Methods and compositions for controlling or reducing pests

Abstract

The present invention provides pharmaceutical compositions or biocide compositions for controlling or mitigating mite infestations, and methods for controlling and / or mitigating mite infestations in vectors. [Solution] A pharmaceutical composition or biocide composition suitable for controlling or reducing mite infestation, comprising a bacterial preparation of at least one strain of Bacillus thuringiensis (B. thuringiensis) as an active ingredient, characterized in that the bacterial preparation contains an effective amount of live spores of at least one strain of B. thuringiensis.

Description

[Technical Field]

[0001] The present invention relates to methods, preferably for veterinary use, and compositions comprising live spores of the Bacillus thuringiensis (Bt) strain. The methods and compositions are for the prevention and treatment of mite infestations, particularly for the control or reduction of mite infestations caused by external parasites, such as the control and reduction of infestations caused by Dermanyssus gallinae (poultry red mite), which particularly affects birds. [Background technology]

[0002] Mites are small invertebrates, mostly free-living, but some are parasitic. Mites that invade and parasitize animals cause invasion and / or infection in non-human animals, resulting in enormous losses. Mites are extremely diverse, and their classification is quite complex. Mites and ticks are classified separately from insects (of the class Insecta), for example. However, mites and ticks have completely different ecologies from each other.

[0003] Animal mites can cause serious types of skin diseases, and some mites invade other organs. Mites infest various parts of an animal's body, including the surface layer of the skin, between dead cells in the stratum corneum (e.g., Psoroptes ovis), and the hair follicles of the host (e.g., Demodex). They can infect various parts of the body, or invade the host during brief feeding (so-called blood-sucking mites, e.g., Dermanisus galinae). Some mites are adapted to invade the lungs and air sacs of birds or the lungs of mammals (e.g., Cytodites nudus). .

[0004] The ectoparasitic mites Varroa destructor and von Anderson and Truman Destructor Anderson and Trueman (formerly known as Varroa Jacobsoni Audemans) Jacobsoni Oudemans is a type of honeybee (Apis mellifera L). It is a major pest. This hemolymph-feeding mite not only weakens adult and larval bees, but also acts as a vector and inducer of viral infection in varroysis, causing significant damage to bee populations worldwide (Non-Patent Literature 1). Another important pathogenic mite causing serious and global invasions is the so-called "red mite," "mite," or Dermanisus gallinae (D. gallinae). The first concern associated with red mite invasions is the extremely high and ever-increasing prevalence of this disease in Europe. Recent epidemiological studies have reported that D. gallinae invades 83% of European farms. This prevalence reaches 94% in the Netherlands, Germany, and Belgium. Mite invasions affect all production methods, from backyard or organic farms to more intensive enriched cage or barn systems. The impact of mite invasions has increased in Europe over the past few decades and is expected to increase further.

[0005] One of the primary factors contributing to this increase is the recent transformation of housing systems for laying hens in EU member states. Directive 1999 / 74 / EC on egg production and egg trade has prohibited the use of conventional cages for poultry since 2012. While this law aims to improve the welfare of laying hens, it has led to a shift to housing systems that incorporate more complex environments, which are considered to be favorable for mite proliferation and exacerbate the problem of red mite infestation. For example, enriched cages provide red mites with far more hiding places to avoid effective treatment. It has been noted that mite infestation rates in hens raised in conventional cage systems are much lower compared to alternative systems. In 2009, before conventional cages were first banned (Austria and Germany had... Despite the ban on cages since 2010, 74.4% of laying hen housing systems in the European Union were still conventional cages. By 2013, all member states had completed the transition process from conventional cages to primarily enriched cages, housing systems, and free-range housing systems. In other words, within four years from 2009, the vast majority of laying hens had been moved from systems unfavorable to mite proliferation to systems favorable to it.

[0006] Another environmental factor expected to contribute to an increase in red mite infestations is climate warming. During the extreme heat of the summer of 2003, a major weather event, a massive outbreak of red mites was involved in the death of many hens.

[0007] Finally, concerns about safety led to the removal of several mite control chemicals from the domestic market, and the lack of new and effective control methods for a long period may have contributed to the worsening prevalence of D. gallinae in Europe. Therefore, there is a need to provide methods and compositions for controlling or mitigating mite infestations, particularly those caused by Dermanisus gallinae (mites).

[0008] In this sense, Non-Patent Literature 2 provides an overview of various possible strategies for controlling D. gallinae. According to Non-Patent Literature 2, several strains of B. thuringiensis have shown insecticidal activity against lepidopteran and some diptera pests. However, according to Non-Patent Literature 2, the use of Bacillus thuringiensis (B. thuringiensis) in vertebrates is hindered by its toxicity at the cellular level.

[0009] Bacillus thuringiensis is an aerobic, spore-forming, Gram-positive, and entomopathogenic bacterium. The B. thuringiensis species generally consists of a large family of various subspecies (such as B. thuringiensis subspecies kurstaki and B. thuringiensis subspecies aizawai) that are classified based on phylogenetic and serotyping characteristics.

[0010] B. thuringiensis is known as an immediate-acting and host-specific biopesticide in agriculture.

[0011] In the current state of the art (see, for example, Non-Patent Document 3, a recent review), basically, the toxicity of B. thuringiensis is considered to be due to various toxins produced by the bacterium and the toxins described in Non-Patent Document 3 that play an important role in targeting insects and other invertebrates. The above toxins include insecticidal Cry proteins, vegetative insecticidal protein (Vip) toxins, and cytotoxin (Cyt) proteins.

[0012] The toxins identified in B. thuringiensis are continuously updated. See, for example, www.lifesci.sussex.ac.uk / Home / Neil_Crickmore / Bt / . In B. thuringiensis strains isolated from various regions of the world, more than 900 toxin genes encoding various insecticidal protein toxins have been identified and characterized.

[0013] In addition to the above toxins, B. thuringiensis produces other compounds that also exhibit toxic effects, such as chitinase (an enzyme protein) and metalloprotease. B. thuringiensis also secretes other toxic compounds, such as thuringiensin (Thu, beta-exotoxin), a thermostable secondary metabolite.

[0014] Non-Patent Document 4 evaluated the effect of Bacillus thuringiensis subsp. kurstaki (Btk) on the larvae of the wax moth using a contact bioassay method. Non-Patent Document 4 used the commercial product Dipel (trademark) in the study. The product label of Dipel (trademark) According to this, the insecticidal activity obtained by using the product is due to four proteins of B. thuringiensis subsp. kurstaki, Cry1Aa, Cry1Ab, Cry1Ac and Cry2 toxin, which are produced by B. thuringiensis subsp. kurstaki. Based on the content of Cry, Cyt and Vip proteins, each strain may be specifically active against phytophagous insects (insect plants) such as pests of Lepidoptera, Diptera or Coleoptera, and furthermore against other invertebrates such as mites and nematodes present in both plants and animals. It may have activity specifically against mites and other invertebrates such as nematodes.

[0015] Therefore, as already mentioned, the biological activity of B. thuringiensis is mainly due to thuringiensin and furthermore to parasporal crystal proteins (or δ-endotoxins) Cry toxins. These Cry proteins are toxic to a wide range of pests such as Lepidoptera, Coleoptera and Diptera. This crystal is estimated to account for about 30% of the total cell proteins of B. thuringiensis (Non-Patent Document 5).

[0016] Therefore, as explained above, the literature mainly focuses on the toxicity of the above-mentioned toxins, especially for the treatment of pests in agriculture.

[0017] [[ID=I4]]However, the current state of knowledge regarding the effect of B. thuringiensis on available animal mites is poor. There are few in vitro and in vivo studies reporting on the acaricidal activity of B. thuringiensis against animals, let alone the biological activity of B. thuringiensis strains against animal mites.

[0018] [[ID=I7]] Currently, no B. thuringiensis products are approved on the global market for controlling tick infestations in veterinary practice. Control remains primarily through the use of synthetic mite control agents such as foxim, avermectin, pyrethroids, amitraz, and other chemicals, with widespread reports of resistance issues and treatment failures. Therefore, safer and more effective control measures are needed for controlling tick infestations in animal production, such as D. galinae in poultry.

[0019] In conclusion, there remains a need in the art to find solutions to the problem of controlling mites, particularly poultry mites, without harming animals or humans. The use of environmentally friendly microbial acaricides as a substitute for harmful chemical pesticides, as an alternative to large-scale control of invasive / pests in non-human animals, offers a favorable and novel solution to this problem. In this regard, this application is the first to demonstrate that B. thuringiensis is suitable for the production of novel and beneficial acaricide compositions and is a promising alternative for the biological control of these types of invasive / pests in non-human animal production. [Prior art documents] [Non-patent literature]

[0020] [Non-Patent Document 1] Ball & Allen 1988 [Non-Patent Document 2] Chauve C. et al., 1998, The poultry red mite Dermanyssus gallinae (De Geer, 1778): current situation and future prospects for control. Vet. Parasitol. 79 (1998) 239-248 [Non-Patent Document 3] Zhen et al., Comparative genomics of Bacillus thuringiensis reveals a path to specialized exploitation of multiple invertebrate hosts, American Society for Microbiology, 2017, 8(4) [Non-Patent Document 4] Torres, EC et al., 2018. Actividad acaricida de Bacillus thuringiensis sobre el acaro rojo de las aves, Dermanyssus gallinae. Rev. Vet 29 (2): 128-132, 2018 [Non-Patent Document 5] Den DH et al., 1984 [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows the average mite mortality rate over 24 hours for five different formulations (Formulations 1-5) described in Example 1. The vertical axis shows the average percentage of mite mortality over 24 hours for the different formulations on the horizontal axis. [Figure 2] This figure shows the average mite mortality rates over 24 and 48 hours for three different formulations (Formulations 1-3) described in Example 2. The vertical axis shows the average percentage of mite mortality over 24 and 48 hours for the different formulations, with the horizontal axis representing the average percentage. [Figure 3] This figure shows the average mite mortality rates over 24 and 48 hours for nine different groups (groups 1-9) described in Example 3. The horizontal axis shows the average percentage of mite mortality over 24 and 48 hours for the nine different groups, and the vertical axis shows that. [Figure 4]This figure shows the average mite mortality rates over 24 and 48 hours for six different formulations (Formulations 1-6) described in Example 5. The vertical axis shows the average percentage of mite mortality rates over 24 and 48 hours for the six different formulations, with the horizontal axis representing the average percentage. [Modes for carrying out the invention]

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the invention pertains at the time of filing. However, in the event of any potential ambiguity, the definitions presented herein shall prevail over any other definitions. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and likewise plural terms shall include singular forms.

[0023] In this disclosure, "comprises," "comprising," "containing," and "having" have the meanings assigned to them under European and US patent law, and may mean "includes," "including," etc. Similarly, "consisting essentially of" or "consists essentially" The term has the meaning given in European and US patent law, and is open-ended, allowing for the existence of things other than those enumerated, provided that the fundamental or novel characteristics of those enumerated are not altered by the existence of things other than those enumerated, but excluding embodiments of the prior art.

[0024] "To be in contact with" means to be touching, being bound to, or being in close proximity to the composition. Such composition is, but is not limited to, a medicinal bath. It can be applied by spraying, injecting, coating, spraying, immersion and / or dispensing.

[0025] To “control or mitigate” mite infestation, “suitable for controlling or mitigate” or “for controlling or mitigate” means to inhibit or prevent mite survival, or to reduce, slow, or stabilize the growth of a mite colony. In the context of the present invention, mites can be at any stage of their life cycle known to those skilled in the art, such as juvenile development, larval stage, and adult stage.

[0026] "Biological function" refers to any physiological or behavioral activity of an organism. Examples of biological functions, though not limited to, include reproduction, respiration, neural activity, and movement.

