Honeybee vaccine and methods of use

Abstract

This disclosure provides compositions and methods for treating and immunizing insects and insect populations against bacterial diseases. This disclosure further provides compositions and methods for prophylactic immunization of beehives to protect them from larval foulbrood infection. In embodiments, this disclosure further provides methods for prophylactic immunization of beehives to protect them from infection by Bacillus larvae (Bacillus subtilis). Paenibacillus larvae Compositions and methods for causing American foulbrood infection in larvae.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 901,988, filed September 18, 2019, pursuant to 35 USC § 119(e), the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Honeybees are commercially used as a vital species for pollination and honey production. In the United States, commercial beekeepers operate approximately 2.8 million hives, and honeybees are responsible for pollinating more than one-third of all crops, including nuts, berries, fruits, and flowering vegetables. In addition, honeybees pollinate a wide variety of wildflowers, thus contributing to the biodiversity of many ecosystems.

[0004] Unfortunately, bee colonies have suffered severe decline and loss in recent years. For example, in the year from April 2018 to April 2019, more than 40% of the 2.8 million commercially operated beehives in the United States died; this equates to a loss of approximately $700 million in income for beekeepers in the United States alone, not including income losses for orchard owners and other farmers due to a lack of pollinating insects.

[0005] The main causes of bee colony loss are pests and pathogens, including parasites, as well as viral and bacterial infections.

[0006] One particularly destructive bacterial pathogen is American foulbrood (Afb). American foulbrood (Afb) is caused by the spore-forming bacterium *Bacillus larvae* (…). Paenibacillus larvae This bacterial disease is caused by [a bacterium name - missing from original text] (Genersch et al., J. Invertebr. Pathol. 103, (2010b)). This disease can be catastrophic for beekeepers and is difficult to eradicate. The bacterium is present in approximately 50% of all hives, and its spores can survive for up to 70 years. Between 4% and 20% of hives will develop clinical symptoms at any given time, resulting in millions of dollars in economic losses for farmers and beekeepers annually.

[0007] Unfortunately, there is currently no safe and effective preventative solution available to protect beehives from American foulbrood. In acute cases, antibiotics are approved for treating bees and can be administered to the hive in the early stages of clinical symptoms to limit the spread of the bacteria. However, this is largely ineffective because the disease is caused by spores, not bacteria. Therefore, burning the beehive is often the only way to control the disease.

[0008] Therefore, there is a need in the art for safe and effective measures to control and prevent American foulbrood and other related insect diseases. This disclosure meets this need and also provides related advantages.

[0009] Overview

[0010] This disclosure relates to novel compositions, vaccine formulations, and methods for improving the efficacy against Bacillus subtilis (B. spp.) Paenibacillus sp. The immune response to and / or vaccination treatment and / or prevention of larval foulbrood caused by *Bacillus larvae*, including American foulbrood (AFB) in bees and other insects, which is caused by the administration and / or inoculation of dead, non-pathogenic *Bacillus larvae* to the queen bee, including but not limited to *Bacillus vesicularis* (…). Paenibacillus alvei PA) and dendritic spores ( Paenibacillus dentriiform (PD). On the one hand, the bee mentioned is a honeybee.

[0011] Contrary to what is known in the art, the applicant describes administration and / or vaccination by applying stimulation from a non-disease pathogen to treat and / or preventively immunize bees against diseases caused by Bacillus subtilis (e.g., Afb). No specific matching between the disease pathogen and the vaccine strain is required.

[0012] Therefore, in one aspect, this disclosure provides a method for protecting insects from disease by applying or administering an immune stimulus derived from a pathogen different from the disease pathogen to provide broad-spectrum, universal protection.

[0013] In one exemplary embodiment, this document provides a composition comprising, in combination or individually, dead Bacillus subtilis (e.g., PD and PA) for protection against infection by Bacillus larvae (PL). The composition may also contain, for example, bee feed, such as honeybee food.

[0014] In other embodiments, this disclosure provides a method for preventing American foulbrood in a colony of bees (e.g., honeybees). The method may include administering or feeding the queen a vaccine formulation consisting of dead PB or PA, or a combination of both. On one hand, approximately 1.5 x 10⁻⁶ ppm of vaccine may be provided or fed to the queen, worker bees, nurse bees, or larvae. 6 To approximately 1.5 x 10 8 One antigen unit per gram of insect food.

[0015] In one embodiment, this document provides a composition comprising, or substantially comprising, whole cell or cell wall fragments of at least one dead, non-pathogenic bacterial species of a genus (e.g., dead Bacillus subtilis, such as PD or PA, or a combination thereof), and a carrier. In one aspect, the composition comprises, or substantially comprises, whole cell or cell wall fragments of at least two, three, or four or more dead, non-pathogenic bacterial species of a genus (e.g., dead Bacillus subtilis, such as PD or PA, or a combination thereof), and a carrier. Non-limiting examples of non-pathogenic bacterial species used in the formulation of the composition are species of the genus Bacillus subtilis, *Bacillus auris* (PA) and *Bacillus denticulatus* (PD), or combinations thereof. In a further aspect, the composition comprises one or more, two or more, three or more, or four or more dead Bacillus subtilis species, wherein said species is selected from one or more of the following: *Bacillus auris* (PA) Paenibacillus alvei ), Bacillus dendriticis ( Paenibacillus dentritiformis ), Amylolytic Bacillus ( Paenibacillus amylolytic ), Campinas Bacillus ( Paenibacillus campinasensis ), Bacillus chondroitinibacterium ( Paenibacillus chondroitinus ), Korean-Chinese Bacillus ( Paenibacillus chungangensis ), Paenibacillus doosanensis Bacillus dextrin ( Paenibacillus glucanolyticus ), humic spore-forming bacteria ( Paenibacillus humicus Lactobacillus ( Lactobacillus lactis ), Bacillus splendidus ( Paenibacillus lautus ), Bacillus subtilis ( Paenibacillus lentimorbus ), Bacillus subtilis ( Paenibacillus maceran ), Bacillus subtilis ( Paenibacillus soaking -like), Bacillus macrocarpa ( Paenibacillus macquariensis ), Paenibacillus from Motobu , feed-grade Bacillus ( Paenibacillus pabuli ), Paenibacillus phoenicis, Polymyxin Bacillus ( Paenibacillus polymyxa ), Japanese scarab beetle ( Paenibacillus baby's legs ), Paenibacillus puldeungensis , Paenibacillus residuum , Paenibacillus star life Thiamine-like Bacillus ( Paenibacillus thiaminolyticus ), Paenibacillus validus and xylolytic Bacillus ( Paenibacillus xylanisolvens(or combinations thereof). In a further aspect, the dead Bacillus species in the composition include, alone or in combination with, Bacillus apis and Bacillus dendriticis, or consist essentially of, or consist of, them.

[0016] Non-limiting examples of the carrier can be solid or liquid carriers, and may include preservatives, insect nutrients, or other colorants as needed. In one specific embodiment, the carrier is insect food, such as queen bee wafers or sugar feed.

