, and optionally wherein the insect is a bee and / or the insect population is a bee population.

[0320] 5. The method according to any one of embodiments 1-4, wherein the insect is a honeybee or a bumblebee, or the colony is a honeybee or bumblebee colony or community, and wherein the viral disease or infection is caused by teratomavirus A, teratomavirus B, teratomavirus C, acute bee paralysis virus, Israeli acute bee paralysis virus, Kashmir bee virus, slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, chronic bee paralysis virus, sac larva virus, or black queen cell virus.

[0321] 6. The method according to embodiment 5, wherein the virus causing the disease or infection is pterygoid virus B (DWV-B).

[0322] 7. The method according to any one of embodiments 1-6, wherein the dead and / or inactivated non-pathogenic bacteria or cell wall fragments thereof are applied in combination with a carrier, optionally wherein the insect is a bee or a bumblebee and / or the colony is a bee colony or a bumblebee colony, and the carrier contains bee food or bee feed.

[0323] 8. The method according to any one of embodiments 1-7, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated bacterial species of the genus Paenibaccilus.

[0324] 9. The method according to embodiment 8, wherein the dead and / or inactivated bacterial species are selected from *P. agarexedens*, *P. agaridevorans*, *P. alginolyticus*, *P. alkaliterrae*, *P. alvei*, *P. amylolyticus*, *P. anaericanus*, *P. antarcticus*, *P. apiarius*, *P. assamensis*, *P. azoreducens*, *P. azotofixans*, *P. barcinonensis*, *P. borealis*, *P. brasilensis*, and *P.* brassicae[1], P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. cookii, P. curdlanolyticus, P. daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P. favisporus, P. glucanolyticus, P. glucanolyticus. *Pseudomonas glycanilyticus*, *P. gordonae*, *P. graminis*, *P. granivorans*, *P. hodogayensis*, *P. illinoisensis*, *P. jamilae*, *P. kobensis*, *P. koleovorans*, *P. koreensis*, and *P. jelly-like*.*P. kribbensis*, *P. lactis*, *P. larvae*, *P. lautus*, *P. lentimorbus*, *P. macerans*, *P. macquariensis*, *P. massiliensis*, *P. mendelii*, *P. motobuensis*, *P. naphthalenovorans*, *P. nematophilus*, *P. odorifer*, *P. papuli*, *P. peoriae*, *P. phoenicis*, *P. phyllosphaerae*, *P. polymyxa*. *P. polymyxa*, *P. popilliae*, *P. pulvifaciens*, *P. rhizosphaerae*, *P. sanguinis*, *P. stellifer*, *Paenibacillus stellife*, *P. terrae*, *P. thiaminolyticus*, *P. timonensis*, *P. tundrae*, *P. turicensis*, *P. tylopili*, *P. validus*, *P. vortex*, *P. vulneris*, *P. wynnii*, and / or *P. xylanolyticus*.xylanilyticus, or a combination of two or more thereof; or optionally selected from Paenibacillus alvei, Paenibacillus dentritiformis, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chondroitinus, Paenibacillus chungangensis, Paenibacillus doosanensis, Paenibacillus glucanolyticus, Paenibacillus humicus, Paenibacillus lactis, Paenibacillus larvae, Paenibacillus splenicus. The following are listed: Paenibacillus lautus, Paenibacillus lentimorbus, Paenibacillus maceran, Paenibacillus macerans-like, Paenibacillus macquariensis, Paenibacillus motobuensis, Paenibacillus papuli, Paenibacillus phoenicis, Paenibacillus polymyxa, Paenibacillus popilliae, Paenibacillus puldeungensis, Paenibacillus residui, Paenibacillus stellife, Paenibacillus sthiaminolyticus, Paenibacillus validus, and Paenibacillus xylanisolvens, or combinations of two or more thereof. .

[0325] 10. The method according to any one of embodiments 1-9, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated Paenibacillus larvae (PL) or cell wall fragments thereof.

[0326] 11. The method according to any one of embodiments 1-10, wherein the effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprises about 1.5 x 10^6 per gram or carrier. 4 Or 1.5 x 10 7 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0327] 12. The method according to embodiment 11, wherein each dose to the insect is approximately 1.5 x 10 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0328] 13. The method according to embodiment 12, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of 1.5 x 10 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0329] 14. A method for vaccinating, treating, or immunizing bees, bee offspring, or bee colonies against one or more viral infections or viral diseases, comprising administering to the bees, queen bees, or bee colonies an effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof, and optionally a carrier, thereby vaccinating, treating, or immunizing the bees, offspring, or colonies against the viral disease or infection.

[0330] 15. The method according to embodiment 14, wherein the bee is a honeybee or the bee colony is a honeybee colony or honeybee community, and the virus causing the viral disease or infection is selected from teratogenic wing virus A, teratogenic wing virus B, teratogenic wing virus C, acute honeybee paralysis virus, Israeli acute honeybee paralysis virus, Kashmir honeybee virus, slow honeybee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, chronic honeybee paralysis virus, sac larvae virus, and black queen cell virus.