[0027] "Effective dose" means the effective amount required in a single dose or as part of a series of doses to prevent or treat tick infestation in animals, that is, to improve or reduce the number of parasites in the animals and / or their environment (cages, grounds, vectors, etc.), and / or to inhibit the onset of tick infestation in animals, for example, by controlling and / or mitigating tick populations, either entirely or partially. The effective dose or effective amount is required for the treatment or prevention of such disease. For either reason, it is also possible to control and / or reduce the severity of secondary infections or the incidence of disease in animals. This amount varies depending on various factors, including the health status of the subject, and can be easily determined by those skilled in the art. In any case, the effective amount can preferably be easily determined by observing or detecting the number of mites on animals and / or vectors both before and after administration of the composition of the present invention, for example, the number of mites is reduced by 10% to 100%, preferably more than 50%, more preferably more than 70%, more preferably more than 80%, even more preferably more than 90%, even more preferably more than 95%, and even more preferably more than 99% after the first administration. Factors that affect the effective amount include, for example, the parasitic species to be treated and the developmental stage of the parasite, the type of affected animal (e.g., species and breed), age, size, sex, diet, activity and condition, and environmental conditions such as temperature and humidity, and the preferred effective amount of the composition according to the present invention may vary.

[0028] "Invasion" means colonization of a site or parasitism of a living organism by a pest. Colonization of a vector (an inanimate object that, when contaminated with infectious agents such as bacteria, viruses, or parasites, can transmit disease to a new host) is also included in the context of this invention. A pest is any organism, whether animal, bacteria, parasite, virus, plant, or fungus, that invades or is troublesome to plants or animals, humans or things related to humans, livestock, or artifacts. The most important animal groups as pests (in order of economic importance) are insects, mites, nematodes, and gastropods. Such invasion may, in some cases, induce infection in the subject or host, exacerbate and / or worsen their health condition, and / or cause disease in such subject. A relationship has been observed between the invasion of D. gallinae and the mortality rate of hens (Cosoroaba I., 2001. Massive Dermanyssus gallinae invasion in battery-husbandry raised fowls. Rev. Med. Vet. Toulouse 152:89-96).

[0029] The term "mite" refers to arachnid parasites that invade vertebrates and invertebrates, particularly poultry, including mammals, fish, insects (e.g., bees), and birds. Examples of commercially important poultry mites include species of the genera Dermanyssus, Ornithonyssus, Allopsoroptoides galli, Neocnemidocoptes gallinae, Knemidocoptes mutans, Laminosioptes cysticola, Megninia cubitalis, and Megninia gingrimula. ginglymura), Pterolichus obtus, Syringophilus bipectinatus, Columbiphilus polonica, Deroglyphus elongates and gau This is a Gaudoglyphus minor.

[0030] In the context of the present invention, mites can be at any stage of their life cycle known to those skilled in the art, such as larval development, larval stage, and adult stage.

[0031] Dermanisus galinae, also known as chicken mite, red mite, or roost mite, is a small, ectoparasitic mite, approximately 1.5 mm in length, whose body color varies from gray to brown / red depending on its feeding status. Dermanisus galinae has four life cycle stages: eggs, larvae, first nymphs, second nymphs, and adults. Larvae hatch with six legs and do not feed. After the first molt, they develop eight legs in both the nymph and adult stages. Female first and second nymphs and adults feed regularly on the host's blood, while males feed only occasionally. After parasitizing a host, red mites feed for a short period of up to one hour every two to four days, usually in the dark, though not always. The complete development of Dermanisus gallinae from egg to adult, consisting of one larval stage and two nymphal stages, typically takes two weeks. Dermanisus gallinae densities generally reach up to 50,000 mites per bird in a cage system, but in severe cases, the density can reach... The number of mites can reach up to 500,000. D. gallinae can be present throughout the year, but the highest densities occur during hot and humid seasons. D. gallinae is found worldwide, and is particularly problematic in temperate and warm regions. D. gallinae is an ectoparasitic mite, considered a transient arthropod because it feeds on its host but does not permanently parasitize it, although it spends most of its adult life in the host's environment. The mites remain on the host only to feed, then move to nearby cracks and crevices to lay eggs. Mites occur in both battery cages and floor systems. However, this problem is more prevalent and widespread in floor and "enriched" cage systems established in Europe due to animal welfare concerns, due to the presence of numerous suitable hiding places for the mites. Controlling transient parasites such as Dermanisus gallinae is particularly difficult because they can be found in both the host animal and the environment.

[0032] A "mite killer" refers to a substance that has acaricidal activity and therefore causes harmful effects on the biological functions of mites. The effects may be, for example, ovicidal, larvicidal, and / or adulticidal, or a combination thereof.

[0033] "Acaricidal activity" means any activity that inhibits, prevents, stops, and / or reduces the growth, reproduction, or survival of mites or other dust mites. The effect may be, for example, ovicidal, larvicidal, and / or adulticidal, or a combination thereof.

[0034] To “prevent,” “in order to prevent,” or “prevent” a mite infestation means, but is not limited to, reducing, mitigating, or improving the risk of a mite infestation being established at a site by killing adult parasites and / or any developmental stage / larval stage that are capable of invading a host before they invade the host, or by killing or inhibiting mites when they invade a pre-treated animal, or by preventing the development of mite offspring, for example, by reducing the number of eggs laid and / or the hatching rate. Furthermore, it means preventing and / or protecting animals from harmful effects caused by pests, preferably mite infestations. The effects may be, for example, ovicidal, larvicidal, and / or adulticidal, or a combination thereof. The effects may be direct, i.e., by killing mites immediately or after a period of time, for example by destroying eggs, or indirect, for example, by reducing the number of eggs laid and / or the hatching rate.

[0035] To “treat,” “to treat,” or “to treat” a mite infestation means, but is not limited to, suppressing, limiting, reducing, stabilizing, or delaying the growth of a mite population at a particular site by killing adult parasites and / or any developmental stage / larval stage capable of invading a host before they invade the host, or by killing or inhibiting mites when they invade a pre-treated animal. Furthermore, it means treating and protecting an animal from harmful effects caused by pests, preferably mite infestations, such as pre-existing symptoms, clinical signs, disorders, conditions, and / or diseases. The effects may be, for example, ovicidal, larvicidal, and / or adulticidal, or a combination thereof. The effects may be direct, i.e., by killing mites immediately or after a delay, for example by destroying eggs, or indirect, for example by reducing the number of eggs laid and / or the hatching rate.

[0036] "Pharmaceuticals" or "medicines" means, as is widely recognized, any pharmaceutical or veterinary composition (also referred to as drugs, medications or simply pharmacologics) used to cure, treat or prevent disease in animals, including humans. Drugs are classified in various ways. One important distinction is between conventional small molecule drugs, usually obtained by chemical synthesis, and biological preparations or biopharmaceuticals, which include, but are not limited to, live or dead microorganisms, recombinant proteins, vaccines, blood products used for treatment (e.g., IVIG), gene therapies, monoclonal antibodies, and cell therapies (e.g., stem cell therapies). In this invention, pharmaceuticals are Preferably, it is a veterinary medicine, and more preferably, a composition for veterinary use in animals and / or vectors.

[0037] "Pharmaceutical composition" means an active substance or combination of active substances intended to prepare a final pharmaceutical product for preventive and / or therapeutic use. In the present invention, the pharmaceutical or drug is preferably a veterinary drug, and more preferably a pharmaceutical composition for veterinary use. The pharmaceutical composition may be applied directly to an animal or indirectly to the environment in which the animal is present.

[0038] "Pharmacopoeia-acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject with the composition of the present invention without causing any undesirable biological effects or adversely interacting with any of the components of such composition. As used herein, the terms "pharmacopoeia-acceptable carrier" and "pharmacopoeia-acceptable vehicle" are synonymous and refer to a vehicle for containing the active substance of a pharmaceutical composition that can be administered to a subject and / or the environment without adverse effects. Suitable pharmacopoeia-acceptable carriers include, but are not limited to, sterile water, purified water, saline, glucose, dextrose, or buffer solutions. The carrier may also contain, but are not limited to, diluents, stabilizers, preservatives, wetting agents, dispersants, emulsifiers, pH buffers (e.g., phosphate buffer), viscosity additives, and the like.

[0039] "Biocide composition" means, chemically or biologically speaking, a chemical substance, organism, or microorganism intended to destroy, suppress, neutralize, or exert a control effect against any harmful organism. In the context of the present invention, a biocide composition preferably refers to an organism, or an organism such as a microorganism, preferably a bacterial microorganism, more preferably a compound naturally produced by Bacillus thuringiensis. A biocide composition may encompass a diverse group of harmful substances, including insecticides, disinfectants, preservatives, and pesticides, used to control organisms that are harmful to human or animal health or that damage natural products or products. A biocide product contains one or more biocidal substances and other non-active co-formulants that ensure the effectiveness of the final biocide product as well as the desired pH, viscosity, color, aroma, etc. Alternatively, the biocide composition may be applied directly to animals or indirectly to the environment in which animals are present.

[0040] "Bacterial preparation" means, as is known in the art, a bacterial culture, a part of a bacterial culture, or a preparation containing a post-processed bacterial culture. A bacterial preparation may contain a particular strain of bacteria or two or more strains of the same bacteria. In the present invention, a bacterial preparation may contain one or more species of Bacillus thuringiensis and its subspecies.

[0041] "Active ingredient" or "active substance" means a component and / or substance in a biologically active pharmaceutical composition or biocide composition.

[0042] An “adjuvant” or “acceptable adjuvant” is understood, as is known in the art, to be a non-nonspecific stimulant of the immune system administered with an active substance to make the immune response more effective. Some examples of adjuvants are aluminum hydroxide, aluminum phosphate, aluminum oxide, muramyl dipeptide, vitamin E, squalane, squalene, ginseng, zymosan, glucan, dimethylaminoethyl dextran, dextran, nonionic block polymers, monophosphoryl lipid A, vegetable oils, saponins, complete Freund's adjuvants, incomplete Freund's adjuvants, W / O, O / W, W / O / W emulsions, and mixtures thereof. Adjuvants are components used to enhance the effectiveness and / or improve the ability of pesticides and / or biocides, such as herbicides, insecticides, fungicides, acaricides, and other active ingredients that control or eliminate undesirable pests, to penetrate, target, or protect target organisms (stabilizers, preservatives and It can also be understood as a coloring agent, etc.

[0043] "Subject" means an individual. In one embodiment, the subject is a mammal, such as a primate, including humans. In another embodiment, mammals are non-human primates, particularly marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons. The term "subject" also includes domesticated animals such as cats and dogs; livestock, such as cattle, horses, pigs, sheep, and goats; laboratory animals, such as ferrets, chinchillas, mice, rabbits, rats, gerbils, and guinea pigs; and birds, such as chickens, turkeys, ducks, pheasants, pigeons, doves, parrots, cockatoos, and geese. Subjects may also include, but are not limited to, fish (such as zebrafish, goldfish, tilapia, salmon, and trout), amphibians, and reptiles. As used herein, “subject” is synonymous with “patient,” “host,” or “affected subject,” and these terms may be used interchangeably.

[0044] In the context of the present invention, the terms “bird,” “bird species,” “bird subject,” or “bird host” are understood to encompass all birds that are prone to or susceptible to tick infestation and / or infection, preferably D. gallinae infection or infestation. Birds included in the present invention include, for example, those collectively referred to as poultry or wild chickens. In other embodiments, these terms are broadened to include domesticated birds or game birds, such as chickens, turkeys, pheasants, geese, and / or ducks. In one embodiment, the terms “bird,” “bird species,” “bird subject,” or “bird host” extend to commercially important or domesticated birds, such as poultry.