[0017] In one aspect, the composition contains whole cells or cell wall fragments of at least one dead, non-pathogenic bacterial species of the genus *Bacteria* at a concentration of approximately 1.5 x 10-1 per gram of the composition or food. 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 8 To approximately 1.5 x 10 11 One antigen unit, or approximately 1.5 x 10 8 To approximately 1.5 x 10 10 One antigen unit, or approximately 1.5 x 10 8 To approximately 1.5 x 10 9 One antigen unit, or at least 1.5 x 10 7 One antigen unit, or at least about 1.5 x 10 8 One antigen unit, or at least about 1.5 x 10 9 One antigen unit, or at least about 1.5 x 10 10 The total amount of antigen units exists.

[0018] In one exemplary embodiment, this document provides a composition comprising, in combination or individually, dead Bacillus subtilis (e.g., PD and PA) for protecting bee larvae and queen bee offspring from infection with Bacillus larvae (PL). The composition may also contain, for example, insect feed, such as bee feed. The range of whole cells or cell wall fragments of Bacillus subtilis is within the scope provided herein.

[0019] In other embodiments, this disclosure provides a method for preventing American foulbrood in a colony of bees (e.g., honeybees). The method may include administering to worker bees, larvae, nurse bees, and / or the queen bee a composition and / or vaccine formulation as disclosed herein, comprising, or substantially consisting of, dead Bacillus subtilis (e.g., PD or PA, or a combination of both). As will be apparent to those skilled in the art, the queen bee may feed on food, or worker bees or nurse bees may ingest food and feed the queen. Furthermore, bee larvae may also feed on the composition. The range of whole cells or cell wall fragments of Bacillus subtilis is within the scope provided herein.

[0020] In other embodiments, this disclosure provides a treatment for diseases caused by Bacillus subtilis (e.g., American foulbrood) in colonies of bees (e.g., honeybees). The method may include administering to worker bees, nurse bees, larvae, and / or the queen a composition or formulation comprising, or substantially comprising, dead, non-pathogenic Bacillus subtilis (e.g., PD or PA, or a combination of both). In one aspect, the composition comprises dead PD or PA to treat diseases caused by Bacillus subtilis larvae (e.g., Afb). The range of whole cells or cell wall fragments of Bacillus subtilis is within the scope provided herein.

[0021] In other embodiments, this disclosure provides a method for preventing American foulbrood in bee colonies, such as bee larvae or offspring from the queen treated by the methods disclosed herein. The method may include administering to worker bees, nurse bees, and / or the queen bee a formulation comprising, or substantially comprising, dead, non-pathogenic Bacillus subtilis (e.g., PD or PA or a combination of both). The range of whole cells or cell wall fragments of Bacillus subtilis is within the scope provided herein.

[0022] This article also provides a method for treating, immunizing, or enhancing the immune response in bee larvae against diseases caused by Bacillus subtilis (such as larval foulbrood known to be caused by PL), the method comprising, or substantially comprising, the following steps, or comprising the following steps: applying an effective amount of the composition described herein to nurse bees, worker bees, or queen bees, or bee larvae, thereby immunizing the queen bee, and bee larvae and offspring produced by the queen bee. On the one hand, larval foulbrood is caused by Bacillus subtilis (… Paenibacillaceae Caused by any species in the family ( ). Unbound by theory, antigens that will be passed on to the larvae can be detected in the queen bee's ovary. This protects the larvae from disease.

[0023] This method can be used to protect larvae—both queen bees and worker bees—from larval foulbrood caused by members of the Bacillusaceae family (such as PL, PA, and PD) to provide larval populations that have been immunized against larval foulbrood by queen bees that have ingested a vaccine composition.

[0024] This article further provides a method for treating and / or preventing larval foulbrood (e.g., Afb) in a colony of bees (e.g., honeybees), the method comprising, or substantially comprising, the following steps: administering to worker bees and the queen bee a composition as described herein, wherein, in one aspect, the composition comprises, or substantially comprises, a dead, non-disease-free whole or fragment from a Bacillus species, or comprises, or comprises, a dead, non-disease-free Bacillus species. In another aspect, the dead, non-disease-free Bacillus species is selected from Bacillus apiae (Bacillus apiae). Paenibacillus alvei ), Bacillus dendriticis ( Paenibacillus dentritiformis ), Amylolytic Bacillus ( Paenibacillus amylolyticus ), Campinas Bacillus ( Paenibacillus campinasensis ), Bacillus chondroitinibacterium ( Paenibacillus chondroitin ), Korean-Chinese Bacillus ( Paenibacillus chungangensis ), Paenibacillus from Doosan Bacillus dextrin ( Paenibacillus glucanolyticus ), humic spore-forming bacteria ( Paenibacillus humicus Lactobacillus ( Lactobacillus lactis ), Bacillus splendidus ( Paenibacillus lautus ), Bacillus subtilis ( Paenibacillus lentimorbus ), Bacillus subtilis ( Paenibacillus maceran ), Bacillus subtilis ( Paenibacillus macerans -like), Bacillus macrocarpa ( Paenibacillus macquariensis ), Paenibacillus motobuensis , feed-grade Bacillus ( Paenibacillus pabuli ), Paenibacillus phoenicis, Polymyxin Bacillus ( Paenibacillus polymyxa ), Japanese scarab beetle ( Paenibacillus popilliae ), Paenibacillus puldeungensis , Paenibacillus residuum , Paenibacillus stellifer Thiamine-like Bacillus ( Paenibacillus thiaminolyticus ), Paenibacillus validus and xylolytic Bacillus ( Paenibacillus xylanisolvens ( ), or combinations thereof. In another aspect, the composition comprises one or two of dead, non-disease-like Bacillus species, namely dead Bacillus auris and Bacillus denticulatus, or a combination of both with other Bacillus species or other inactivated cell or bacterial fragments of the Bacillus family.

[0025] In one aspect of the method, the application comprises feeding worker bees, nurse bees, larvae, and / or the queen bee an appropriate amount of queen bee sugar cake or sugar feed, wherein each dose of vaccine, composition, or preparation is administered in an amount comprising approximately 1.5 x 10⁻⁶ ppm. 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 units. 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 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-disease-like Bacillus species or fragments thereof per gram of food containing one antigen unit.

[0026] A further method is provided for preparing insect vaccines and / or compositions and / or formulations, 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 of the genus *Bacillus*. In one embodiment, the method further comprises mixing the isolated antigen units with insect food or a carrier (e.g., queen bee cake or gel). In one aspect, the non-pathogenic bacterial species is a species of the genus *Bacillus*, such as *Bacillus vesicularis* and *Bacillus denticulatus*, or a combination thereof. In another aspect, the dead *Bacillus* species is selected from *Bacillus vesicularis* (…). Paenibacillus alvei ), Bacillus dendriticis ( Paenibacillus dentritiformis ), Amylolytic Bacillus ( Paenibacillus amylolyticus ), Campinas Bacillus ( Paenibacillus from the plains ), Bacillus chondroitinibacterium ( Paenibacillus chondroitinus ), Korean-Chinese Bacillus ( Paenibacillus chungangensis ), Paenibacillus doosanensis Bacillus dextrin ( Paenibacillus glucanolyticus ), humic spore-forming bacteria ( Paenibacillus humicus Lactobacillus ( Lactobacillus lactis), Bacillus splendidus ( Paenibacillus lautus ), Bacillus subtilis ( Paenibacillus lentimorbus ), Bacillus subtilis ( Paenibacillus maceran ), Bacillus subtilis ( Paenibacillus macerans -like), Bacillus macrocarpa ( Paenibacillus Macquarie ), Paenibacillus motobuensis , feed-grade Bacillus ( Paenibacillus fodder ), Paenibacillus phoenicis, Polymyxin Bacillus ( Paenibacillus polymyxa ), Japanese scarab beetle ( Paenibacillus popilliae ), Paenibacillus puldeungensis , Paenibacillus residuum , Paenibacillus stellifer Thiamine-like Bacillus ( Paenibacillus thiaminolytic ), Paenibacillus validus and xylolytic Bacillus ( Paenibacillus xylanol-dissolving (or combinations thereof). In another respect, dead Bacillus species are Bacillus apis and Bacillus dendriticus.