[0331] 16. The method according to embodiment 14, wherein the bee is a bumblebee or a bumblebee colony or community, and wherein the viral disease or infection is caused by Deformia wingvirus A, Deformia wingvirus B, Deformia wingvirus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, or Black queen cell virus.

[0332] 17. The method according to embodiment 14, wherein the bee is a honeybee and the colony is a honeybee colony or community, and the virus causing the disease or infection is Deformed Wing Virus B (DWV-B).

[0333] 18. The method according to any one of embodiments 14-17, wherein the carrier comprises bee food or bee feed.

[0334] 19. The method according to any one of embodiments 14-18, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated bacterial species of the genus Bacillus.

[0335] 20. The method according to embodiment 19, wherein the dead and / or inactivated bacterial species are selected from Bacillus agaricus, Bacillus edibleus, Bacillus alginate, Bacillus alkalophilus, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobicus, Bacillus antarcticus, Bacillus vesicularis, Bacillus assamum, Bacillus reductive azotobacter, Bacillus nitrogen-fixing, Bacillus barbarus, Bacillus northernus, Bacillus brasiliensis, and P. brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus sttellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or a combination of two or more thereof.

[0336] 21. The method according to any one of embodiments 14-19, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0337] 22. The method according to any one of embodiments 14-21, wherein the effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprises about 1.5 x 10^6 mg / g or carrier. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0338] 23. The method according to embodiment 21, wherein approximately 1.5 x 10 g of the product is applied to the bees or bee colony per dose. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0339] 24. The method according to embodiment 21, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of 1.5 x 104 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0340] 25. A method for treating or immunizing a bee colony against one or more of the viral infections or diseases caused by viruses, the method comprising introducing an uninfected queen bee into the bee colony and administering to the queen bee an effective amount of dead and / or inactivated whole cells or cell wall fragments of Gram-positive bacteria and a carrier, thereby treating or immunizing the bee colony against the viral disease or infection.

[0341] 26. The method according to embodiment 25, wherein the bee colony is a bee colony or community, and the virus causing the viral disease or infection is selected from Aberrant wing virus A, Aberrant wing virus B, Aberrant wing virus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, and Black queen cell virus.

[0342] 27. The method according to embodiment 25, wherein the population or community is a bumblebee population or community, and wherein the viral disease or infection is caused by Deformed Wing Virus A, Deformed Wing Virus B, Deformed Wing Virus C, Acute Bee Paralysis Virus, Israel Acute Bee Paralysis Virus, Kashmir Bee Virus, Slow Bee Paralysis Virus, Lake Sinai Virus 1, Lake Sinai Virus 2, Chronic Bee Paralysis Virus, Sacbryovirus, or Black Queen Cell Virus.

[0343] 28. The method according to embodiment 25, wherein the colony is a bee colony or community, and the virus causing the disease or infection is Deformed Wing Virus B (DWV-B).

[0344] 29. The method according to any one of embodiments 25-28, wherein the carrier comprises bee food.

[0345] 30. The method according to any one of embodiments 25-29, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated bacterial species of the genus Bacillus.

[0346] 31. The method according to embodiment 30, wherein the dead and / or inactivated bacterial species are selected from Bacillus agaricus, Bacillus edibleus, Bacillus alginate, Bacillus alkalophilus, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobicus, Bacillus antarcticus, Bacillus vesicularis, Bacillus assamum, Bacillus reductive azotobacter, Bacillus nitrogen-fixing, Bacillus barbarus, Bacillus northernus, Bacillus brasiliensis, and P. brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus sttellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or a combination of two or more thereof.

[0347] 32. The method according to any one of embodiments 25-31, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

[0348] 33. The method according to any one of embodiments 12-19, wherein the effective amount of dead and / or inactivated Gram-positive bacterial whole cells or cell wall fragments comprises approximately 1.5 x 10^6 cells per gram of bee food or carrier. 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0349] 34. The method according to embodiment 33, wherein approximately 1.5 x 10 g of the product is applied per dose to the queen, worker bees, caretaker bees, or larvae in the colony. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0350] 35. The method according to embodiment 34, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of approximately 1.5 x 10⁻⁶ are administered to the queen bee, worker bees, caretaker bees, or bee larvae in the colony. 7 Or approximately 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0351] 36. A composition comprising whole cells or cell wall fragments of dead and / or inactivated Gram-negative and / or Gram-positive bacteria, and a carrier, wherein optionally the carrier is a phosphate-buffered saline or water.

[0352] 37. The composition according to embodiment 36, wherein the Gram-negative bacteria comprises, is substantially composed of, or is composed of bacterial species of the genus Vibrio.