[0045] A "laying hen" or "layer" refers to a chicken that is raised primarily for egg production. It refers to a mature female chicken (Gallus domesticus). Such eggs are generally used for consumption as human food. The term “laying hen” in this invention includes breeding stock raised to produce eggs that will hatch into future laying hens.

[0046] "Broiler" refers to domesticated wild chickens of the pheasant species that have been specially bred and raised for meat production.

[0047] "A site where animals are kept" means a place where animals are permanently or temporarily housed and can access the site for a limited time (temporarily, such as a free-range hen coop) or permanently, such as a conventional cage for laying hens or a broiler chicken coop.

[0048] In the context of this invention, the term “non-spore protein” refers to a set of proteins synthesized by vegetative Bacillus thuringiensis cells that are not enclosed by a spore shell and generally not by an outer membrane, and in particular include accessory spore crystalline proteins. The outer membrane is the outer layer of a mature spore and defines the boundary between the spore and the environment or host with which it interacts. For pathogens, this interaction includes the initial point of contact between the spore and cells of the host's immune system. Crystalline proteins are usually located outside the outer membrane, although in some strains of Bacillus thuringiensis there are a few cases where the outer membrane can enclose the crystalline proteins. Therefore, in the context of this invention, accessory spore crystalline proteins are considered non-spore proteins regardless of their location inside or outside the outer membrane.

[0049] In the context of this invention, the term “accessory spore crystalline protein” is understood as the proteins Cry and Cyt, which are delta (δ)-endotoxins synthesized by the vegetative Bacillus thuringiensis bacterium. These proteins aggregate during the spore formation process to form crystalline inclusions, known as crystals, which are formed by the same type of proteins as crystals but surrounded by a different conformational casing. Inclusion bodies consist mainly of one or more polypeptides known as protoxins. Intomopathogenic Bacillus thuringiensis strains have the ability to form one or more crystalline inclusion bodies, i.e., accessory spore crystalline proteins.

[0050] A complete list of delta-endotoxins can be found, for example, at http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / (Crickmore et al., 1998, 2016).

[0051] In the context of this invention, the term "live bacterial spore" is understood as a Bacillus thuringiensis spore that has the ability to passively detect favorable environmental changes, germinate, and produce physiologically active vegetative cells. In contrast, the term "non-viable spore" refers to a spore that cannot germinate even under favorable conditions. The number of live bacterial spores can be determined by standard microbiological methods such as the known colony-forming unit (CFU / ml) method based on the number of viable cells (Goldman, Emanuel; Green, Lorrence H. Practical Handbook of Microbiology. 3 rd Edition, published 4 June, 2015; Chapter 2. Quantification of Microorganisms. Page 19, Plate Count Method by Peter S. Lee, page 24. CRC Press, 1055 pages. ISBN 9781466587397). Colony counting is usually performed manually using a pen and click counter. Alternatively, semi-automatic (software) and automatic (hardware + software) systems can be used.

[0052] In the context of the present invention, live bacterial spores of B. thuringiensis (Bt) isolated from a farm are preferred, live Bt spores isolated from a poultry farm are more preferred, live Bt spores isolated from a poultry farm with a low or high degree of mite infestation are even more preferred, and live Bt spores isolated from a poultry farm having favorable conditions that result in a low degree of mite infestation but ultimately a high infestation rate of Dermanisus gallinae are most preferred.

[0053] In the context of the present invention, the "non-crystallized (non-crystallized liferous)" of B. thuringensis The term "B. thuringiensis strain" refers to a strain having a genome (chromosomes and plasmid DNA) lacking the Cry protein. The absence of the Cry protein can be assessed by extracting DNA from B. thuringiensis strains using standard methods such as the InstaGene® Matrix, performing whole-genome sequencing, and then checking for any agreement with the Cry protein in well-known genome databases such as http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt (Crickmore et al., 1998, 2016). These databases contain the complete Cry gene or genes with partial homology to the Cry protein. Non-crystalline B. thuringiensis strains must lack the Cry gene with any degree of homology to these genes (complete or partial sequences). Therefore, no results (complete or partial to the described Cry protein) can be found in these databases for non-crystalline B. thuringiensis strains.

[0054] Whole genome sequencing of B. thuringiensis strains can also be performed using standard methods such as Illumina's HiSeq platform. The sequences are CLC Genomic. The assembly can be done using software tools such as Workbench (CLC Bio, Denmark). Reads are usually trimmed to remove low-quality reads and extreme cases such as reads shorter than 30 base pairs. The resulting reads are then used to create contigs. Novo-assemble. The resulting contigs will be paired with the reference sequence and will have at least 95% identity percentage, containing at least 95% lectures of the aligned sequence. After the contig construction, the lectures will be reassigned to the contig. The resulting contigs will be analyzed using BLAST (Altschul et al.) with a database of amino acid sequences of Bacillus thuringiensis toxins, such as those available at http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt (Crickmore et al., 1998, 2016). The analysis is performed using (al., 1990). BLAST is not limited to the use of this database, and is applicable to those skilled in the art. This may include other known and available databases.

[0055] In the context of this invention, the terms “mutant protein” or “mutant toxin” are understood to refer to a protein product encoded by a gene having a non-silent mutation. Mutant proteins may have a single amino acid change (which is often minor but frequently a significant change leading to disease) or extensive amino acid changes, such as cleavage of the C-terminus after the introduction of an immature stop codon.

[0056] explanation To our surprise, the authors of this invention discovered that the activity of various Bacillus thuringiensis (Bt) strains on the mortality rate of mites such as D. gallinae is mainly associated with the presence of effective amounts of live bacterial spores, rather than with the presence of non-spore proteins such as accessory spore crystal proteins and / or other toxins produced by the Bt strains. To our knowledge, this is the first time that such activity has been clearly associated with live bacterial spores rather than with non-spore proteins such as accessory spore crystal proteins produced by the Bt strains.

[0057] Experimental evidence supporting this discovery can be seen through the examples described herein. In this sense, Example 1 shows that an amorphous strain of B. thuringiensis (GR-S5-8), which does not produce any accessory spore crystal protein and was deposited under the Budapest Convention on June 20, 2019, by HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) with accession number DSM 33175, yielded a higher mortality rate of 83% (Composition 3) than a formulation containing both spore and accessory spore crystal protein (Composition 2). Furthermore, in this example, experimental formulation 4, enriched with purified live spores, showed increased activity compared to other formulations, resulting in a more significant reduction in the D. gallinae population (80% mite mortality rate) compared to unpurified formulation 2, which contained both spores and accessory spore crystal proteins, while formulation 2 resulted in a mite mortality rate of 67%. It was certainly unexpected that the purified formulation enriched with live spores exhibited better activity against mite mortality than that observed in compositions containing accessory spore crystal proteins. In addition, when live spores and accessory spore crystal proteins were subjected to inactivation by heat treatment, no mite mortality rate was observed at all (formulation 5). That is, accessory spore crystal proteins in the absence of live spores do not show activity against mites. To the best of our knowledge, this is the first time that anti-mite activity, such as anti-D. gallinae activity, has been clearly linked to live Bt spores, rather than to the presence of non-spore proteins such as accessory spore crystal proteins produced by the Bt strain.

[0058] Furthermore, as shown in Example 3, it was further observed that the lower the amount of live spores present in the composition, the lower the mortality rate of D. gallinae mites both 24 and 48 hours after treatment with the experimental formulation. Similarly, higher doses of live spores resulted in higher mortality rates, regardless of the non-spore protein content of the composition. These results further demonstrate the role of live spores in generating anti-mite activity. Similarly, all Bt strains tested were 3.1 × 10⁶ 9 The formulation showed a significant mortality rate when combined with 100% live spores (CFU) / ml. In particular, the deposited DSM 33035 Bt strain DE2-S2-8 showed higher anti-mite activity, exhibiting a 66% mortality rate 48 hours after treatment (Group 9).

[0059] Furthermore, unexpectedly, the mixture of B. thuringiensis preparation and mineral oil did not adversely affect the viability of B. thuringiensis strains or their anti-mite activity. On the contrary, it significantly increased the effectiveness of the experimental formulation, resulting in a synergistic effect on mite elimination rates. In this sense, as shown in Example 5, the mixture of mineral oil and 3.1 × 10⁻¹⁰ 9 individual spores Formulation 3 (Group 3), prepared using (CFU) / ml, achieved a mite mortality rate of over 90% in just 48 hours. It was further observed that a mixture of B. thuringiensis formulations mixed with mineral oil preparations allowed for a reduction in the dose of B. thuringiensis bacteria used in the experimental formulations. For example, 3.4 × 10 8 A mineral oil preparation (Group 1) containing a dose of 100 mite spores (CFU) / ml resulted in a mite mortality rate of 70.10% after 48 hours. A similar mortality rate was obtained when the B. thuringiensis preparation was not mixed with mineral oil, but in this case, the rate was 3.1 × 10⁻⁶. 9 The formulation was prepared at a dose of 10

[0060] Therefore, a synergistic effect between the Bt preparation and the mineral oil was confirmed. In additional experiments, it was observed that the synergistic effect was maintained even with the mixing of the FS Bt sample and an emulsion containing a very low percentage of mineral oil, such as 1.25%.

[0061] Based on the results given so far, a first aspect of the present invention is a pharmaceutical composition or biocide composition suitable for controlling or reducing mite infestation, comprising a bacterial preparation of at least one strain of Bacillus thuringiensis (B. thuringiensis) as an active ingredient, wherein the bacterial preparation contains an effective amount of vegetative spores of at least one strain of the above B. thuringiensis. The bacterial preparation preferably contains at least 1×10 4 cfu of vegetative spores per ml of the composition, preferably at least 1×10 5 cfu of vegetative spores per ml of the composition, preferably at least 1×10 6 cfu of vegetative spores per ml of the composition, more preferably at least 1×10 7 cfu of vegetative spores per ml of the composition. More preferably, the bacterial preparation contains an effective amount of at least 3×10 8 cfu of vegetative spores per ml of the composition. Even more preferably, the bacterial preparation contains an effective amount of at least 3×10 9 cfu of vegetative spores per ml of the composition.

[0062] In a preferred embodiment of the first aspect of the present invention or any of its preferred embodiments, the bacterial preparation comprises an effective amount of a purified formulation enriched with live spores of at least one strain of Bacillus thuringiensis (B. thuringiensis). It should be noted that such purified population may include any of the strains identified throughout the present invention, including Bacillus thuringiensis subspecies crustaki (Btk). In particular, the purified population of live spores contains a significantly higher proportion of live spores than the crude population of bacteria from which the live spores are isolated. For example, the purification procedure should result in at least a 5-fold increase in live spores compared to the crude population, preferably at least a 10-fold increase, more preferably at least a 15-fold increase, most preferably at least a 20-fold increase, and optimally at least a 25-fold increase.

[0063] Therefore, the purified population of the present invention contains significantly higher levels of viable spores than those found in nature, as described above.

[0064] In a first aspect of the present invention or another preferred embodiment thereof, the bacterial preparation contains an effective amount of live spores of at least one strain of B. thuringiensis suitable for controlling or mites mite infestation caused by Dermanisus gallinae (mite mite).