[0027] In one aspect, each dose provides approximately 1.5 x 10⁻⁶ whole cells or cell wall fragments of at least one, two, three, four, or more dead, non-pathogenic bacterial species of the genus *Bacteria*. 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 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 containing a dead, non-disease-like Bacillus species or fragments thereof.

[0028] This document also provides treatments and / or vaccine compositions prepared by the methods described herein, as well as bee or insect larvae or colonies of larvae from queen bees that have been immunized by ingestion of the treatments and / or vaccine compositions described herein. The compositions and / or formulations may be provided in kits comprising the compositions and instructions for use as described herein.

[0029] Other features, objects and advantages of the present invention will become apparent from the following detailed description. Attached Figure Description

[0030] Figure 1A Small pictures 1-3 and Figure 1B This illustrates the mechanism of Afb vaccination in bees and provides examples of vaccination or treatment against other diseases caused by Bacillus subtilis. (Figure 1) Treatment and Vaccination: Worker bees are fed the compositions disclosed herein. Regular queen bee feed (royal sugar) containing the composition (oral vaccine) is ingested by worker bees or nurse bees and added to royal jelly in the mandibular glands. The queen bee or queen larvae are fed the composition (e.g., vaccine) containing royal jelly by worker bees and nurse bees. Figure 2 The vaccine is stored in the fat bodies of larvae and the queen bee, and is transported to the queen's ovary once she is fully developed. This transfer is mediated by vitellogenin, a protein found in egg yolk. Figure 3 shows the larval treatment. The Y-axis shows the percentage of larvae surviving 72 hours post-infection. Vaccination of the queen bee with dead American foulbrood larvae produced larvae that were more resistant to subsequent infection with the pathogen (grey bars, vaccine: 46% larval survival vs. placebo: 23% larval survival, χ² = 4.32, p = 0.004). There was no negative impact on larval survival under uninfected control conditions (black bars, vaccine: 35% larval survival vs. placebo: 32% larval survival, χ² = 3.57, p = 0.06). Figure 1B It also provides an illustration of the vaccination mechanism.

[0031] Figure 2 The illustration depicts 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.

[0032] Figure 3A and 3BThe results of queen bee administration and / or vaccination with *Bacillus dendriticus* are shown. Vaccination with PD protects offspring from PA infection (Figure 3A) (survival regression comparison between placebo (light gray) and PD (dark gray) when infected with PA). Figure 3B shows the results of PL infection (survival regression comparison between placebo (light gray) and PD (dark gray) when infected with PL). Queen bee vaccination with dead *Bacillus dendriticus* produces larvae that are more resistant to subsequent pathogens. (PD vs. PA gray bars ± SD, vaccine: approximately 60% larval survival vs. placebo: 40% larval survival, p = 0.08) and (PD vs. PL gray bars ± SD, 35% larval survival vs. 25% larval survival, p = 0.18). There was no negative impact on larval survival under the infection-free control condition (grey bar ± SD, vaccine: 15% larval survival vs. placebo: 5% larval survival, p>0.05).

[0033] Detailed description

[0034] definition

[0035] As used herein and in the appended claims, singular articles such as “a”, “an”, and “the”, as well as similar designations in the context of describing an element, shall be construed as covering both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context.

[0036] As used herein, “about” is as understood by one of ordinary skill in the art and may vary to some extent depending on the context in which it is used. If the context in which the term “about” is used contains a term that is not clear to one of ordinary skill in the art, then “about” will mean up to 10% positive or negative of that particular term.

[0037] As those skilled in the art will understand, for any and all purposes, all scopes disclosed herein also include any and all possible subscopes and combinations thereof. Furthermore, as those skilled in the art will understand, the scope includes each individual member.

[0038] As used herein, the term “exemplary” means “used as an example, instance, or illustration”, rather than “preferred” or “superior to other implementations”.

[0039] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In particular, this disclosure utilizes conventional techniques in the field of beekeeping.

[0040] As used herein, the term "American foulbrood" or "Afb" refers to a fatal bacterial disease of bee larvae caused by the spore-forming bacterium Bacillus larvae. Because Bacillus larvae causes American foulbrood in bees, it is referred to herein as a "pathogenic" bacterium or "disease species".

[0041] As used herein, the term "non-pathogenic" species or "non-pathogenic species" refers to a bacterial species that may or may not be pathogenic but does not cause the target disease. For example, for American foulbrood caused by Bacillus larvae, exemplary non-pathogenic Bacillus species, which may be found in the environment but not in humans or human wounds, may include, for example, Bacillus vesicularis (…). Paenibacillus alvei ), Bacillus dendriticis ( Paenibacillus dentriiform ), Amylolytic Bacillus ( Paenibacillus amylolyticus ), Campinas Bacillus ( Paenibacillus campinasensis ), Bacillus chondroitinibacterium ( Paenibacillus chondroitinus ), Korean-Chinese Bacillus ( Paenibacillus chungangensis ), Paenibacillus doosanensis Bacillus dextrin ( Paenibacillus glucanolyticus ), humic spore-forming bacteria ( Paenibacillus humus Lactobacillus ( Lactobacillus lactis ), Bacillus splendidus ( Paenibacillus clean ), Bacillus subtilis ( Paenibacillus lentimorbus ), Bacillus subtilis ( Paenibacillus maceran ), Bacillus subtilis ( Paenibacillus macerans -like), Bacillus macrocarpa ( Paenibacillus macquariensis ), Paenibacillus motobuensis , feed-grade Bacillus ( Paenibacillus pabuli ), Paenibacillus phoenicis, Polymyxin Bacillus ( Paenibacillus polymyxa ), Japanese scarab beetle ( Paenibacillus popilliae ), Paenibacillus puldeungensis , Paenibacillus residuum , Paenibacillus stellifer Thiamine-like Bacillus ( Paenibacillus thiaminolyticus ), Paenibacillus validus and xylolytic Bacillus ( Paenibacillus xylanisolvens For example, Bacillus vesicularis and Bacillus dendriticis are pathogenic and cause larval diseases in larvae, but the disease is not American foulbrood caused by Bacillus larvalis, and is therefore a non-pathogenic species.