[0353] 38. The composition according to embodiment 36 or 37, wherein the Gram-negative bacteria is selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio campbelli, Vibrio damsela, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio penaeicida, Vibrio vulnificus, Vibrio nereis, Vibrio tubiashi, Vibrio fluvialis, Vibrio splendidus, or Vibrio nigripulchritudo.

[0354] 39. The composition according to any one of embodiments 36-38, wherein the Gram-positive bacteria comprises, is substantially composed of, or is composed of bacterial species of the genus Bacillus.

[0355] 40. The composition according to any one of embodiments 36-39, wherein the Gram-positive bacteria are selected from Bacillus agaris, Bacillus edibleus, Bacillus alginate, Bacillus alkalophilus, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobicus, Bacillus antarcticus, Bacillus vesicularis, Bacillus assamum, Bacillus reductive azotobacter, Bacillus nitrogen-fixing, Bacillus barbarus, Bacillus northernus, Bacillus brasiliensis, and P. brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus *Residui*, *Paenibacillus sttellife*, thiamine-derived Bacillus, *Paenibacillus validus*, and xylolytic Bacillus, or a combination of two or more thereof.

[0356] 41. The composition according to any one of embodiments 36-40, wherein the carrier comprises feed for invertebrates.

[0357] 42. The composition according to embodiment 41, wherein the invertebrate is a crustacean.

[0358] 43. The composition according to embodiment 41, wherein the invertebrate is a shrimp, and the carrier is selected from one or more of phosphate-buffered saline, water, shrimp food, or shrimp feed.

[0359] 44. The composition according to embodiment 41, wherein the invertebrate is identified in Tables 1-4 or Experiment 4.

[0360] 45. The composition according to embodiment 41, wherein the invertebrate is a shrimp, and the carrier is shrimp food or feed.

[0361] 46. ​​The composition according to any one of embodiments 41-45, wherein the composition is formulated for oral administration, injection administration, submersion administration, or any combination of two or more thereof.

[0362] 47. A method for vaccinating, treating, or immunizing an invertebrate or its offspring against one or more of an infection or disease caused by a viral or bacterial infection, comprising administering an effective amount of the composition according to any one of embodiments 41-46 to the invertebrate, thereby vaccinating, treating, or immunizing the invertebrate or its offspring against a viral or bacterial disease or infection in the animal.

[0363] 48. The method according to embodiment 47, wherein the invertebrate is identified in Tables 1-4 or Experiment 4.

[0364] 49. The method according to embodiment 48, wherein the bacteria comprises, or is substantially composed of, larvae-like Bacillus and thiaminolyticus-like Bacillus, the insect is a black soldier fly, and the pathogen is Hermetia illucens totivirus.

[0365] 50. The method according to embodiment 1 or 2, wherein the bacterial or viral infection is caused by a vector-borne pathogen as shown in Table 2, and the dead and / or inactivated non-pathogenic Gram-positive bacteria are identified in the corresponding row of Table 2, and the insects are shown in the corresponding row of Table 2.

[0366] 51. The method according to any one of embodiments 1, 2 or 50, wherein the insect is a mosquito species, optionally Culexinae and Anophelesinae, and further optionally wherein the mosquito species is selected from Aedes albopictus, Aedes aegypti or Aedes polynesiensis.

[0367] 52. A method for vaccinating, treating, preventing, or immunizing an insect carrying a vector-borne pathogen against one or more of a viral infection, bacterial infection, bacterial disease, viral disease, and / or disease transmission, the method comprising administering to the insect or insect host an effective amount of a dead and / or inactivated Gram-positive bacterium or a fragment of its cell wall thereof, optionally in combination with a vector, thereby vaccinating, treating, or immunizing the insect or insect host against the viral or bacterial disease or infection, optionally wherein the insect is a mosquito or a tick, and further optionally wherein the insect is a mosquito.

[0368] 53. The method according to embodiment 52, wherein the insect or tick is identified in Table 2, optionally wherein the insect is a mosquito selected from the Culexinae or Anophelesinae subfamily, and further optionally wherein the mosquito family is selected from Aedes albopictus, Aedes aegypti, or Aedes polynesiana.