[0065] In another preferred embodiment of the first aspect of the present invention or any of its preferred embodiments, at least one strain of B. thuringiensis is preferably a strain suitable for controlling or miting mite infestation caused by Dermanisus galinae (mite mite), and is a strain that produces less non-spore proteins, such as accessory spore crystalline proteins, per 1 ml of composition than live spores. At least one strain of B. thuringiensis is preferably suitable for controlling or miting mite infestation caused by Dermanisus galinae (mite mite), and Bacillus thuringiensis Preferably, the strain produces less non-spore proteins such as accessory spore crystalline proteins than the subspecies Btk, Aizawai, Islaerensis, and Morisoni. More preferably, at least one strain of B. thuringensis is suitable for controlling or mites mite infestation caused by Dermanisus galinae (mite mite), and produces 3.1 × 10⁶ per ml. 9 The strain produces less non-spore proteins, such as accessory spore crystal proteins of B. thuringiensis, per viable spore than the naturally occurring microorganism Bacillus thuringiensis subspecies crustaki (Btk). More preferably, at least one strain of B. thuringiensis is suitable for controlling or mites mite infestations caused by Dermanisus galinae (mite mite), and produces 3.1 × 10⁶ per ml. 9 Non-spore proteins such as B. thuringiensis accessory spore crystal protein at a concentration of less than 1.7 mg / ml per viable spore, preferably 3.1 × 10⁶ per ml. 9 Non-spore proteins such as B. thuringiensis accessory spore crystal protein at a concentration of less than 1.5 mg / ml per living spore, or 3.1 × 10⁶ per ml. 9 Non-spore proteins such as B. thuringiensis accessory spore crystal protein at a concentration of less than 1.3 mg / ml per viable spore, or 3.1 × 10⁶ per ml. 9 Non-spore proteins such as B. thuringiensis accessory spore crystal protein at a concentration of less than 0.8 mg / ml per living spore, or 3.1 × 10⁶ per ml. 9 Non-spore proteins such as B. thuringiensis accessory spore crystal protein at a concentration of less than 0.5 mg / ml per living spore, or 3.1 × 10⁶ per ml. 9 Non-spore proteins such as B. thuringiensis accessory spore crystal protein at a concentration of less than 0.3 mg / ml per living spore, or 3.1 × 10⁶ per ml. 9 This strain produces non-spore proteins, such as B. thuringiensis accessory spore crystalline protein, at a concentration of less than 0.1 mg / ml per viable spore.

[0066] More preferably, non-spore proteins such as the B. thuringiensis accessory spore crystal proteins described in the above paragraph are selected from the group consisting of delta(δ)-endotoxins such as Cry and Cyt proteins synthesized by vegetative Bacillus thuringiensis bacteria. These proteins aggregate during the spore formation process, forming crystal inclusions known as crystals, which are formed by the same type of protein as crystals but surrounded by a different conformational casing. A non-restrictive selection of non-spore proteins is possible, including delta(δ)-endotoxins such as Cry (Cry1, Cry2, Cry3, etc.) and Cyt (Cyt1, Cyt3, etc.) family proteins (a detailed list of delta-endotoxins is available on the following website as of July 2019: http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / ) (Crickmore et al., 1998, 2016). Vegetative insecticidal proteins such as Vip family proteins (Vip1, Vip2, Vip3, and Vip4); secretory insecticidal proteins such as Sip family proteins; β-exotoxins such as turingiencin, sphericolysins, and albe Proteins related to cholesterol-dependent cell lysins, such as alveolysins; These include enhancer-like proteins such as Bel enhancer; helper proteins such as P19 and P20 proteins; or non-protein β-exotoxins such as Bt 41.9kDa protein (Palma et al., 2014. Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity. Toxins 2014, 6, 3296-3325; doi:10.3390 / toxins6123296). .

[0067] In a first aspect of the present invention or another preferred embodiment thereof, at least one strain of Bacillus thuringiensis is, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of June 20, 2019 Bacillus thuringiensis strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33175. HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under accession number DSM 33034, and, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33035. Selected from the group consisting of .

[0068] In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the pharmaceutical composition or biocide composition further comprises at least one further active or functional component selected from the group consisting of insecticides, mite control agents, fungicides, nematicides, antibiotics, cleaning agents, immunogenic substances, animal feed, essential oils, mineral oils, nutritional supplements, probiotics, prebiotics, synbiotics, polysaccharides, and combinations thereof. Preferably, further active ingredients include avian infectious bronchitis virus (IBv), Newcastle disease virus (NDV), adenovirus, egg-low syndrome virus (EDS), bursal disease virus (IBDV), chicken anemia virus, avian encephalomyelitis virus, fowlpox virus, turkey rhinotracheitis virus, duck plague virus, pigeonpox virus, Marek's disease virus (MDV), avian leukemia virus, infectious laryngotracheitis virus (ILTV), avian pneumovirus, reovirus, Escherichia coli, Salmonella sp., Ornithobacterium rhinotracheale, Haemophilus paragallinarum, and Pasteurella multofida. Erysipelothrix rhusiopathiae, Erysipela sp., Multocida Icoplasma sp., Clostridium sp., A group consisting of species of the genera Eimeria and Aspergillus. It is an immunogenic substance derived from selected microorganisms.

[0069] In a first aspect of the present invention or another preferred embodiment thereof, the pharmaceutical composition or biocide composition is a dosage form selected from the group consisting of powders, granules, microcapsules, lotions, ointments, gels, creams, pastes, suspensions, stocks, liquids, and emulsions. The pharmaceutical composition or biocide composition is preferably suitable for application by medicinal bath, spray, injection, application, spray, immersion, or dispensing.

[0070] In a further preferred embodiment of the first aspect of the present invention or any of its preferred embodiments, the composition further comprises one or more pharmaceutically acceptable vehicles and / or one or more acceptable adjuvants.

[0071] A second aspect of the present invention relates to a pharmaceutical composition or biocide composition, as defined in either the first aspect of the present invention or a preferred embodiment thereof, for use as a pharmaceutical or biocide.

[0072] A third aspect of the present invention relates to a pharmaceutical composition or biocide composition, as defined in either the first aspect of the present invention or a preferred embodiment thereof, for use in controlling and / or mitigating mite infestation.

[0073] A fourth aspect of the present invention is the first aspect of the present invention or a preferred aspect thereof, which is preferably used for controlling and / or mitigating mite infestations caused by external parasitic mites in animals or vectors. The present invention relates to a pharmaceutical composition or biocide composition as defined in any of the embodiments. Preferably, the external parasitic mite is a species of the genus Dermanyssus, Ornithonyssus, Argus, Allopsoroptoides galli, or Neocnemidocoptes gallinae. *Knemidocoptes mutans*, *Laminosioptes cysticola*, *Megninia cubitalis* ), Megninia ginglymura, Pterolichus obtus, Syringophilus bipectinatus, Columbiphilus polonica, Deroglyphus elongates, Gaudoglyphus minor, O The group is selected from Otodectes cynotis, Cheyletiella yasguri, Demodex sp., Notoederes cati, Cheyletiella sp., Psoroptes sp., Chorioptes sp., Psorergates ovis, Sarcoptes scabiei, Psorobia ovis, Railietia auris, and Varroa sp., as well as combinations thereof. More preferably, the ectoparasitic mite is Dermanisus gallinae (mite).

[0074] In a preferred embodiment of a fourth aspect of the present invention, the animal is selected from the group consisting of birds, pigs, cattle, horses, cats, dogs, sheep, rabbits, and bees. The birds are preferably domesticated.

[0075] In another preferred embodiment of the fourth aspect of the present invention or any of its preferred embodiments, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 25%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fourth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 40%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fourth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 50%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fourth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 60%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fourth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 70%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors.

[0076] In a fourth aspect of the present invention or another preferred embodiment of any of its preferred embodiments, the pharmaceutical composition or biocide composition is applied by a medicinal bath, spray, injection, coating, spray, immersion, or spraying.

[0077] In a fourth aspect of the present invention or another preferred embodiment thereof, at least one strain of Bacillus thuringiensis is, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of June 20, 2019 Bacillus thuringiensis (Bt) strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33175. HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under accession number DSM 33034, and, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33035. Selected from the group consisting of .

[0078] A fifth aspect of the present invention relates to a method for controlling and / or mitigating tick infestation in a vector, comprising using a pharmaceutical composition or biocide composition as defined in either the first aspect of the present invention or a preferred embodiment thereof, wherein the vector is preferably located on a site where animals are raised, and more preferably the animals are selected from the group consisting of birds, pigs, cattle, horses, cats, dogs, sheep, rabbits and bees.

[0079] In a preferred embodiment of the fifth aspect of the present invention or any of its preferred embodiments, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 25%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fifth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 40%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fifth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 50%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fifth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 60%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the fifth aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 70%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In a fifth aspect of the present invention or another preferred embodiment of any of its preferred embodiments, the pharmaceutical composition or biocide composition is applied to a vector located on a site where animals are kept.

[0080] In a fifth aspect of the present invention or another preferred embodiment of any of its preferred embodiments, the pharmaceutical composition or biocide composition is applied by medicinal bath, spray, injection, coating, spray, immersion, or spraying.

[0081] In a fifth aspect of the present invention or another preferred embodiment thereof, at least one strain of Bacillus thuringiensis is, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of June 20, 2019 Bacillus thuringiensis (Bt) strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33175. HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under accession number DSM 33034, and, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33035. Selected from the group consisting of .

[0082] In a sixth aspect of the present invention, the pharmaceutical composition or biocide composition defined in any first aspect of the present invention or a preferred embodiment thereof comprises a further active ingredient, the further active ingredient being an essential oil or mineral oil. Preferably, the composition is incorporated into the essential oil or mineral oil. More preferably, the further active ingredient is a mineral oil, the mineral oil being liquid paraffin.

[0083] A seventh aspect of the present invention relates to a pharmaceutical composition or biocide composition as defined in the sixth aspect of the present invention, preferably used for controlling and / or mitigating tick infestation in animals or vectors. Preferably, the control and / or mitigation of tick infestation is carried out in the vector, which is preferably located on the premises where the animals are raised. More preferably, the control and / or mitigation of tick infestation is carried out in the animal, which is selected from the group consisting of birds, pigs, cattle, horses, cats, dogs, sheep, rabbits and bees. The birds are preferably poultry.

[0084] In a preferred embodiment of the seventh aspect of the present invention or any of its preferred embodiments, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 25%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the seventh aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 40%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the seventh aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 50%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the seventh aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 60%, preferably occurring within 48 hours from the moment the composition is applied to and / or comes into contact with the mites and / or vectors. In another preferred embodiment of the seventh aspect of the present invention, the external parasitic mites are selected such that the control and / or reduction of mite infestation results in a mite mortality rate of at least 70% The composition is selected to result in a rate of at least 70% mites being killed, preferably within 48 hours from the moment the composition is applied to and / or brought into contact with the mites and / or vectors. In another preferred embodiment of the seventh aspect of the present invention, the external parasitic mites are selected so that the control and / or reduction of mite infestation results in a rate of at least 80% mites being killed, preferably within 48 hours from the moment the composition is applied to and / or brought into contact with the mites and / or vectors. In another preferred embodiment of the seventh aspect of the present invention, the external parasitic mites are selected so that the control and / or reduction of mite infestation results in a rate of at least 90% mites being killed, preferably within 48 hours from the moment the composition is applied to and / or brought into contact with the mites and / or vectors.

[0085] In an alternative embodiment of the seventh aspect of the present invention, the present invention relates to a pharmaceutical composition or biocide composition as defined in the first or sixth aspect of the present invention, used for the prevention and / or treatment of diseases caused by external parasitic mites in animals that require such prevention and / or treatment. Preferably, the diseases are caused by species of the genera Dermanisus, Ornithonisus, Argus, Allopsoroptoides galli, Neocnemidocoptes galinae, Cnemidocoptes mutans, Laminosioptes cysticola, Megninia capitaris, Megninia gingrimula, Pterolicus obtus, Syringophyllus bipectonatus, Colombiphyllus polonica, Delogriffus elongates, The infestation is caused by external parasitic mites selected from the group consisting of Gaudgriffus minnow, Otodectes sinuthis, Cheiretiera yasuguri, species of Demodex, Notoedleres cati, species of Cheiretiera, species of Psoroptes, species of Coryoptes, Psorelgates obis, Sarcoptes scabiei, Psolovia obis, Laylietia auris, and species of Barroa, and combinations thereof. More preferably, the prevention and / or treatment of the mite infestation is carried out in animals, which are selected from the group consisting of birds, pigs, cattle, horses, cats, dogs, sheep, rabbits, and bees. Birds are preferably domesticated.