[0042] As used in this article, the term "bee" refers to any bee belonging to the genus *Apis*, distinguished primarily by its honey production and storage, and the use of wax to build perennial colony nests. For example, there are two species of bees: A. mellifera or A. cerana indica Bees are typically maintained by beekeepers. Bees include, but are not limited to, those that are kept by beekeepers. Microbes Subgenus Bee andreniform and Flower bee , Megapis Subgenus Dorsal bee ,as well as Bee Subgenus Bee ceramic , Apis koschevnikovi , Honey bee and Black-banded bee .

[0043] As used herein, the term "bee colony" or "honeybee community" refers to a social unit comprising a colony of bees (such as honeybees). This social unit can be any systematic organization utilized by bees for the purpose of promoting the survival of the group or community. Typically, a "bee colony" consists of thousands of bees that cooperate in nest building, food gathering, and brood rearing. Each member of a "bee colony" has a specific task to perform, and the collective effort of the entire colony is required for survival and reproduction. In late spring and summer, a colony typically includes a queen bee, thousands of worker bees, and hundreds of drones. Therefore, a bee colony is a "swarm of honeybees."

[0044] Typically, a bee colony reaches its peak from late spring to summer and its lowest point in winter. The social structure of the colony is maintained by the queen bee and worker bees and depends on an effective communication system. Domesticated bees are raised in a "beehive" or "honeycomb." Therefore, the term "beehive" or "honeycomb" refers to a structure that serves as the habitat of a bee colony (such as a honeybee colony).

[0045] As used in this article, the term "bee" refers to a honeybee ( Bee Any bee that is a member of the genus *Honeybee*. There are two species of honeybees. A. mellifer, A. cernan indica、A. andrreniformis、A. florea、A. koschevnikovie and A. black belt This is an example of what happens.

[0046] The terms "effective amount" or "amount effective against," or any grammatically equivalent terms or expressions, refer to an amount sufficient to treat or prevent a disease or ailment (e.g., larval foulbrood) when applied in any way to, for example, the queen bee, larvae, worker bees, nurse bees, or a bee colony for the treatment or prevention of such disease or ailment. Typically, the effective dose for treating and / or immunizing the queen bee and its respective larvae with antigens is approximately 1.5 x 10^6 doses per gram of food. 7 – 1.5x10 11Individual antigens and the range thereof. In one embodiment, an "effective amount" refers to an amount of the composition sufficient, when fed to the queen bee, to vaccinate the queen and her larvae such that, compared to larvae from an unvaccinated queen, the larvae from the vaccinated queen have 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 at least 10%, or at least 5% increased resistance to larval foulbrood infection. To measure this, the following protocol will be followed. The queen bee will be placed in a queen cage containing a pancake containing the antigen, according to the queen bee provider. Each queen bee will be provided with 6-10 worker bees and kept in a laboratory at an ambient temperature of approximately 23-26°C, protected from light, for 8 days. Survival rates will be recorded daily. After 8 days, the queen bee will be inserted into a recipient hive in her cage (the queen cells will be pre-examined—and removed if any are found), and the cage door (depending on the cage design) will be opened to allow access to the hive. If the queen bee is released, all hives will be allowed to acclimatize for 5 days before inspection. Afterward, the presence and absence of the queen and eggs will be recorded again. Ten days after queen placement, the presence of eggs in all hives will be monitored. Under suitable egg-laying conditions (approximately 60-100 eggs per hive), infection experiments to study vaccine efficacy can be conducted 2 weeks after queen placement.

[0047] 15-30 larvae per hive (L1) from each hive will be prepared under laboratory conditions and exposed to toxic spores for 48 hours, then transferred to a new larval feed free of spores. The spores will be mixed into the larval feed, which consists of 50% royal jelly, 6% glucose, 6% fructose, 1% yeast extract, and 37% distilled water. New feed will be given every 2 days (Day 2 = 700 μl, Day 4 = 800 μl, Day 6 = 1000 μl, Day 7 = 1000 μl, Day 8 = end of monitoring), and survival rate will be monitored daily; dead larvae will be removed during the observation period. The larvae will be prepared into 379 μL larval feed droplets (Day 0), with 5 larvae per drop. A parallel group without spores in the feed will serve as an environmental control. Vaccine efficacy will be calculated based on larval survival rates in different groups. [Please indicate how this will be measured.]

[0048] As used in this article, the term "nursery bee" refers to a bee that feeds worker bee larvae with worker bee jelly secreted from glands that produce royal jelly.

[0049] The term "worker bee" as used in this article refers to any female (normally social) honeybee that lacks the full reproductive capacity of a queen bee in a colony.

[0050] The term "brood" refers to the three developmental stages of a bee, collectively known as the brood stage. Bees begin as eggs, which hatch into larvae (plural). The larvae have no legs and are solely responsible for feeding.

[0051] The terms "preventive" or "vaccine" refer to agents used to prevent diseases, such as bee diseases, such as American foulbrood caused by Bacillus larvae in larvae.

[0052] As used in this article, the term “(vaccine) vaccination” refers to a means of generating immunity against a disease, such as generating immunity against larval spore-forming bacteria, thereby preventing the occurrence of disease or symptoms in larvae, offspring, or communities (preventive treatment) or inhibiting the spread of disease (slowing down or stopping its development).

[0053] The term "treatment" aims to enhance the immune response in the queen bee and then pass it on to her offspring.

[0054] The term "enhancing immune response" refers to the production of non-pathogenic antigens in the ovaries or developing eggs of a queen bee in response to a vaccine or treatment.

[0055] "Antigen unit" refers to the number of cells or antigen fragments that are not disease pathogens.

[0056] The term "dosage" refers to the amount of food given to the queen bee, worker bees, nurse bees, or larvae in a single feeding or unit of food.

[0057] Methods for carrying out the present invention

[0058] Honeybees, especially Western honeybees ( Honey bee Honeybees are the primary pollinators of most economic crops in North America and are among the most actively managed pollinators in the world. Therefore, honeybees have a significant economic impact, and maintaining healthy bee colonies is an important aspect of many agricultural practices.

[0059] Recently, commercial bee colonies have suffered widespread losses due to diseases such as American foulbrood (Afb) caused by Bacillus larvae, and larval diseases caused by PA and PD that have similar symptoms to Afb.

[0060] American foulbrood (Afb) is a devastating colony disease that is highly contagious and extremely difficult to treat. It can wipe out an entire colony. Typically, the most effective treatment for Afb is to burn all hives and equipment. Fortunately, this disclosure provides novel compositions and methods for treating and / or immunizing queen bees and their offspring against American foulbrood, thereby providing preventative immunization to the bee colony to prevent the disease.

[0061] Previously described methods for immunizing bees against Afb can be employed by inoculating the queen bee with dead bacteria of the same species (e.g., PL) to protect against diseases caused by PL (e.g., Afb) (see, for example, U.S. Patent Application Publication 2018 / 021529). The applicant discloses for the first time that other non-pathogenic bacterial species can be used to prepare vaccines for prophylactic treatment and / or immunization of bees against American foulbrood. Furthermore, this protection applies to a variety of larval diseases, where inoculation with one strain of Bacillus (pathogenic and non-pathogenic) can prevent infection by another strain of Bacillus. The applicant discloses for the first time that a specific match between the disease pathogen and the vaccine strain is not required. This distinguishes this disclosure from prior art that is neither conceived nor disclosed.