[0369] 54. The method according to any one of embodiments 50-53, wherein the Gram-positive bacteria or cell wall fragments thereof are species of the genus *Paenabacillus*, optionally selected from *Paenabacillus agaricus*, *Paenabacillus edibleus*, *Paenabacillus alginate*, *Paenabacillus alkalophilus*, *Paenabacillus apigensis*, *Paenabacillus amyloliquefaciens*, *Paenabacillus anaerobicus*, *Paenabacillus antarcticus*, *Paenabacillus apigensis*, *Paenabacillus assamum*, *Paenabacillus reductiveis*, *Paenabacillus nitrogenophilus*, *Paenabacillus Barcelona*, *Paenabacillus northernus*, *Paenabacillus brasiliensis*, and *P.* brassicae[1], Campinas spore-forming bacteria, Jinju spore-forming bacteria, chitinous spore-forming bacteria, chondroitin spore-forming bacteria, ash spore-forming bacteria, Cook spore-forming bacteria, coagulating spore-forming bacteria, Ota spore-forming bacteria, dendritic spore-forming bacteria, sclerosing spore-forming bacteria, Ehime spore-forming bacteria, Egi spore-forming bacteria, honey comb spore-forming bacteria, dextran spore-forming bacteria, polysaccharide spore-forming bacteria, Gordon spore-forming bacteria Poaceae Bacillus, Particle-eating Bacillus, P. hodogayensis, Illinois Bacillus, Garmina Bacillus, Kobe Bacillus, Coleoptile Bacillus, Korean Bacillus, Gel-like Bacillus, Lactobacillus, Larval Bacillus, Brilliant Bacillus, Slow-developing Bacillus, Hemp-seated Bacillus, Macole Bacillus, Marseille Bacillus, Monsei Bacillus, P. *Motobuensis*, *Naphthalene-eating Bacillus*, *Nematotrophic Bacillus*, *Odor-eating Bacillus*, *Feed-eating Bacillus*, *P. phoenicis*, *Leucobacterium*, *Polymycosis*, *Bacillus japonicus*, *Dust-eating Bacillus*, *Rhizosphere*, *Hematologic Bacillus*, *P. stellifer*, *Paenibacillus stellife*, *Land-eating Bacillus*, *Thiamine-degrading Bacillus*, *Timon*, *P. tundrae*, *Zurich*, *P. tylopili*, *P.*Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum, and / or Bacillus xylolyticus, and further optionally selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextranis, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculiformis, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus Stellife, thiamine-derived Bacillus, Paenibacillus validus, and xylolytic Bacillus, or a combination of two or more thereof.

[0370] 55. The method according to any one of embodiments 50-54, wherein the Gram-positive bacteria or cell wall fragments thereof comprise or consist substantially of PL or cell wall fragments thereof.

[0371] 56. The method according to any one of embodiments 50-55, wherein the dead and / or inactivated Gram-positive bacteria are applied in combination with the carrier.

[0372] 57. The method according to any one of embodiments 50-56, wherein the effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprises about 1.5 x 10^6 mg / g or carrier. 4 Or 1.5 x 10 7 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0373] 58. The method according to embodiment 57, wherein each dose to the insect is approximately 1.5 x 10 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

[0374] 59. The method according to embodiment 58, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of 1.5 x 10⁻⁶ are applied to insects carrying vector-borne diseases. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 The whole cell or cell wall fragment of a dead and / or inactivated Gram-positive bacterium containing one antigen unit.

[0375] Equivalent scheme

[0376] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain.

[0377] The technology described herein can be practiced appropriately in the absence of any element or elements, limitations or multiple limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” “containing,” etc., should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein have been used as descriptive rather than restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications can be made within the scope of the claims made herein.

[0378] Therefore, it should be understood that the materials, methods, and examples provided herein are representative of preferred aspects, are exemplary, and are not intended to limit the scope of the technology.

[0379] This technique has been described broadly and generally herein. Each narrower group of species and subgenus falling within the general description also constitutes part of this technique. This includes the general description of this technique, with additional conditions or negative restrictions for the removal of any subject from that genus, regardless of whether the removed material is specifically described herein.

[0380] Furthermore, when the features or aspects of this technology are described in accordance with the Markush Group, those skilled in the art will recognize that this technology is also described in accordance with any single member or subgroup of the Markush Group.

[0381] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety by reference as if each were individually incorporated by reference. In the event of conflict, this specification (including definitions) shall prevail.

[0382] Other aspects are set forth in the following claims. References

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[0420] Ramsey, S. D., Ochoa, R., Bauchan, G., Gulbronson, C., Mowery, J. D.,Cohen, A., Lim, D., Joklik, J., Cicero, J. M., Ellis, J. D., Hawthorne, D., &vanEngelsdorp, D. (2019). Varroa destructor feeds primarily on honey bee fatbody tissue and not hemolymph. Proceedings of the National Academy ofSciences of the United States of America, 116(5), 1792–1801. https: / / doi.org / 10.1073 / pnas.1818371116

[0421] Ryabov, E. V., Childers, A. K., Lopez, D., Grubbs, K., Posada‐Florez,F., Weaver, D., Girten, W., vanEngelsdorp, D., Chen, Y., & Evans, J. D.(2019). Dynamic evolution in the key honey bee pathogen deformed wing virus:Novel insights into virulence and competition using reverse genetics. PLOSBiology, 17(10), e3000502. https: / / doi.org / 10.1371 / journal.pbio.3000502

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Claims

1. A method for vaccinating, treating, or immunizing an invertebrate, a population of invertebrates, its offspring, or an invertebrate host against a bacterial or viral infection or disease, the method comprising administering to the invertebrate or population an effective amount of a dead and / or inactivated nonpathogenic Gram-positive bacterium or a fragment of its cell wall thereof, thereby vaccinating, treating, or immunizing the invertebrate against the bacterial or viral disease or infection, optionally wherein the invertebrate is a crustacean or an insect.