[0086] In a seventh aspect of the present invention or another preferred embodiment of any of its preferred embodiments, the pharmaceutical composition or biocide composition is applied by a medicinal bath, spray, injection, coating, spray, immersion, or spraying.

[0087] In a seventh aspect of the present invention or another preferred embodiment thereof, at least one strain of Bacillus thuringiensis is, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of June 20, 2019 Bacillus thuringiensis (Bt) strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33175. HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under accession number DSM 33034, and, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) as of February 21, 2019 B. thuringiensis strains and their variants, respectively, deposited under the Budapest Convention by Amer, Girona, Spain, to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33035. Selected from the group consisting of .

[0088] In a seventh aspect of the present invention or another preferred embodiment of any of its preferred embodiments, the mite is an external parasitic mite. Preferably, the external parasitic mites are selected from the group consisting of species of the genera Dermanisus, Ornithonisus, Argus, Allopsoroptoides galli, Neocnemidocoptes gallinae, Cnemidocoptes mutans, Laminosioptes cysticola, Megninia cupitalis, Megninia gingrimula, Pterolicus obtus, Siringophilus bipectonatus, Colombiphyllus polonica, Delogriphus elongates, Gaudgriffus minnow, Otodectes sinotis, Cheiretiera yasuguri, Demodex species, Notoedleres cati, Cheiretiera species, Psoroptes species, Coryoptes species, Psorelgates obis, Sarcoptes scabiei, Psolovia obis, Lailletia auris, and Barroa species. More preferably, the external parasitic mite is Dermanisus galinae (mite).

[0089] In a seventh aspect of the present invention or another preferred embodiment of any of its preferred embodiments, the composition is applied by a medicinal bath, spray, injection, coating, spray, immersion, or spraying. It is preferable to apply the composition by spraying.

[0090] The present invention will be further illustrated in light of the following examples, which are illustrative and not limiting. Furthermore, the present invention covers all possible combinations of the specific embodiments and preferred embodiments described herein. [Examples]

[0091] Example 1: Assessment of spore activity. Bioassay after ingestion. The objective of this example was to study the activity of different Bacillus thuringiensis (Bt) strains formulated under different conditions against Dermanisus gallinae mites using bioassays performed by ingestion.

[0092] Bacillus thuringiensis (Bt) DSM 33173 strain isolated in Spain (as of June 20, 2019, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170) (Amer, Girona, Spain) deposited with the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under the Budapest Convention with accession number DSM 33173, and B. thuringiensis DSM 33175 (as of June 20, 2019, HIPRA SCIENTFIC, The strain used in this assay was deposited under the Budapest Convention by the SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) at the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) with accession number DSM 33175. The DSM 33175 strain was specifically isolated from a poultry farm where a high incidence of D. galinae was recorded. The DSM 33175 strain is a Bt strain that lacks the gene encoding the accessory spore crystal protein. Therefore, one of the strains included in the active bioassay was an amorphous Bt strain, specifically the DSM 33175 strain. On the other hand, the DSM 33173 Bt strain was characterized as a strain containing both spore and accessory spore crystal proteins (sequencing of the Bt DSM 33173 strain genome using standard methods detected the Cry21 and Cry55 genes).

[0093] A person skilled in the art can use common sense to determine whether the Bt strain is an amorphous strain. As is known, amorphous strains of B. thuringiensis are strains that have a genome (chromosomes and plasmid DNA) lacking the Cry protein. The absence of the Cry protein can be determined by extracting DNA from the B. thuringiensis strain using standard methods such as the use of InstaGene® Matrix, performing whole-genome sequencing, and then checking for any match with the Cry protein in well-known genome databases such as http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt (Crickmore et al., 1998, 2016). This can be assessed by verifying whether or not the gene is present. These databases contain genes that have partial homology to the complete Cry gene or the Cry protein. Non-crystalline strains of B. thuringiensis must lack the Cry gene to any degree of homology to these genes. Therefore, no results (complete or partial homology to the described Cry protein) can be retrieved from these databases in non-crystalline B. thuringiensis strains.

[0094] Whole genome sequencing of B. thuringiensis strains can also be performed using standard methods such as Illumina's HiSeq platform. The sequences are CLC Genomic. The assembly can be done using software tools such as Workbench (CLC Bio, Denmark). Reads are usually trimmed to remove low-quality reads and extreme cases such as reads shorter than 30 base pairs. The resulting reads are then used to create contigs. Novo-assemble. The resulting contigs are paired with the reference sequence and contain at least 95% of the lectures of the aligned sequence, having at least 95% identity percentage. After the contig construction, the lectures are reassigned to the contig. The resulting contigs are then analyzed using BLAST (Altschul et al., 1990) with a database of amino acid sequences of Bacillus thuringiensis toxins, such as those available at http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt (Crickmore et al., 1998, 2016). The analysis is performed using this database. BLAST is not limited to the use of this database and may include other available databases known to those skilled in the art.

[0095] In this example, individual colonies of both DSM 33173 and DSM 33175 strains were inoculated into different Erlenmeyer culture vessels as follows. The colonies were cultured in 250 ml Erlenmeyer culture vessels containing 80 ml of CCY medium (Stewart, GSA, K. Johnstone, E. Hagelberg, and DJ Ellar. 1981. Commitment of bacterial spores to germinate. Biochem. J. 198:101-106) at 28°C for 72 hours while being stirred at 220 rpm in an incubator shaker. After 72 hours, spore formation was confirmed by visual inspection using a light microscope at 1000x magnification, and it was confirmed that spores had been completely formed. At this point, 1 M NaCl and 10 mM EDTA were added to the culture. Subsequently, the culture was centrifuged at 11200 G for 10 minutes at 4°C. The pellet was then resuspended in Mili-Q water, and the supernatant was discarded. The resuspended pellet was centrifuged a second time at 11200G for 10 minutes at 4°C. The supernatant was discarded again, and the pellet was resuspended in Mili-Q water. The resuspended pellet was centrifuged a third time at 11200G for 10 minutes at 4°C, the resulting supernatant was discarded again, and the pellet was finally resuspended in Mili-Q water to a final volume of 1.5 ml to 1.8 ml to obtain the final suspension of the Bt sample for bioassay (this was named the FS Bt sample).

[0096] Next, the non-spore protein content in the final suspension of the Bt sample was quantified. To perform this quantification, the FS Bt sample was diluted 10-fold with purified water and then homogenized. Subsequently, 940 ml of pH 11.3 carbonate buffer, 10 μl of 1 M dithiothreitol (DTT), and 50 μl of the 10-fold diluted FS Bt sample suspension were added to a 1.5 ml Eppendorf tube. The Eppendorf tube was then incubated at 37°C for 2 hours with stirring at 220 rpm in an incubator shaker. At this point, the Eppendorf tube was centrifuged at 13200 G for 5 minutes. Finally, the non-spore protein was quantified using the supernatant. The quantification was performed using standard protein quantification methods such as the Bradford protein assay described in Bradford, MM 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

[0097] To perform the bioassay, use the following solution: 10 ml of fetal bovine serum (FBS) Modinate, 50 mg of D-glucose, 25 mg of ATP, and 100 μl of a color development solution (35 g / l quinoline yellow E-104 and 40 g / l patent blue E-131 in PBS until the total volume reaches 50 ml) were prepared in advance and referred to as the final FBS solution.

[0098] Next, the following five different experimental formulations were prepared using the final FBS solution: Composition 1: Negative control: Final FBS solution mixed with the same amount of purified water in a 1:1 (v / v) ratio. Composition 2 (Bt DSM 33173): Final FBS solution prepared by mixing the final suspension of the Bt sample (FS Bt sample), which was prepared with purified water to contain 0.4 mg / ml of total non-spore protein from B. thuringiensis strain DSM 33173, in a 1:1 (v / v) ratio with the final FBS solution. The FS Bt sample of strain DSM 33173 was not subjected to any purification process. Composition 3 (Bt DSM 33175): A final FBS solution prepared by mixing the final suspension of the Bt sample (FS Bt sample), which was prepared with purified water to contain 0.4 mg / ml of total non-spore proteins from B. thuringiensis strain DSM 33175, in a 1:1 (v / v) ratio with the final FBS solution. The FS Bt sample of DSM 33175 did not contain accessory spore crystalline proteins. Composition 4 (DSM 33173, purified spores): The final suspension of the Bt sample of the DSM 33173 strain (FS Bt sample) was subjected to a further purification process to enrich the FS Bt sample with live spores. For the purification process, the B. thuringiensis final suspension (FS Bt sample) was solubilized in a pH 9.0 carbonate buffer solution. After solubilization, the suspension was centrifuged and the pellet was resuspended in Mili-Q water. The supernatant containing the solubilized protein was then discarded, leaving the pellet. The pellet was then adjusted with purified water until the non-spore protein content of B. thuringiensis strain DSM 33173 was 0.4 mg / ml. Finally, this was mixed with the FBS final solution at a 1:1 (v / v) ratio for bioassay. Composition 4 had a high live spore content. Formulation 5 (DSM 33173, heat-treated): The final FBS solution was prepared by mixing the final suspension of the Bt sample (FS Bt sample), which was heat-treated at 120°C for 20 minutes and adjusted with purified water to a total non-spore protein content of B. thuringiensis strain DSM 33173 of 0.4 mg / ml, in a 1:1 (v / v) ratio. The heat treatment performed on the FS Bt sample was carried out to inactivate both non-spore proteins such as crystalline proteins and living spores.

[0099] The color-developing solution contained in the final FBS solution was used as an indicator to clearly identify mites that had ingested one of the five experimental formulations.

[0100] Adult and nymph samples of Dermanisus gallinae were collected from poultry (egg-laying chicken) farms with high rates of Dermanisus gallinae mite infestation. Only healthy, mobile mites were selected for this study. The mites were maintained in 50 ml ventilated containers at 24°C to 26°C for one week, without any contact with food sources.

[0101] The mites were divided into five different groups of approximately 20 individuals each. Each group was given one of five different experimental formulations. The mites from each group were introduced into methacrylate tubes approximately 10 cm long and 0.5 cm wide. One end of the methacrylate tube was sealed with a 20 μm mesh fitted with a 1 ml micropipette cut tip. The other end was For this experiment, 2 x 2 cm skin samples from one-day-old chickens were fixed using a 1 ml micropipette tip containing 0.6 ml of experimental formulation (1-5, one for each group).

[0102] The mites from each group were left at 30°C and high relative humidity of 80% to 100% for 2 hours, allowing them to freely ingest the experimental formulation. Subsequently, colored mites that had ingested the experimental formulation were selected. Then, the colored mites were introduced into new methacrylate tubes, one end of which was completely sealed and the other end sealed with a 20 μm mesh fitted with a 1 ml micropipette cut tip.

[0103] The activity of five different experimental formulations was evaluated by monitoring the mite mortality rate in each group. Mortality rates were confirmed after 24 hours using a stereomicroscope at 20x to 40x magnification.