[0062] Surprisingly, non-pathogenic bacteria can protect host organisms from disease. Disease-causing pathogens possess highly specific virulence factors, such as Bacillus thuringiensis (Bt). Bacillus thyringiensis The Cry toxin in *Cry-2A* is found in *Cry4B*, an insect pathogen that infects different insect groups; however, infectivity depends on the type of toxin produced. For example, Cry-2A infects only lepidopteran hosts, Cry4B infects mosquitoes, and mCry-3A infects beetles. This high host specificity, even within the same bacterial species, makes the disclosed finding—that we can protect hosts from disease by vaccinating them with non-pathogenic bacteria—unexpected and novel.

[0063] Therefore, one aspect of this disclosure is a method for treating and / or protecting insects from disease, which provides broad-spectrum protection by applying therapeutic and / or immune stimulation derived from a Bacillus species that does not cause the disease. Non-limiting examples of insects that can be protected using the vaccine formulations disclosed herein include, for example, bees, honeybees (…). Bee sp.), bumblebee ( Bumblebee These can protect bees from infections and diseases caused by, for example, Bacillus larvae (the pathogen of American foulbrood). As used herein, the term "applying immune stimulation" refers to prevention of disease caused by pathogenic Bacillus larvae. The composition, formulation, or vaccine can be administered to bees or larvae once or more per year, such as once, twice, three times, or more per year, before or after hibernation. It can be administered as a single dose or multiple doses.

[0064] As used herein, the term “genus” has its conventional meaning as known in the art. Generally, a genus is defined as a taxonomic rank used in the biological classification of organisms; in the hierarchical structure of biological classification, a genus is above a species and below a family. For example, the genus *Bacillus* is a facultative anaerobic, endospore-forming bacterium, classified by Ash et al. 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 its host, characterized by certain features, such as interfering with the function of the organism and severely damaging the host, including its death. Broad-spectrum, universal protection is achieved when the host is protected from infection by immunization with bacteria of a specific genus or multiple species of that genus (i.e., a “species”) and then encounters a disease-causing pathogen not included in the vaccine formulation.

[0065] In an exemplary embodiment, this disclosure provides a novel vaccine composition and / or formulation and method for treating and / or preventing American larval foulbrood (AFB) in bees by inoculating the queen bee with dead, non-larval Bacillus subtilis (e.g., Bacillus auris (PA) and Bacillus denticulatus (PD)).

[0066] Preparation of compositions for preventing bacterial diseases in insect populations

[0067] General methods

[0068] This disclosure utilizes conventional techniques in the field of beekeeping. The basic text of the disclosed beekeeping terminology and methods includes, for example... Queen Rearing and Bee Breeding Harry H. Laidlaw and Robert E. Page (1997). The following methods are merely exemplary and can be applied more broadly to other insects, diseases, and bacteria.

[0069] Preparation of oral vaccines

[0070] In its embodiments, this disclosure provides compositions comprising organisms exhibiting a specific phenotype. These compositions can prevent not only diseases caused by the species in the vaccine formulation, but also similar diseases caused by other species within the same genus. Such species capable of preventing diseases caused by other species are referred to as non-disease organisms.

[0071] For example, all Bacillus species cause larval diseases, and they look very similar. However, only Bacillus larvae infection leads to American foulbrood. Fortunately, this disclosure provides the ability to prevent Bacillus larvae infection as well as larval infections caused by other bacteria. Therefore, the terms "pathogenic / causing disease" and / or "non-pathogenic / not causing disease" are relative terms in relation to the target disease. For example, in the case of American foulbrood, where the pathogen is Bacillus larvae, Bacillus larvae is a "pathogenic" bacterium. In this case, other Bacillus species, such as PA and / or PD, are "non-pathogenic".

[0072] In the implementation scheme, a vaccine composition comprising a non-disease organism is prepared as disclosed in Example 1 herein. In short, the non-pathogenic bacteria are grown on a liquid or solid culture medium (agar plate, fermenter, flask) and collected by methods known in the art. Both the liquid and solid culture media can be of plant or animal origin.

[0073] Non-limiting examples of inactivating collected bacteria include the use of formalin, diethyleneimine (BEI), or heat and pressure (autoclaving). Any method known in the art for killing bacteria can be used. After killing, cells can be lysed and large fragments separated by centrifugation at approximately 10,000 g to approximately 60,000 g for 1–4 hours (or longer, depending on volume, size, and concentration). The supernatant is recovered and further fractionated as disclosed in Example 1. Alternatively, whole-cell formulations can be used.

[0074] The supernatant can be freeze-dried, lyophilized, or kept in solution and can be applied to bees in various forms, such as feed, spray, or injection. One method of application includes, but is not limited to, preparations of royal jelly, which can be purchased pre-packaged at beekeeping supply stores or prepared by methods known in the art.

[0075] In one exemplary embodiment, the composition and / or vaccine formulation is given to worker bees, who will incorporate the vaccine into 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 brood colony against pathogenic bacterial diseases (such as American foulbrood), for example, vaccines prepared with Bacillus subtilis can protect against American foulbrood for 3-12 months.

[0076] Use of composition

[0077] The oral vaccine technology disclosed herein is administered via queen bee feed (“queen sugar”), any other bee feed or supplement ingested by the queen bee, nurse bees, or worker bees and fed to the queen bee or queen bee larvae via royal jelly. It is based on the concept of transgenerational immune initiation (see, for example, Salmela H, Amdam GV, Freitak D (2015) Transfer of Immunity from Mother to Offspring Is Mediated via Egg-Yolk Protein Vitellogenin. PLoSPathog 11(7):e1005015.https: / / doi.org / 10.1371 / journal.ppat.1005015.), which immunizes the queen bee and then transfers the antigens and acquired innate immune signals to the eggs, thereby protecting newly hatched larvae and bees from harmful pathogens.

[0078] As is known in the art, the insect immune system can recognize specific pathogens and trigger immunity in offspring. Highly specific immune initiation can be achieved when female insects transfer immune inducers to developing oocytes. The molecular mechanism behind this transfer occurs through vitellogenin, a protein that binds to bacteria or bacterial fragments that make up the cell wall and recognizes pathogen-associated molecular patterns to deliver immune initiation signals (see, for example, Salmela H, Amdam GV, Freitak D (2015), ibid.).

[0079] Unbound by theory, it is believed that certain cell wall markers, for example within bacterial genera, are shared by the genus *Bacillus*. Exposure to these cell wall markers, whether as part of the whole cell or a fragment of the cell wall, stimulates immunity to other members of the genus, not just to the species from which the cell wall marker is derived. Therefore, exposure to a cell wall marker from a species (e.g., a non-pathogenic species) of the genus stimulates immunity to other members of the genus, including pathogenic members. Thus, in an exemplary embodiment, using at least... P. alvei or P. larvae Immunization with the composition can prevent P. alvei Infection and prevention P. larvae Infect.

[0080] In one implementation, but not limited to, the oral vaccine composition is delivered as part of normal queen bee rearing practice. The queen bee is placed in a "queen cage" with 8-10 worker bees and provided with sufficient feed (queen sugar) to sustain her for one to two weeks. The oral vaccine is added to the queen sugar, and the queen bee and worker bees will be fed on it for 3-7 days before being placed in a new hive to prepare for laying eggs and creating new protected bee colonies.