2. The method of claim 1, further comprising administering an effective amount of dead and / or inactivated Gram-negative bacteria or cell wall fragments thereof to the invertebrate or the population of invertebrates, and optionally wherein the invertebrate is a crustacean or an insect.

3. The method according to claim 1 or 2, wherein the bacterial or viral infection is caused by a single row of any of the tables 1-4 or by a pathogen as shown in Experiment 4, and the dead and / or inactivated non-pathogenic Gram-positive bacteria are identified in the corresponding row of the tables 1-4 or as shown in Experiment 4.

4. The method according to claim 1 or 2, wherein the invertebrate is an insect and / or the invertebrate population is an insect or an insect population or community, and the virus causing the viral disease or infection is selected from Aberrant wing virus A, Aberrant wing virus B, Aberrant wing virus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, Queen cell virus, and optionally wherein the insect is a bee and / or the insect population is a bee population.

5. The method according to claim 1 or 2, wherein the insect is a honeybee or a bumblebee, or the colony is a honeybee or bumblebee colony or community, and wherein the viral disease or infection is caused by Deformia wingvirus A, Deformia wingvirus B, Deformia wingvirus C, Acute honeybee paralysis virus, Israel acute honeybee paralysis virus, Kashmir honeybee virus, Slow honeybee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic honeybee paralysis virus, Sacbryovirus, or Black Queen Cell virus.

6. The method of claim 5, wherein the virus causing the disease or infection is pterygoid virus B (DWV-B).

7. The method according to claim 1 or 2, wherein the dead and / or inactivated non-pathogenic bacteria or cell wall fragments thereof are administered in combination with a carrier, optionally wherein the insect is a bee or bumblebee and / or the colony is a bee colony or bumblebee colony, and the carrier contains bee food or bee feed.

8. The method according to claim 1 or 2, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated bacterial species of the genus Paenibaccilus.

9. The method according to claim 8, wherein the dead and / or inactivated bacterial species are selected from *P. agarexedens*, *P. agaridevorans*, *P. alginolyticus*, *P. alkaliterrae*, *P. alvei*, *P. amylolyticus*, *P. anaericanus*, *P. antarcticus*, *P. apiarius*, *P. assamensis*, *P. azoreducens*, *P. azotofixans*, *P. barcinonensis*, *P. borealis*, *P. brasilensis*, and *P.* brassicae[1], P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. cookii, P. curdlanolyticus, P. daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P. favisporus, P. glucanolyticus, P. glucanolyticus. *P. glycanilyticus*, *P. gordonae*, *P. graminis*, *P. granivorans*, *P. hodogayensis*, *P. illinoisensis*, *P. jamilaae*, *P. kobensis*, *P. koleovorans*, *P. koreensis*, and *P. jelly-like*.*P. kribbensis*, *P. lactis*, *P. larvae*, *P. lautus*, *P. lentimorbus*, *P. macerans*, *P. macquariensis*, *P. massiliensis*, *P. mendelii*, *P. motobuensis*, *P. naphthalenovorans*, *P. nematophilus*, *P. odorifer*, *P. pabuli*, *P. peoriae*, *P. phoenicis*, *P. phyllosphaerae*, *P. polymyxa*, *P. jatropha*. *P. popilliae*, *P. pulvifaciens*, *P. rhizosphaerae*, *P. sanguinis*, *P. stellifer*, *Paenibacillusstellife*, *P. terrae*, *P. thiaminolyticus*, *P. timonensis*, *P. tundrae*, *P. turicensis*, *P. tylopili*, *P. validus*, *P. vortex*, *P. vulneris*, *P. wynnii*, and / or *P. xylanolyticus*.xylanilyticus, or a combination of two or more thereof; or optionally selected from Paenibacillus alvei, Paenibacillus dentritiformis, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chondroitinus, Paenibacillus chungangensis, Paenibacillus doosanensis, Paenibacillus glucanolyticus, Paenibacillus humicus, Paenibacillus lactis, Paenibacillus larvae, Paenibacillus splenicus. The following are listed: Paenibacillus lautus, Paenibacillus lentimorbus, Paenibacillus maceran, Paenibacillus macerans-like, Paenibacillus macquariensis, Paenibacillus motobuensis, Paenibacillus papuli, Paenibacillus phoenicis, Paenibacillus polymyxa, Paenibacillus popilliae, Paenibacillus puldeungensis, Paenibacillus residui, Paenibacillus stellife, Paenibacillus sthiaminolyticus, Paenibacillus validus, and Paenibacillus xylanisolvens, or combinations of two or more thereof. .