[0104] The results of the mortality rate are summarized in Figure 1. The assay showed that the amorphous strain DSM 33175, which does not produce any crystalline protein, yielded a higher mortality rate of 83% (group 3) than the formulation containing both spore and accessory spore crystalline protein (67%, group 2), thus demonstrating that spores have a clearer activity than accessory spore crystalline protein in reducing the mortality rate of D. gallinae mites.

[0105] Furthermore, it was observed that experimental formulation 4 (group 4), enriched with purified live spores, exhibited increased activity compared to other formulations (particularly compared to formulation 2), resulting in a more significant reduction in the D. gallinae population (80% mite mortality rate) compared to unpurified formulation 2 (group 2), which contained both spore and accessory spore crystalline proteins, while formulation 2 resulted in only a 67% mite mortality rate.

[0106] In addition, when the live spores and accessory spore crystal proteins were subjected to heat treatment for inactivation, no mite mortality was observed (Group 5).

[0107] Given that the toxic effects of B. thuringiensis strains used as insecticides in agriculture are clearly disclosed in the prior art, the inventors did not anticipate that the purified formulation enriched with live spores would have better activity in killing mites than that observed in compositions containing crystalline proteins. Therefore, these results are considered surprising and unexpected.

[0108] Example 2: Assessment of spore activity using plate bioassay A second assay was performed to demonstrate the activity of live spores on mite mortality. This time, instead of the ingestion model used in Example 1, a new experimental model based on an agar plate bioassay was used. The plate bioassay more closely resembles future field conditions.

[0109] Various experimental formulations were prepared for plate bioassays.

[0110] For this purpose, 7.5 g of European bacteriological agar was homogenized in 500 ml of Mili-Q water. The resulting homogenized agar was autoclaved at 121°C for 20 minutes. Then, it was plated (in a Petri dish, e.g., a Greiner bio-one) A 25 ml agar solution was placed in one Petri dish (model number 633181, with lid, outer diameter 93 mm, effective capacity 15 ml, maximum capacity 80 ml).

[0111] Three different experimental formulations were prepared as follows: Formulation 1 (unpurified spore preparation): The final suspension of the Bt sample described in Example 1 (FS Bt sample) was prepared with purified water so that the non-spore protein content of B. thuringiensis strain DSM 33173 was approximately 2.5 mg / ml. Formulation 2 (Purified Spore Preparation): The final suspension (FS) of the Bt sample described in Example 1. The Bt sample was subjected to a purification process to enrich the FS Bt sample with live spores. For the purification process, the final suspension of B. thuringiensis (FS Bt sample) was solubilized in a pH 9.0 carbonate buffer solution. After solubilization, the suspension was centrifuged, and the pellet was resuspended in Mili-Q water. The supernatant was then discarded, leaving the pellet. The pellet was then adjusted with purified water until the non-spore protein content of B. thuringiensis strain DSM 33173 was approximately 2.5 mg / ml. Composition 3 (negative control): Mili-Q water was used as the negative control.

[0112] Each of the above experimental formulations (1-3) was spread onto an agar plate at a concentration of 50 μl / plate.

[0113] Adult and nymph samples of Dermanisus gallinae were collected from poultry (egg-laying chicken) farms with high rates of Dermanisus gallinae mite infestation. Only healthy, mobile mites were selected for this study. The mites were kept in 50 ml ventilated containers at 24°C to 26°C for one week, without any contact with food sources. The mites were divided into three different groups of approximately 20 individuals each.

[0114] Mites from each group were placed on agar plates prepared in advance using the experimental formulations (1-3) to be tested.

[0115] The petri dishes were sealed using two-face double-sided bonding tape to prevent mites from escaping. During the study, the petri dishes were maintained at 26°C and 70% relative humidity. The activity of three different experimental formulations was evaluated by monitoring the mite mortality rate in each plate group. Mortality rates were confirmed at 24 and 48 hours using a stereomicroscope at 20x to 40x magnification.

[0116] The mortality rate results are summarized in Figure 2. The results obtained using the plate bioassay in Example 2 supported the effect observed using the ingestion bioassay experimental model in Example 1.

[0117] The inventors believe that the lower mortality rate percentage obtained in Example 2, which was based on an agar plate bioassay instead of the ingestion model used in Example 1, is normal. Although the plate bioassay more closely resembles future field conditions, the mortality rate is always higher when using the ingestion bioassay. This is because, in the ingestion bioassay, after the mites ingest the experimental formulation, only the mites that ingested the formulation are selected to confirm their survival and mortality rates.

[0118] Plate bioassays demonstrated that formulation 2 (group 2), based on the purified spore preparation, yielded a higher mite mortality rate than formulation 1 (group 1), based on the unpurified spore preparation, with mortality rates of 48.81% and 27.77%, respectively, after 48 hours. Surprisingly, the inventors found that compositions rich in accessory spore crystal proteins were inferior to purified compositions rich in live spores in terms of mite mortality rates.

[0119] Example 3: Study on dose-response of various Bt compositions This example was conducted to verify the activity of various B. thuringiensis (Bt) compositions containing live spores at different doses.

[0120] Adult and nymph samples of Dermanisus gallinae were collected from poultry (egg-laying chicken) farms with high rates of Dermanisus gallinae mite infestation. Healthy, mobile mites were selected for dose / response assays. The assay was performed using a model based on an agar plate bioassay (Example 2), but a petri dish (e.g., Greiner bio-one petri dish, model 63) was used. Instead of the 3181, lidded dish, outer diameter 93mm, effective capacity 15ml, maximum capacity 80ml, use a cell culture flask (e.g., Falcon flask, model number 353107, 12.5cm). 2 A 25 ml container with a screw cap and ventilation hole was used. The cell culture flasks were pre-prepared using European bacteriological agar as described in Example 2. In this case, 11 ml of agar solution was placed in each flask instead of 25 ml per petri dish. Mites were collected from the farm, with approximately 20 mites each. They were divided into nine different groups.

[0121] The B. thuringiensis strains used in this study were GR-S5-8, an isolate corresponding to a Bt strain isolated from a poultry farm in Spain where a high invasiveness rate of D. galinae was detected; GR-P14-3, similarly an isolate corresponding to a Bt strain isolated from a poultry farm in Spain where a high invasiveness rate of D. galinae was detected; and DE2-S2-8, an isolate corresponding to a Bt strain isolated from a poultry farm in Germany where a low invasiveness rate of D. galinae was detected. The GR-S5-8 isolate was, as mentioned above, submitted to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen by HIPRA SCIENTIFIC, SLU on June 20, 2019. This corresponds to DSM 33175 shares deposited with und Zellkulturen under the Budapest Convention. Furthermore, the DE2-S2-8 Bt isolate was certified by HIPRA SCIENTIFIC, SLU on February 21, 2019, by the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Deposited in Zellkulturen under the Budapest Convention with accession number DSM 33035 It was done.

[0122] All B. thuringiensis strains were grown as described in Example 1 to obtain final suspensions of Bt samples (FS Bt samples) for dose-response assays. The number of viable spores per 1 ml of B. thuringiensis final suspension (FS Bt sample) was determined by standard microbiological methods such as colony-forming units (CFU) / ml.

[0123] To elaborate, it is important to emphasize that, as explained above in Examples 1 and 2, the inventors decided to determine the number of viable spores per 1 ml of B. thuringiensis final suspension instead of determining or quantifying the concentration of the accessory spore crystal protein (non-spore protein), based on the results that showed the desired effect is related to spores rather than accessory spore crystal protein.

[0124] Next, the final suspensions of Bt samples from strains GR-S5-8 and GR-P14-3 were adjusted with purified water to four different constant doses (CFU / ml) of live spores per 1 ml of preparation. The four constant doses of live spores were 3.8 × 10⁶ per 1 ml of preparation. 7 pieces, 1.1×10 8 pieces, 3.4×10 8 pieces and 3.1 × 10 9 This resulted in 10⁴ condensed spores (CFUs). These different doses correspond to groups 1-4 for the GR-P14-3 strain and groups 5-8 for the GR-S5-8 strain. On the other hand, the final suspension of the Bt sample of B. thuringiensis strain DE2-S2-8 was 3.1 × 10⁴. 9 The dose was adjusted to one condensed spore (CFU) / ml. This constant dose corresponds to group 9.

[0125] 50 μl of various Bt preparations, adjusted to different live spore / ml doses, were introduced into cell culture flasks. During the study, the flasks were maintained at 26°C and 70% relative humidity. The activity of the various spore preparations was assessed by monitoring the mite mortality rate in each flask. Mortality rates were confirmed at 24 and 48 hours using a stereomicroscope at 20x–40x magnification.

[0126] The results are summarized in Figure 3. The results clearly demonstrate the role of live spores in generating anti-mite activity. It was observed that the lower the amount of live spores in the composition, the lower the mortality rate of D. gallinae mites observed both 24 hours and 48 hours after treatment with the experimental formulation. Similarly, the higher the dose of live spores, the higher the mortality rate observed, regardless of the non-spore protein content of the composition.

[0127] Furthermore, all Bt strains tested showed 3.1 × 10⁻⁶ 9 The formulation showed a significant mortality rate when combined with 100% live spores (CFU) / ml. In particular, the deposited DSM 33035 Bt strain DE2-S2-8 showed higher anti-mite activity, exhibiting a 66% mortality rate 48 hours after treatment (Group 9).

[0128] Example 4: Comparative assay of the activity of various Bt strains against mites In this example, the activity of various B. thuringiensis (Bt) strains isolated from poultry farms against various types of pests was evaluated and compared with Bt strains isolated from commercially available agricultural products.

[0129] Seven different B. thuringiensis strains were included in the comparative assay. The Bt strains were as follows: DE2-S2-8 strain (isolated from a poultry farm in Germany with a low D. galinae infestation rate, DSM 33035), DE1-S2-4 strain (isolated from a poultry farm in Germany with a high D. galinae infestation rate, DSM 33034), DE2-S1-1 strain (isolated from a poultry farm in Germany with a low D. galinae infestation rate), GR_P1_4 strain (isolated from a poultry farm in Spain with a high rate of D. galinae invasion), MA_S15_3 strain (isolated from a poultry farm in Spain with a low D. galinae invasion rate), DE2-S3-4 strain (isolated from a poultry farm in Germany with a low D. galinae infestation rate), and, B. thuringiensis subspecies Krustaki strain ABTS-351 (a strain found in various commercially available agricultural pest products). In this case, the commercially available product bioMAX 32 manufactured by Valent BioScience LLC. (Trademark) was isolated.

[0130] All seven Bt strains were cultured as described in Example 1 to obtain the final suspension of Bt samples (FS Bt sample) for comparative assay. The number of viable spores per 1 ml of the B. thuringiensis final suspension (FS Bt sample) was expressed as colony-forming units (CFU) / ml (Goldman, E.; Green, L. Practical Handbook of Microbiology. 3). rd The count was determined using standard microbiological methods such as those described in (Edition, published 4 June 2015; Chapter 2. Quantification of Microorganisms. Page 19, Plate Count Method by Peter S. Lee, page 24. CRC Press, 1055 pages. ISBN 9781466587397). Colony counting was performed manually using a pen and click counter.

[0131] Adult and nymph samples of Dermanisus gallinae were collected from poultry (egg-laying chicken) farms with high rates of Dermanisus gallinae mite infestation. Healthy, mobile mites were selected for comparative assays. Mites were collected from the farms and divided into seven different groups of approximately 20 mites each. The assays were performed using the flask bioassays described in Example 3 (all pre-prepared using European bacteriological agar as described in Example 2). 11 ml of agar solution was placed in each flask.

[0132] 50 μl of the final suspension (FS Bt sample) of the Bt sample prepared using each specific strain was introduced into the cell culture flasks of each group. The specific strains were grown as described in Example 1, and 3.1 × 10⁶ of the preparation per 1 ml was added using purified water. 9 The content of live spores (CFUs) was adjusted to one unit. The content of non-spore proteins was further determined as described in Example 1.