[0081] In another implementation, the vaccine is placed in royal jelly in the queen bee transport box or in the queen bee or queen bee rearing box, where the nurse bees will consume the vaccine along with the royal jelly and transport it to their royal jelly glands, where it is mixed with the royal jelly fed to the queen bee larvae or queen bee.

[0082] In another implementation, the vaccine is fed to the developing queen bee larvae during the larval rearing stage in the queen bee rearing hive. Example

[0083] Example 1

[0084] The following examples illustrate exemplary methods for preparing compositions comprising an effective amount of more than one type of dead, fragmented Bacillus subtilis.

[0085] Prepare Bacillus aureus (PA) and Bacillus dendriticus (PD) (or other antigens) to provide vaccines or treatments for queen bees. The methods disclosed below provide consistency for all vaccine preparations.

[0086] Seed cultures were inoculated from frozen glycerol stock solutions of Bacillus auris (PA), Bacillus dendriticis (PD), or other Bacillus species. Inoculated plates were grown in a dark growth chamber at 35°C for 4–12 days.

[0087] Harvest Bacillus colonies by washing plates with 5 mL of ice-cold H2O and scraping them into centrifuge tubes (or glass bottles). Measure the optical density (OD600) at 600 nm using standard methods. Mix equal volumes of the harvested bacterial culture and H2O in two separate cuvettes. Prepare the antigen solution to the desired concentration—at least 1.5 x 10⁻⁶—based on the OD600 reading. 9 100 bacterial cells / mL. Autoclave the bacterial solution at 121°C or for 15 minutes to kill the bacteria.

[0088] Methods for obtaining bacterial fragments to be used as antigens

[0089] Figure 2The diagram illustrates an exemplary method for obtaining bacterial fragments to be used as antigens. aBugBuster® can be used for fresh or frozen cell pellets. Harvest cells from liquid culture 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. Discard the liquid and allow the pellet to drain, removing as much liquid as possible. Weigh the pellet. Once the cells have been harvested and are not autoclaved, they can be granulated 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 of culture. Incubate the cell suspension on a shaking platform or rotary mixer at a slow setting at room temperature for 10–20 minutes. Incubate for 1 hour, shaking, at +25°C. Remove insoluble cell debris by centrifugation at 16,000 × g for 20 minutes at 4°C. Transfer the supernatant to a new test 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, transferring the supernatant to a new centrifuge tube.

[0090] Centrifugation steps used (at 4°C):

[0091] 1) 800xg in 10 minutes

[0092] 2) 1500xg in 10 minutes

[0093] 3) 10000xg for 60 minutes

[0094] 4) 3 hours 20000xg

[0095] Add 500 µl PBS to each precipitate and vortex.

[0096] The collected fragments are used as antigens in pancakes to inoculate or treat the queen bee and her offspring.

[0097] Example 2

[0098] The following examples illustrate an exemplary method of immunizing beehives with non-pathogenic Bacillus subtilis to resist American foulbrood.

[0099] Use a cake containing a non-pathogenic Bacillus subtilis composition to inoculate or treat at least seven queen bees. The cake contains 80 g of royal jelly (e.g., commercial bee feed - Nordzucker), 1 mL of antigen (containing 1.5 x 10^6 mmol / L) in 1x phosphate-buffered saline (PBS; 1x dilution, pH 6.6–7.2, 137 mM NaCl, 2.7 mM KCl, 9.5 mM phosphate buffer). 8 – 1.5x10 10 (Individual antigens). The concentration of the antigen was measured using a spectrophotometric method (OD600).

[0100] The unit of treatment or inoculation is the queen bee, prior to placement in the hive. As is known in the art, to introduce the queen bee into the hive, she is first placed alone in a queen cage and fed queen sugar. In this embodiment, approximately one week before placement in the hive, the queen bee is fed commercial bee feed cakes containing a composition of non-pathogenic Bacillus antigens prepared as disclosed in Example 1 herein.

[0101] After feeding the queen bee a queen sugar biscuit containing non-pathogenic Bacillus subtilis antigen, the queen bee is placed in the hive.

[0102] After being placed in the hive, the queen bee will begin laying eggs within 3-5 days and continue until the end of the brooding period. The larvae laid by the queen bee will have at least 30% greater resistance to American foulbrood (a bacterium that causes larval decay).

[0103] Resistance to infection was determined by transplanting freshly hatched (12–36 hours) bee larvae from vaccinated and placebo-treated hives into laboratory artificial rearing conditions. All larvae were then subjected to oral infection treatment, including inoculation with live... P. The larvae will be fed with larvae spores, or a control treatment (food without spores) will be used. Survival rates will be monitored for 8 days, and resistance to infection will be measured by comparing the survival rates of immunized and non-immunized larvae.

[0104] Example 3

[0105] Figure 3A and 3B The results of queen bee administration and / or vaccination with Bacillus dendriticis are shown. To test PD antigen resistance to PL infection, one queen bee was inoculated with 1.5 x 10 g of Bacillus dendriticis in 8 g of bee feed. 8The CFU antigen was administered, with one queen bee serving as a placebo control and 8 grams of bee feed containing no antigen. The queen bee was kept in a standard queen cage with 8 worker bees for 8 days at room temperature (approximately 23°C) and 60% relative humidity. Afterward, they were placed in donor hives and allowed to lay eggs. Three weeks after placing the queen bee in the hive, L1 larvae were transplanted in the laboratory and infected with spores of *P. larvae*, with spore-free larval food serving as an environmental control. Fifteen larvae per queen bee were transplanted into each treatment, and their survival was monitored in the laboratory for 8 days. To test for PD antigen resistance to PA, a similar protocol as described above was followed, except that 3 placebo hives and 1 hive for PD antigen were used, with all hives sampled 3 times. Efficacy: Vaccination of queen bees with dead Bacillus subtilis produced larvae with greater resistance to subsequent pathogen infection (PD vs. PA gray bars, vaccine: approximately 60% larval survival vs. placebo: 40% larval survival, p = 0.08) and (PD vs. PL gray bars: 35% larval survival vs. placebo: 25% larval survival, p = 0.18). There was no negative impact on larval survival in the uninfected control condition (gray bars, vaccine: 15% larval survival vs. placebo: 15% larval survival, p > 0.05).

[0106] The mortality rate of infected larvae was monitored throughout the experiment up to day 8. Day 8 is a critical point in bee development as it marks the pupation event. Laboratory handling of bee pupae is extremely cumbersome because they are surrounded by a very thin cuticle that can rupture even with the gentlest touch. Therefore, monitoring of the impact of infection on the pupation event was discontinued. Furthermore, larval foulbrood kills bee larvae by day 5, so once a larva pupates on day 8, it has already survived the infection, rendering monitoring of infection survival rate obsolete.