10. The method according to claim 1 or 2, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

11. The method according to claim 1 or 2, wherein the effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprises about 1.5 x 10⁻⁶ per gram or carrier. 4 Or 1.5 x 10 7 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

12. The method of claim 11, wherein each dose administered to the insect is approximately 1.5 x 10⁻⁶. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

13. The method of claim 12, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of 1.5 x 10 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

14. A method for vaccinating, treating, or immunizing bees, bee offspring, or bee colonies against one or more viral infections or viral diseases, comprising administering to the bees, queen bees, or bee colonies an effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof, and optionally a carrier, thereby vaccinating, treating, or immunizing the bees, offspring, or colonies against the viral disease or infection.

15. The method of claim 14, wherein the bee is a honeybee or the bee colony is a honeybee colony or honeybee community, and the virus causing the viral disease or infection is selected from Aberrant wing virus A, Aberrant wing virus B, Aberrant wing virus C, Acute honeybee paralysis virus, Israel acute honeybee paralysis virus, Kashmir honeybee virus, Slow honeybee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic honeybee paralysis virus, Sacbryovirus, and Black Queen Cell virus.

16. The method of claim 14, wherein the bee is a bumblebee or a bumblebee colony or community, and wherein the viral disease or infection is caused by Deformia wingvirus A, Deformia wingvirus B, Deformia wingvirus C, Acute Bee Paralysis Virus, Israel Acute Bee Paralysis Virus, Kashmir Bee Virus, Slow Bee Paralysis Virus, Lake Sinai Virus 1, Lake Sinai Virus 2, Chronic Bee Paralysis Virus, Sacbryovirus, or Black Queen Cell Virus.

17. The method of claim 14, wherein the bee is a honeybee and the colony is a honeybee colony or community, and the virus causing the disease or infection is Deformed Wing Virus B (DWV-B).

18. The method of claim 14 or 15, wherein the carrier comprises bee food or bee feed.

19. The method according to claim 14 or 15, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated bacterial species of the genus Bacillus.

20. The method according to claim 19, wherein the dead and / or inactivated bacterial species are selected from Bacillus agarophilus, Bacillus edibleus, Bacillus alginate, Bacillus alkalophilus, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobicus, Bacillus antarcticus, Bacillus vesicularis, Bacillus assamum, Bacillus reductive azo, Bacillus nitrogen-fixing, Bacillus Barcelonas, Bacillus northernus, Bacillus brasiliensis, and P. brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextrin, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus Stellife, thiamine-derived Bacillus, Paenibacillus validus, and xylolytic Bacillus, or a combination of two or more thereof.

21. The method according to claim 14 or 15, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

22. The method according to claim 14 or 15, wherein the effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprises about 1.5 x 10⁻⁶ per gram or carrier. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

23. The method of claim 21, wherein each dose is applied to the bees or bee colony at a concentration of about 1.5 x 10⁻⁶. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

24. The method of claim 21, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

25. A method for treating or immunizing a bee colony against one or more of the viral infections or diseases caused by viruses, the method comprising introducing an uninfected queen bee into the bee colony and administering to the queen bee an effective amount of dead and / or inactivated whole cells or cell wall fragments of Gram-positive bacteria and a carrier, thereby treating or immunizing the bee colony against the viral disease or infection.

26. The method of claim 25, wherein the bee colony is a bee colony or community, and the virus causing the viral disease or infection is selected from Aberrant wing virus A, Aberrant wing virus B, Aberrant wing virus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, and Queen Cell virus.

27. The method of claim 25, wherein the population or community is a bumblebee population or community, and wherein the viral disease or infection is caused by Aberrant wing virus A, Aberrant wing virus B, Aberrant wing virus C, Acute bee paralysis virus, Israel acute bee paralysis virus, Kashmir bee virus, Slow bee paralysis virus, Lake Sinai virus 1, Lake Sinai virus 2, Chronic bee paralysis virus, Sacbryovirus, or Queen Cell virus.

28. The method of claim 25, wherein the colony is a bee colony or community, and the virus causing the disease or infection is Deformed Wing Virus B (DWV-B).

29. The method of claim 25 or 26, wherein the carrier comprises bee food.

30. The method of claim 25 or 26, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated bacterial species of the genus Bacillus.

31. The method according to claim 30, wherein the dead and / or inactivated bacterial species are selected from Bacillus agarophilus, Bacillus edibleus, Bacillus alginate, Bacillus alkalophilus, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobicus, Bacillus antarcticus, Bacillus vesicularis, Bacillus assamum, Bacillus reductive azotobacter, Bacillus nitrogen-fixing, Bacillus Barcelonas, Bacillus northernus, Bacillus brasiliensis, and P. brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextrin, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus Stellife, thiamine-derived Bacillus, Paenibacillus validus, and xylolytic Bacillus, or a combination of two or more thereof.

32. The method according to claim 25 or 26, wherein the dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprise dead and / or inactivated larval Bacillus (PL) or cell wall fragments thereof.