[0133] During the study, the flasks were maintained at 26°C and 70% relative humidity. The activity of various spore preparations was evaluated by monitoring the mite mortality rate in each flask. The mortality rate was confirmed at 24 and 48 hours using a stereomicroscope at 20x to 40x magnification.

[0134] The mortality rate results are summarized in Table 1.

[0135] [Table 1]

[0136] All preparations resulted in mite mortality within 48 hours. However, the mite mortality rates observed with commercially available Bt products used for agricultural pest control, such as those of Dermanisus gallinae, were significantly reduced when using preparations containing B. thuringiensis subspecies Krustaki strain ABTS-351, which is commonly found in commercially available agricultural products, as described above. On the other hand, the activity in mite mortality doubled for Bt strains other than the commercially available Bt subspecies Krustaki. The above relationship with activity in mite mortality was further demonstrated with other preparations tested that have high non-spore protein content, such as B. thuringiensis subspecies Aizawai (3.09 mg / ml; 21.33% mortality rate), B. thuringiensis subspecies Morisoni (1.24 mg / ml; 22.50% mortality rate), or B. thuringiensis subspecies Islaerensis (0.91 mg / ml; 30.36% mortality rate).

[0137] Therefore, surprisingly, it was observed that the Bt strain, which generally yielded high mite mortality rates, produced a low amount of non-spore protein in response to the live spores in the preparation. The higher the non-spore protein content, the lower the observed activity.

[0138] Example 5: Activity of a Bt composition containing mineral oil In this example, the activity of B. thuringiensis compositions containing mineral oil was evaluated. For this study, a suspension of Bt samples (FS Bt samples) based on Bt strain DE2-S2-8 (DSM 33035) was prepared as described in Example 1. Subsequently, six different formulations containing different amounts of live spores per 1 ml, with and without mineral oil, were prepared as follows: Formulation 1. A DE2-S2-8 strain FS Bt sample was mixed with an 86.2% (v / v) PBS emulsion containing 20% ​​(v / v) mineral oil (Marcol 52), 0.9% polysorbate 80, 0.65% sorbitan monooleate, and 2.4% Simulsol 5100. The final sample content was 3.4 × 10⁻⁶. 8 The concentration was adjusted to one condensed spore (CFU) per ml. Formulation 2. A DE2-S2-8 strain FS Bt sample was mixed with an 86.2% (v / v) PBS emulsion containing 20% ​​(v / v) mineral oil (Marcol 52), 0.9% polysorbate 80, 0.65% sorbitan monooleate, and 2.4% Simulsol 5100. The final content of the sample was 1.03 × 10⁻⁶. 9 The concentration was adjusted to one condensed spore (CFU) per ml. Formulation 3. A DE2-S2-8 strain FS Bt sample was mixed with an 86.2% (v / v) PBS emulsion containing 20% ​​(v / v) mineral oil (Marcol 52), 0.9% polysorbate 80, 0.65% sorbitan monooleate, and 2.4% Simulsol 5100. The final sample content was 3.10 × 10⁻⁶. 9 The content was adjusted to the amount of individual spores (CFU) / ml. Formulation 4. Using purified water, 1.03 × 10 9 FS Bt samples of the DE2-S2-8 strain, adjusted to contain 100 condensed spores (CFU) / ml. Formulation 5. Using purified water, 3.10 × 10 9 FS Bt samples of the DE2-S2-8 strain, adjusted to contain 100 condensed spores (CFU) / ml. Formulation 6. Mili-Q water was used as a negative control.

[0139] Adult and nymph samples of Dermanisus gallinae were collected from poultry (egg-laying chicken) farms with high rates of Dermanisus gallinae mite infestation. Healthy, mobile mites were selected for the assay. Mites were collected from the farms and divided into six different groups of approximately 20 mites each. The assay was performed using the flask bioassays described in Example 3 (all pre-prepared using European bacteriological agar as described in Example 2). 11 ml of agar solution was placed in each flask.

[0140] 50 μl of the final suspension (FS Bt sample) of Bt samples prepared using each formulation (1-5) was introduced into the cell culture flasks of each group. Group 6 was given 50 μl of Mili-Q water as a negative control. The flasks were maintained at 26°C and 70% relative humidity throughout the study. The activity of each formulation was evaluated by monitoring the mite mortality rate in each flask. The mortality rate was confirmed at 24 and 48 hours using a stereomicroscope at 20x to 40x magnification.

[0141] The results of the mite mortality rate are summarized in Figure 4. Unexpectedly, the mixture of B. thuringiensis preparation and mineral oil did not adversely affect the viability of B. thuringiensis strains or their anti-mite activity. On the contrary, it significantly increased the effectiveness of the experimental formulation, resulting in a synergistic effect on the mite mortality rate. 9 Formula 3 (Group 3), prepared using 100% live spores (CFU) / ml, achieved an excellent mite mortality rate of over 90% in just 48 hours.

[0142] It was further observed that a mixture of B. thuringiensis formulations added to mineral oil preparations allowed for a reduction in the dose of B. thuringiensis bacteria used in the experimental formulations. For example, 3.4 × 10⁻⁶ 8 A mineral oil preparation (Group 1) containing a dose of 100 mite spores (CFU) / ml resulted in a mite mortality rate of 70.10% after 48 hours. A similar mortality rate was obtained when the B. thuringiensis preparation was not mixed with mineral oil, but in this case, the rate was 3.1 × 10⁻⁶. 9 The formulation was prepared at a dose of 10

[0143] Therefore, the inventors confirmed the synergistic effect between the Bt preparation and mineral oil. In additional experiments, it was observed that the synergistic effect was maintained even when the FS Bt sample was mixed with an emulsion containing a very low percentage of mineral oil, such as 1.25%.