[0107] Implementation Plan

[0108] In one aspect, this document provides a composition comprising the whole cell or cell wall fragment of at least one dead, non-pathogenic bacterial species of the genus *Bacteria*, and optionally a carrier, or substantially composed of, or composed of, it. In one aspect, the carrier is insect food. In another aspect, the non-pathogenic bacterial species includes *Bacillus* genus, with non-limiting examples including *Bacillus apis* or *Bacillus dendriticus*, or combinations thereof. In yet another aspect, the one or more dead, non-pathogenic bacterial species are selected from *Bacillus apis* (…). Paenibacillus alvei ), Bacillus dendriticis ( Paenibacillus dentriiform ), Amylolytic Bacillus ( Paenibacillus amylolyticus ), Campinas Bacillus ( Paenibacillus campinasensis ), Bacillus chondroitinibacterium ( Paenibacillus chondroitinus ), Korean-Chinese Bacillus ( Paenibacillus chungangensis ), Paenibacillus doosanensis Bacillus dextrin ( Paenibacillus glucanolyticus ), humic spore-forming bacteria ( Paenibacillus humus Lactobacillus ( Lactobacillus lactis ), Bacillus splendidus ( Paenibacillus clean ), Bacillus subtilis ( Paenibacillus lentimorbus ), Bacillus subtilis ( Paenibacillus maceran ), Bacillus subtilis ( Paenibacillus macerans -like), Bacillus macrocarpa ( Paenibacillus macquariensis ), Paenibacillus motobuensis , feed-grade Bacillus ( Paenibacillus pabuli ), Paenibacillus phoenicis, Polymyxin Bacillus ( Paenibacillus polymyxa ), Japanese scarab beetle ( Paenibacillus popilliae ), Paenibacillus puldeungensis , Paenibacillus residuum , Paenibacillus stellifer Thiamine-like Bacillus ( Paenibacillus thiaminolyticus ), Paenibacillus validus and xylolytic Bacillus ( Paenibacillus xylanisolvens (or combinations thereof). On the other hand, the dead, non-pathogenic bacterial species include, or consist substantially of, Bacillus vesicularis and Bacillus dendriticis.

[0109] In one embodiment, the composition disclosed herein comprises approximately 1.5 x 10^6 grams of carrier (e.g., insect food) per gram. 6 Or 1.5 x 10 7 To approximately 1.5x10 11 An antigen unit is a whole cell or cell wall fragment of a dead, non-pathogenic species, or is essentially composed of or composed of.

[0110] In one embodiment, methods are provided for treating, vaccinating, or immunizing a queen bee or bee larvae against larval foulbrood. In one aspect, the queen bee or larvae is a honeybee. Other bee species are described herein. The method comprises, or consists substantially of, the following steps, or comprises the following steps: applying an effective amount of the composition disclosed herein to one or more of worker bees, nurse bees, larvae, or queen bees (e.g., honeybee queen), thereby immunizing the queen bee or honeybee larvae produced by the queen bee. In one embodiment, larval foulbrood is caused by a species of Bacillus. In one aspect, larval foulbrood is caused by… P. larvae Caused by. In another embodiment, larval foul disease is American foul disease. In another aspect, larval foul disease is caused by P. larvaeThe disease caused is American foulbrood. In yet another embodiment, the applied composition comprises, or is substantially composed of, dead whole cells or cell wall fragments of one or more dead, non-pathogenic Bacillus species (e.g., Bacillus vesicularis and Bacillus dendriticis), and causes American foulbrood. For these embodiments, the queen bee, worker bee, nurse bee, or larvae are administered at approximately 1.5 x 10⁻⁶ per dose. 6 Or 1.5 x 10 7 To approximately 1.5x10 11 One unit of antigen is fed or administered to the whole cell or cell wall fragment. In another embodiment, 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 antigen are administered or fed to the queen bee, worker bee, nurse bee, or larvae. 7 To approximately 1.5 x 10 11 A whole cell or cell wall fragment of an antigen unit.

[0111] In another embodiment, this document provides a method for preventing American foulbrood in a colony of bees or bee offspring, the method comprising, or substantially consisting of, the following steps: administering or feeding to the queen bee, worker bees, nurse bees, or larvae a composition comprising, substantially consisting of, or consisting of, at least one dead, non-disease-like species of Bacillus. In one aspect of this method, American foulbrood is caused by… P. larvae This is caused by the fact that, on the one hand, the queen bee and the bee are honeybees. The queen bee can feed directly on the composition or be fed by worker bees or nurse bees. Alternatively or otherwise, the larvae feed on the composition disclosed herein.

[0112] In one aspect, this disclosure provides a method for preventing American foulbrood in a colony of bees or their offspring, the method comprising, or consisting essentially of, the following steps, or consisting of the following steps: administering or feeding the queen bee approximately 1.5 x 10g of food per dose. 6 Or 1.5 x 10 7 To approximately 1.5x10 11 One antigen unit of PA or PD, or both, in whole cells or cell wall fragments. On one hand, feed the queen bee approximately 1.5 x 10g of food per gram or per dose. 7 To approximately 1.5x10 11 One antigen unit. This dose can be administered to the queen bee's food, thereby protecting the larvae and their offspring.

[0113] In one aspect of this implementation, the dead, non-disease-causing Bacillus species applied or fed is selected from one or more of the following Bacillus species: Bacillus vesicularis ( Paenibacillus alvei ), Bacillus dendriticis ( Paenibacillus dentritiformis ), Amylolytic Bacillus ( Paenibacillus amylolyticus ), Campinas Bacillus ( Paenibacillus campinasensis ), Bacillus chondroitinibacterium ( Paenibacillus chondroitin ), Korean-Chinese Bacillus ( Paenibacillus chungangensis ), Paenibacillus from Doosan Bacillus dextrin ( Paenibacillus glucanolyticus ), humic spore-forming bacteria ( Paenibacillus humicus Lactobacillus ( Lactobacillus lactis ), Bacillus splendidus ( Paenibacillus lautus ), Bacillus subtilis ( Paenibacillus lentimorbus ), Bacillus subtilis ( Paenibacillus maceran ), Bacillus subtilis ( Paenibacillus macerans -like), Bacillus macrocarpa ( Paenibacillus macquariensis ), Paenibacillus motobuensis , feed-grade Bacillus ( Paenibacillus pabuli ), Paenibacillus phoenicis, Polymyxin Bacillus ( Paenibacillus polymyxa ), Japanese scarab beetle ( Paenibacillus popilliae ), Paenibacillus puldeungensis , Paenibacillus residuum , Paenibacillus stellifer Thiamine-like Bacillus ( Paenibacillus thiaminolyticus ), Paenibacillus validus and xylolytic Bacillus ( Paenibacillus xylanisolvens (or combinations thereof). In one specific embodiment, the dead, non-disease-causing Bacillus species are selected from dead Bacillus cellulosus, Bacillus dendriticus, or combinations thereof, and are applied to the queen bee, nurse bee, or worker bee. In one embodiment of the method, the queen bee is fed queen bee feed, said feed comprising approximately 1.5 x 10 mmol per dose. 6 Or 1.5 x 10 7 To approximately 1.5 x 10 11 Each antigen unit comprises at least one dead, non-pathogenic Bacillus species or fragments thereof, or is substantially composed of, or is composed of, them. On the other hand, compositions applied to or fed to one or more of the queen bee, worker bee, nurse bee, or larvae contain whole cells or cell wall fragments of one or more dead Bacillus apiacea and Bacillus dendriticis, or are substantially composed of, them. In a further aspect, one or more of the queen bee, worker bee, nurse bee, or larvae are administered approximately 1.5 x 10¹² units per dose. 7 To approximately 1.5 x 10 11A whole cell or cell wall fragment of a dead, non-pathogenic spore-forming bacillus with an antigenic unit.