33. The method according to claim 25 or 26, wherein the effective amount of dead and / or inactivated Gram-positive bacterial whole cells or cell wall fragments comprises approximately 1.5 x 10⁻⁶ per gram of bee food or carrier. 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

34. The method of claim 33, wherein each dose of about 1.5 x 10 g is administered to the queen, worker bees, caretaker bees, or larvae in the colony. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

35. The method of claim 34, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of about 1.5 x 10⁻⁶ are administered to the queen, worker bees, caretaker bees, or bee larvae in the colony. 7 Or approximately 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

36. A composition comprising whole cells or cell wall fragments of dead and / or inactivated Gram-negative and / or Gram-positive bacteria, and a carrier, optionally wherein the carrier is phosphate-buffered saline or water.

37. The composition of claim 36, wherein the Gram-negative bacteria comprises, or is substantially composed of, a bacterial species of the genus Vibrio.

38. The composition according to claim 36 or 37, wherein the Gram-negative bacteria is selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio campbelli, Vibrio damsela, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio penaeicida, Vibrio vulnificus, Vibrio nereis, Vibrio tubiashi, Vibrio fluvialis, Vibrio splendidus, or Vibrio nigripulchritudo.

39. The composition according to claim 36 or 37, wherein the Gram-positive bacteria comprises, or is substantially composed of, a species of the genus Bacillus.

40. The composition according to claim 36 or 37, wherein the Gram-positive bacteria is selected from *Bacillus agaricus*, *Bacillus edibleus*, *Bacillus alginate*, *Bacillus alkalophilus*, *Bacillus vesicularus*, *Bacillus amyloliquefaciens*, *Bacillus anaerobicus*, *Bacillus antarctica*, *Bacillus vesicularus*, *Bacillus assamica*, *Bacillus reductiveazobia*, *Bacillus nitrogen-fixing*, *Bacillus barbarus*, *Bacillus northernus*, *Bacillus brassica*, *P. brassica*, etc. sicae[1], Campinas spore-forming bacteria, Jinju spore-forming bacteria, chitinous spore-forming bacteria, chondroitin spore-forming bacteria, ash spore-forming bacteria, Cook spore-forming bacteria, coagulating spore-forming bacteria, Oda spore-forming bacteria, dendritic spore-forming bacteria, sclerosing spore-forming bacteria, Ehime spore-forming bacteria, Egi spore-forming bacteria, honey comb spore-forming bacteria, dextran spore-forming bacteria, polysaccharide spore-forming bacteria, Gordon spore-forming bacteria, grass spore-forming bacteria, granule-eating spore-forming bacteria, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Hypertrophic* Bacillus, *Hemibarbus* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycinus* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus* Stellife, Soil-borne Bacillus, Thiamine-derived Bacillus, Timone-derived Bacillus, P. tundrae, Zurich-derived Bacillus, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum and / or Bacillus xylolyticus, or combinations of two or more thereof; or alternatively selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjongensis, Paenibacillus doosanensis, Bacillus dextrin, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus Stellife, thiamine-derived Bacillus, Paenibacillus validus, and xylolytic Bacillus, or a combination of two or more thereof.

41. The composition according to claim 36 or 37, wherein the carrier comprises feed for invertebrates.

42. The composition according to claim 41, wherein the invertebrate is a crustacean.

43. The composition according to claim 41, wherein the invertebrate is a shrimp, and the feed is shrimp food.

44. The composition according to claim 41, wherein the invertebrate is identified in Tables 1-4 or Experiment 4.

45. The composition of claim 41, wherein the invertebrate is a shrimp, and the carrier is selected from one or more of phosphate-buffered saline, water, shrimp food, or shrimp feed.

46. ​​The composition according to claim 41 or 42, wherein the composition is formulated for oral administration, injection administration, submersion administration, or any combination of two or more thereof.

47. A method for vaccinating, treating, or immunizing an invertebrate or its offspring against one or more of an infection or disease caused by a viral or bacterial infection, comprising administering an effective amount of the composition of any one of claims 41-46 to the invertebrate, thereby vaccinating, treating, or immunizing the invertebrate or its offspring against the viral or bacterial disease or infection in the animal.

48. The method of claim 47, wherein the invertebrate is identified in Tables 1-4 or Experiment 4.

49. The method of claim 48, wherein the bacteria comprise, substantially comprise, or comprise, larval Bacillus and thiamine Bacillus, the insect is a black soldier fly, and the pathogen is Hermetiaillucens totivirus.

50. The method according to claim 1 or 2, wherein the bacterial or viral infection is caused by a vector-borne pathogen as shown in Table 2, and the dead and / or inactivated non-pathogenic Gram-positive bacteria are identified in the corresponding row of Table 2, and the insect is shown in the corresponding row of Table 2.

51. The method according to any one of claims 1, 2 or 50, wherein the insect is a species of Culicidae, optionally of the subfamilies Culicinae and Anophelinae, and further optionally wherein the Culicidae species is selected from Aedes albopictus, Aedes aegypti or Aedes polynesiensis.