[0114] In the method described, a dose of 1.5 x 10 is applied or fed to one or more, 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 of the queen bee, worker bee, nurse bee, or larvae. 7 To approximately 1.5 x 10 11 A whole cell or cell wall fragment of an antigen unit.

[0115] In one embodiment of this disclosure, a method for preparing an insect composition or vaccine is provided, comprising isolating whole cells and / or cell wall fragments from at least one dead, non-pathogenic bacterial species of Bacillus subtilis, and then optionally, mixing the isolated antigen units with an insect food. In one aspect, the insect food is selected from royal jelly, sugar eaters, spirulina supplements, royal jelly in shipping containers, queen bee cells, and insect supplements. In another aspect, the non-pathogenic bacterial species is a species of the genus Bacillus subtilis, such as Bacillus auris or Bacillus dendriticis, or a combination thereof. Alternatively, one or more dead Bacillus subtilis species are selected from Bacillus auris (… Paenibacillus channels ), Bacillus dendriticis ( Paenibacillus dentritiformis ), Amylolytic Bacillus ( Paenibacillus amylolyticus ), Campinas Bacillus ( Paenibacillus campinasensis ), Bacillus chondroitinibacterium ( Paenibacillus chondroitinus ), Korean-Chinese Bacillus ( Paenibacillus Chunggang ), Paenibacillus doosanensis Bacillus dextrin ( Paenibacillus glucanolytic ), humic spore-forming bacteria ( Paenibacillus humicus Lactobacillus ( Lactobacillus lactis ), Bacillus splendidus ( Paenibacillus lautus ), Bacillus subtilis ( Paenibacillus lentimorbus ), Bacillus subtilis ( Paenibacillus maceran ), Bacillus subtilis ( Paenibacillus macerans -like), Bacillus macrocarpa ( Paenibacillus Macquarie ), Paenibacillus motobuensis , feed-grade Bacillus ( Paenibacillus fodder ), Paenibacillus phoenicis, Polymyxin Bacillus ( Paenibacillus polymyxa ), Japanese scarab beetle ( Paenibacillus popilliae ), Paenibacillus puldeungensis , Paenibacillus residuum , Paenibacillus stellifer Thiamine-like Bacillus ( Paenibacillus thiaminolytic ), Paenibacillus validus and xylolytic Bacillus ( Paenibacillus xylanol-dissolving (or a combination thereof). In one specific implementation, the dead Bacillus species is Bacillus vesicularis or Bacillus dendriticis or a combination thereof.

[0116] On the other hand, the method also includes formulating isolated whole cells or cell wall fragments to provide approximately 1.5 x 10⁻⁶ insect feed per dose. 6 Or 1.5 x 10 7 To approximately 1.5 x 10 11 One antigen unit. In another aspect, this document provides a composition prepared by the methods disclosed herein. In one aspect, said composition is a vaccine composition.

[0117] On the other hand, a kit is provided which comprises the composition or vaccine as described herein and optional instructions for use.

[0118] equivalent

[0119] 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 this technology pertains.

[0120] The technology exemplarily described herein may be practiced appropriately without any element or elements, limitations or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” “containing,” etc., should be understood broadly and without limitation. Furthermore, the terms and expressions used herein have been used as descriptive rather than restrictive terms, and the use of these 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 are possible within the scope of the claimed technology.

[0121] 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 art.

[0122] This technique has been described broadly and generally herein. Each narrower group of species and subgenus falling within the general disclosure also constitutes part of this technique. This includes the general description of the technique, with any attached conditions or negative limitations on the subject matter removed from the genus, regardless of whether the removed material is specifically cited herein.

[0123] Furthermore, where features or aspects of this technology are described according to the Markush Group, those skilled in the art will recognize that this technology is therefore also described according to any single member or subgroup of the Markush Group.

[0124] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety by reference, to the same extent as each individual reference is incorporated by reference alone. In case of conflict, this specification (including definitions) shall prevail.

[0125] Other aspects are set forth in the following claims.

Claims

1. A composition comprising at least one dead, non-pathogenic whole cell or cell wall fragment of Paenibacillus dentritiformis.

2. The composition according to claim 1, wherein the composition comprises 1.5 x 10⁻⁶ insect food per gram. 7 Up to 1.5x10 11 A whole cell or cell wall fragment of an antigen unit.

3. The use of the composition of claim 1 or 2 in the preparation of a medicament for vaccinating, treating or immunizing a queen bee or bee larvae against American foulbrood, said medicament being formulated for application to bees selected from one or more of worker bees or nurse bees feeding a queen, bee larvae or queen bees, thereby immunizing the queen bee or bee larvae produced by the queen bee.

4. The application according to claim 3, wherein the composition comprises dead, non-pathogenic Paenibacillus dentritiformis.

5. The application according to claim 3 or 4, wherein the drug is formulated for a dose of 1.5 x 10⁻⁶. 7 Up to 1.5x10 11 One antigen unit of whole cell or cell wall fragment is applied to one or more of the queen bee, worker bee, nurse bee, or larva.

6. The application according to claim 5, wherein the drug is formulated for 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 Up to 1.5 x 10 11 One antigen unit of a whole cell or cell wall fragment of a dead, non-pathogenic Paenibacillus dentritiformis species is applied to one or more of the queen bee, worker bee, nurse bee, or larvae.

7. The use of a composition comprising whole cells or cell wall fragments of at least one dead, non-disease-forming species of Paenibacillus dentritiformis in the preparation of a medicament for prevention or vaccination against American foulbrood in bee colonies, wherein the medicament is formulated for administration to the queen bee.

8. The application according to claim 7, wherein the application comprises feeding a suitable amount of queen bee feed to one or more of the queen bee, worker bees, nurse bees, or larvae, said feed comprising 1.5 x 10 mg per dose. 7 Up to 1.5 x 10 11 A whole cell or a fragment of the cell wall of at least one dead, non-disease-like Bacillus species, comprising one antigen unit.

9. The application according to claim 8, wherein the drug is formulated for 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 Up to 1.5 x 10 11 One unit of antigen is applied to one or more of the queen bee, worker bee, nurse bee, or larva.

10. A method for preparing a composition or vaccine, comprising isolating whole cells or cell wall fragments from at least one dead, non-pathogenic bacterial species of Paenibacillus dentritiformis, and mixing the isolated whole cells or cell wall fragments with insect food.

11. The method of claim 10, wherein the insect food is selected from royal jelly, sugar, spirulina supplement, royal jelly in a shipping container, royal jelly cells, and insect supplement.

12. The method of claim 10, further comprising formulating isolated whole cells or cell wall fragments to provide 1.5 x 10⁻⁶ per dose. 7 Up to 1.5 x 10 11 One antigen unit.

13. A composition prepared by the method of any one of claims 10 to 12.

14. A vaccine composition prepared by the method of any one of claims 10 to 12.

15. A kit comprising the composition of claim 1 or 2, and instructions for use.

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