52. A method for vaccinating, treating, preventing, or immunizing an insect carrying a vector-borne pathogen against one or more of a viral infection, bacterial infection, bacterial disease, viral disease, and / or disease transmission, the method comprising administering to the insect or insect host an effective amount of a dead and / or inactivated Gram-positive bacterium or a fragment of its cell wall thereof, optionally in combination with a vector, thereby vaccinating, treating, or immunizing the insect or insect host against the viral or bacterial disease or infection, optionally wherein the insect is a mosquito or a tick, and further optionally wherein the insect is a mosquito.

53. The method of claim 52, wherein the insect or tick is identified in Table 2, optionally wherein the insect is a mosquito selected from the Culexinae or Anophelesinae subfamily, and further optionally wherein the mosquito family is selected from Aedes albopictus, Aedes aegypti, or Aedes polynesiana.

54. The method according to any one of claims 50-53, wherein the Gram-positive bacteria or cell wall fragments thereof belong to the Bacillus species, optionally selected from Bacillus agaris, Bacillus edibleus, Bacillus alginate, Bacillus alkalophilus, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobicus, Bacillus antarcticus, Bacillus vesicularis, Bacillus assamum, Bacillus reductive azotobacter, Bacillus nitrogen-fixing, Bacillus barbarus, Bacillus northernus, Bacillus brasiliensis, and P. brassicae[1], Campinas Bacillus, Jinju Bacillus, Chitin Bacillus, Chondroitin Bacillus, Ash Bacillus, Cook Bacillus, Cough Bacillus, Oda Bacillus, Dendritic Bacillus, Sclerosus Bacillus, Ehime Bacillus, Egi Bacillus, Honeycomb Bacillus, Dextran Bacillus, Polysaccharide Bacillus, Gordon Bacillus, Gramineae Bacillus, P. *Hodogayensis*, *Illinois* Bacillus, *Garmina* Bacillus, *Kobe* Bacillus, *Coleoptile* Bacillus, *Korean* Bacillus, *Gel-like* Bacillus, *Lactobacillus*, *Larva* Bacillus, *Brilliant* Bacillus, *Slow-acting* Bacillus, *Hemp-soaking* Bacillus, *Macrobrachium* Bacillus, *Marseille* Bacillus, *Montenebrio* Bacillus, *P. motobuensis*, *Naphthalene-eating* Bacillus, *Nematotroph* Bacillus, *Odor* Bacillus, *Feed* Bacillus, *P. phoenicis*, *Lepidoptera* Bacillus, *Polymycin* Bacillus, *Japanese Scarab Beetle* Bacillus, *Dust* Bacillus, *Rhizosphere* Bacillus, *Hematologic* Bacillus, *P. stellifer*, *Paenibacillus stellife*, *Land* Bacillus, *Thiamine* Bacillus, *Timon* Bacillus, *P.* tundrae, Zurich Bacillus subtilis, P. tylopili, P.Validus, Bacillus vorticella, Bacillus woundae, Bacillus venereum, and / or Bacillus xylolyticus, and further optionally selected from Bacillus apis, Bacillus dendriticus, Bacillus amylolyticus, Bacillus campinatus, Bacillus chondroitin, Bacillus hanjong, Paenibacillus doosanensis, Bacillus dextran, Bacillus humicus, Bacillus lactis, Bacillus larvae, Bacillus splenicus, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus sclerotiorum, Bacillus maculi, Paenibacillus motobuensis, Bacillus foetida, Paenibacillus phoenicis, Bacillus polymyxa, Bacillus puldeungensis, Paenibacillus residui, Paenibacillus Stellife, thiamine-derived Bacillus, Paenibacillus validus, and xylolytic Bacillus, or a combination of two or more thereof.

55. The method according to any one of claims 50-53, wherein the Gram-positive bacterium or its cell wall fragment comprises PL or its cell wall fragment, or is substantially composed of PL.

56. The method according to any one of claims 50-53, wherein the dead and / or inactivated Gram-positive bacteria are applied in combination with the carrier.

57. The method according to any one of claims 50-56, wherein the effective amount of dead and / or inactivated Gram-positive bacteria or cell wall fragments thereof comprises about 1.5 x 10^6 per gram or carrier. 4 Or 1.5 x 10 7 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

58. The method of claim 57, wherein each dose administered to the insect is approximately 1.5 x 10⁻⁶. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 Whole cells or cell wall fragments of dead and / or inactivated Gram-positive bacteria, each antigen unit.

59. The method of claim 58, wherein at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten doses of 1.5 x 10⁻⁶ are applied to the insect carrying the vector-borne disease. 7 Or 1.5 x 10 4 To approximately 1.5 x 10 11 The whole cell or cell wall fragment of a dead and / or inactivated Gram-positive bacterium containing one antigen unit.

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