[0144] CLAUSES 1. A pharmaceutical composition or biocide composition suitable for controlling or mites invading mites, comprising a bacterial preparation of at least one strain of Bacillus thuringiensis (B. thuringiensis) as an active ingredient, characterized in that the bacterial preparation contains an effective amount of live spores of at least one strain of B. thuringiensis. 2. The pharmaceutical or biocide composition according to item 1, wherein the bacterial preparation contains an effective amount of live spores of at least one strain of B. thuringiensis suitable for controlling or mites mite infestation caused by Dermanisus galinae (mite mite). 3. The pharmaceutical or biocide composition according to item 1 or 2, wherein at least one strain of B. thuringiensis is a strain that produces less of B. thuringiensis non-spore proteins, such as accessory spore crystal proteins, than B. thuringiensis subspecies krustaki, subspecies aizawai, subspecies Israelensis, and subspecies Morrisoni. 4. At least one strain of B. thuringiensis yielded 3.1 × 10⁶ per ml. 9 A pharmaceutical composition or biocide composition according to item 1 or 2, which is a strain of B. thuringiensis that produces non-spore proteins such as accessory spore crystal protein at a concentration of less than 1.7 mg / ml per viable spore. 5. A pharmaceutical or biocide composition according to item 3 or 4, wherein the non-spore proteins of B. thuringiensis are selected from the group consisting of delta (δ)-endotoxin, proteins Cry and Cyt, vegetative stage insecticidal proteins (Vip1, Vip2, Vip3 and Vip4), secretory insecticidal proteins (Sip protein), β-exotoxin (turingiencin), cholesterol-dependent cytolysin-related proteins (sphericollicin and alveolisin), enhancer-like proteins (Bel enhancer protein), helper proteins (P19 and P20 proteins), or non-proteinogenic β-exotoxin (Bt 41.9kDa protein). 6. The bacterial preparation contains at least 1 × 10 per 1 ml of composition. 4A pharmaceutical composition or biocide composition according to any one of claims 1 to 5, comprising an effective amount of live cfu spores. 7. The bacterial preparation contains at least 3 × 10 per 1 ml of composition. 8 The pharmaceutical or biocide composition according to item 6, comprising an effective amount of live cfu spores. 8. The bacterial preparation contains at least 3 × 10 per 1 ml of composition. 9 The pharmaceutical or biocide composition according to item 6, comprising an effective amount of live cfu spores. 9. A pharmaceutical composition or biocide composition according to any one of claims 1 to 8, further comprising at least one further active or functional component selected from the group consisting of insecticides, acaricides, fungicides, nematicides, antibiotics, cleaning agents, immunogenic substances, animal feed, essential oils, mineral oils, nutritional supplements, probiotics, prebiotics, synbiotics, polysaccharides, and combinations thereof. 10. The pharmaceutical composition or biocide composition according to item 9, wherein the further active ingredient is an immunogenic substance derived from a microorganism selected from the group consisting of IBv, NDV, adenovirus, EDS, IBDV, chicken anemia virus, avian encephalomyelitis virus, fowlpox virus, turkey rhinotracheitis virus, duck plague virus, pigeonpox virus, MDV, avian leukemia virus, ILTV, tripneumovirus, reovirus, Escherichia coli, Salmonella species, Ornithobacterium rhinotracheale, Haemophilus paragallinarum, Pasteurella multocida, Erydiperotrix lugiopasiae, Erysipera species, Mycoplasma species, Clostridium species, Eimeria species and Aspergillus species. 11. A pharmaceutical composition or biocide composition according to any one of items 1 to 10, which is in a form selected from the group consisting of powders, powders, granules, microcapsules, lotions, ointments, gels, creams, pastes, suspensions, stock solutions, liquids, and emulsions. 12. A pharmaceutical composition or biocide composition according to any one of items 1 to 11, suitable for application by medicinal bath, spray, injection, application, spray, immersion, or dispensing. 13. A pharmaceutical or biocide composition according to any one of items 1 to 12, further comprising one or more pharmaceutically acceptable vehicles and / or one or more acceptable adjuvants. 14. At least one strain of Bacillus thuringiensis was granted accession number DSM to Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) by HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) on February 21, 2019. A pharmaceutical or biocide composition according to any of items 1 to 13, which is a B. thuringiensis strain and its mutant, respectively, deposited under the Budapest Convention in 33034. 15. On February 21, 2019, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) granted accession number DSM to Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany). A pharmaceutical or biocide composition according to any of items 1 to 13, which is a B. thuringiensis strain and its mutant strain, respectively, deposited under the Budapest Convention in 33035. 16. On June 20, 2019, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) granted accession number DSM to Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany). A pharmaceutical or biocide composition according to any of items 1 to 13, which is Bacillus thuringiensis (Bt) strain GR-S5-8 and its mutant strains, respectively deposited under the Budapest Convention 33175. 17. A pharmaceutical or biocide composition according to any one of items 1 to 16, which is a pharmaceutical or biocide. 18. A pharmaceutical composition or biocide composition according to any one of items 1 to 16 for the control and / or reduction of mite infestation. 19. A pharmaceutical composition or biocide composition according to any one of items 1 to 16, for the prevention and / or treatment of diseases caused by external parasitic mites in animals that require such prevention and / or treatment. 20. The disease affects species of the genera Dermanisus, Ornithonisus, Argus, Allopsoroptoides galli, Neocnemidocoptes galinae, Cnemidocoptes mutans, Laminosioptes cysticola, Megninia capitaris, Megninia gingrimula, Pteroricus obtus, Siringophilus bipectonatus, Colombiphyllus polonica, Delogriffus elongates, Gaudgriffus minnow, and Oto A pharmaceutical or biocide composition according to item 19, caused by an external parasitic mite selected from the group consisting of Dectes sinuthis, Cheiretiera yasuguri, species of Demodex, Nothoedres cati, species of Cheiretiera, species of Psoroptes, species of Coryoptes, Psorelgates obis, Sarcoptes scaviei, Psolovia obis, Laylietia auris and species of Valois, and combinations thereof. 21. A pharmaceutical composition or biocide composition according to any one of items 1 to 16, preferably for controlling and / or mitigating mite infestation caused by external parasitic mites in animals or vectors. 22. External parasitic mites include species of the genera Dermanisus, Ornithonisus, Argus, Allopsoroptoides galli, Neocnemidocoptes gallinae, Cnemidocoptes mutans, Laminosioptes cysticola, Megninia capitaris, Megninia gingrimula, Pteroricus obtus, Syringophyllus bipectonatus, Colombiphyllus polonica, Delogriffus elongates, and Gaud A pharmaceutical or biocide composition according to item 21, selected from the group consisting of Griffus minnow, Otodectes sinuthis, Cheiretiera yasuguri, species of Demodex, Notoedleres cati, species of Cheiretiera, species of Psoroptes, species of Coryoptes, Psorelgates obis, Sarcoptes scaviei, Psolovia obis, Laylietia auris and species of Valois, and combinations thereof. 23. The pharmaceutical or biocide composition according to item 20 or 22, wherein the external parasitic mite is Dermanisus galinae (mite). 24. A pharmaceutical or biocide composition according to any one of items 18 or 21-23, wherein the control and / or reduction of mite infestation results in a mite mortality rate of at least 25%. 25. The pharmaceutical or biocide composition according to item 24, wherein a mortality rate of at least 25% of mites occurs within 48 hours from the moment the composition is applied to and / or comes into contact with the mites. 26. A pharmaceutical composition or biocide composition according to any one of items 17 to 25, applied by medicinal bath, spray, injection, application, spray, immersion or spraying. 27. On February 21, 2019, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) granted accession number DSM to Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany). A pharmaceutical or biocide composition according to any of subsections 17 to 26, which is a B. thuringiensis strain and its mutant, respectively, deposited under the Budapest Convention in 33034. 28. On February 21, 2019, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) granted accession number DSM to Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany). A pharmaceutical or biocide composition according to any of subsections 17 to 26, which is a B. thuringiensis strain and its mutant, respectively, deposited under the Budapest Convention in 33035. 29. On June 20, 2019, HIPRA SCIENTFIC, SLU (Avda de La Selva 135, 17170 Amer, Girona, Spain) granted accession number DSM to Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany). A pharmaceutical or biocide composition according to any of items 17 to 26, which is Bacillus thuringiensis (Bt) strain GR-S5-8 and its mutant strains, respectively deposited under the Budapest Convention in 33175. 30. A pharmaceutical composition or biocide composition according to any one of items 19 to 29, wherein the animal is selected from the group consisting of birds, pigs, cattle, horses, cats, dogs, sheep, rabbits and bees. 31. The pharmaceutical or biocide composition described in item 30, wherein the birds are poultry. 32. A method for controlling and / or reducing mite infestation in a vector, comprising using a composition described in any one of sub-sub 33. The method according to paragraph 32, wherein the control and / or reduction of mite infestation results in a mite mortality rate of at least 25%. 34. The method according to claim 33, wherein a mites euthanasia rate of at least 25% occurs within 48 hours from the moment the composition is applied to and / or brought into contact with the mites and / or vectors. 35. The method according to any one of claims 32 to 34, wherein the composition is applied by a medicated bath, spray, injection, coating, spray, immersion or spray. 36. The method described in any one of subparagraphs 32 to 35, wherein the vector is located on the premises where the animals are kept. 37. A pharmaceutical composition or biocide composition according to any one of items 1 to 16, wherein the composition comprises a further active ingredient, the further active ingredient being an essential oil or mineral oil. 38. A pharmaceutical composition or biocide composition according to item 37, which is incorporated into the essential oil or mineral oil described above. 39. The pharmaceutical or biocide composition according to item 37 or 38, wherein a further active ingredient is mineral oil, preferably liquid paraffin. 40. A pharmaceutical composition or biocide composition according to any one of claims 37 to 39, preferably used for controlling and / or mitigating tick infestation in animals or vectors. 41. A pharmaceutical or biocide composition according to any one of claims 37 to 40, wherein at least one strain is selected so that the control and / or reduction of mite infestation results in a mite mortality rate of at least 50%. 42. The pharmaceutical or biocide composition according to item 41, wherein a 50% mortality rate of mites occurs within 48 hours from the moment the composition is applied to and / or brought into contact with the mites and / or vectors. 43. Preferably, the pharmaceutical composition or biocide composition according to item 41 or 42, wherein a 50% mortality rate of mites within 48 hours from the moment the composition is applied to the mites or vector is achieved by medicinal bath, spray, injection, coating, spray, immersion or spraying. 44. A pharmaceutical composition or biocide composition according to any of items 37 to 43, or a pharmaceutical composition or biocide composition for use, wherein the mite is an external parasitic mite. 45. External parasitic mites include species of the genera Dermanisus, Ornithonisus, Argus, Allopsoroptoides galli, Neocnemidocoptes gallinae, Cnemidocoptes mutans, Laminosioptes cysticola, Megninia capitaris, and Megninia gin A pharmaceutical composition or biocide composition according to item 44, or a pharmaceutical composition or biocide composition for use, selected from the group consisting of Grimla, Pteroricus obtus, Siringophilus bipectonatus, Colombiphyllus polonica, Delogriphus elongates, Gaudgriphus minou, Otodectes sinuthis, Cheiretiera yasuguri, species of Demodex, Notoedleres cati, species of Cheiretiera, species of Psoroptes, species of Coryoptes, Psorelgates obis, Sarcoptes scaviei, Psolovia obis, Laylietia auris and species of Baroa, and combinations thereof. 46. ​​A pharmaceutical composition or biocide composition according to item 45, or a pharmaceutical composition or biocide composition for use, wherein the external parasitic mite is Dermanisus galinae (mite). 47. A pharmaceutical composition or biocide composition according to any of items 37 to 46, or a pharmaceutical composition or biocide composition for use, wherein control and / or reduction of mite infestation is achieved in animals selected from the group consisting of birds, pigs, cattle, horses, cats, dogs, sheep, rabbits and bees. 48. A pharmaceutical or biocide composition as described in item 47, or a pharmaceutical or biocide composition for use, wherein the birds are poultry. 49. A pharmaceutical composition or biocide composition as described in any of items 37 to 48, or a pharmaceutical composition or biocide composition for use, suitable for application by medicinal bath, spray, injection, application, spray, immersion or spraying. 50. A pharmaceutical composition or biocide composition as described in any of items 37 to 49, or a pharmaceutical composition or biocide composition for use, suitable for application by spraying. 51. A pharmaceutical composition or biocide composition according to any of sub-sub

Claims

1. A pharmaceutical composition or biocide composition suitable for controlling or mites infestation, comprising a bacterial preparation of at least one strain of Bacillus thuringiensis (B. thuringiensis) as an active ingredient, characterized in that the bacterial preparation contains an effective amount of live spores of at least one strain of B. thuringiensis.

2. The pharmaceutical composition or biocide composition according to claim 1, wherein the bacterial preparation contains an effective amount of live spores of at least one strain of B. thuringiensis suitable for controlling or mites mite infestation caused by Dermanisus galinae (mites).

3. The pharmaceutical composition or biocide composition according to claim 1 or 2, wherein at least one strain of B. thuringiensis is a strain that produces less B. thuringiensis non-spore protein than the B. thuringiensis subspecies crustakii.

4. The aforementioned B. Thuringiensis strain contains 3.1 × 10⁶ per ml. ９ The pharmaceutical composition or biocide composition according to claim 1 or 2, wherein the strain produces less than 1.7 mg / ml of non-spore protein of B. thuringiensis per living spore.

5. The pharmaceutical composition or biocide composition according to claim 3 or 4, wherein the non-spore proteins of B. thuringiensis are selected from the group consisting of delta (δ)-endotoxin, proteins Cry and Cyt, vegetative stage insecticidal proteins (Vip1, Vip2, Vip3 and Vip4), secretory insecticidal proteins (Sip proteins), β-exotoxin (turingiencin), cholesterol-dependent cytolysin-related proteins (sphericollicin and alveolisin), enhancer-like proteins (Bel enhancer protein), helper proteins (P19 and P20 proteins), or non-proteinogenic β-exotoxin (Bt 41.9kDa protein).

6. The bacterial preparation contains at least 1 × 10 per 1 ml of the composition. ４ A pharmaceutical composition or biocide composition according to any one of claims 1 to 5, comprising an effective amount of live spores of CFU.

7. A pharmaceutical composition or biocide composition according to any one of claims 1 to 6, further comprising at least one further active or functional component selected from the group consisting of insecticides, mite control agents, fungicides, nematicides, antibiotics, cleaning agents, immunogenic substances, animal feed, essential oils, mineral oils, nutritional supplements, probiotics, prebiotics, synbiotics, polysaccharides, and combinations thereof.

8. The pharmaceutical composition or biocide composition according to claim 7, wherein the further active ingredient is mineral oil, and the mineral oil is liquid paraffin.

9. At least one strain of the aforementioned Bacillus thuringiensis, a. HIPRA SCIENTFIC, SLU (Avda de La Selva 135) as of February 21, 2019 Deposited by Amer, Girona, Spain in 17170 to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under the Treaty of Budapest with accession number DSM 33034. B. thuringiensis strain and its mutant strains, b. HIPRA SCIENTFIC, SLU (Avda de La Selva 135) as of February 21, 2019 Deposited by Amer, Girona, Spain in 17170 to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under the Treaty of Budapest with accession number DSM 33035. B. thuringiensis strain and its mutant strains, c. HIPRA SCIENTFIC, SLU (Avda de La Selva 135) as of June 20, 2019 Deposited by Amer, Girona, Spain in 17170 to the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under the Treaty of Budapest with accession number DSM 33175. Bacillus thuringiensis (Bt) strain GR-S5-8 and its mutant strains, A pharmaceutical composition or biocide composition according to any one of claims 1 to 8, selected from the group consisting of the following.

10. A pharmaceutical composition or biocide composition according to any one of claims 1 to 9, which is a pharmaceutical or biocide.

11. A pharmaceutical composition or biocide composition according to any one of claims 1 to 9 for controlling and / or mitigating external parasitic mite invasion.

12. A pharmaceutical composition or biocide composition according to any one of claims 1 to 9, used for the prevention and / or treatment of diseases caused by external parasitic mites in animals that require such prevention and / or treatment.

13. The aforementioned external parasitic mites include species of the genera Dermanisus, Ornithonisus, Argus, Allopsoroptoides galli, Neocnemidocoptes gallinae, Cnemidocoptes mutans, Laminosioptes cysticola, Megninia capitaris, Megninia gingrimula, Pteroricus obtus, Silingophyllus bipectonatus, Colombiphyllus polonica, Delogriffus elongates, and Gaudguri. A pharmaceutical composition or biocide composition according to claim 11 or 12, selected from the group consisting of Fus mino, Otodectes sinuthis, Cheiretiera yasuguri, species of Demodex, Notoedleres cati, species of Cheiretiera, species of Psoroptes, species of Coryoptes, Psorelgates obis, Sarcoptes scaviei, Psolovia obis, Laylietia auris and species of Baroa, and combinations thereof.

14. The pharmaceutical composition or biocide composition according to claim 13, wherein the external parasitic mite is Dermanisus galinae (mite mite).

15. A pharmaceutical composition or biocide composition according to any one of claims 1 to 9, further comprising a pharmaceutically acceptable vehicle and / or an acceptable adjuvant, or a pharmaceutical composition or biocide composition according to any one of claims 10 to 14.

16. A pharmaceutical composition or biocide composition according to any one of claims 10 to 15, which is applied by medicinal bath, spray, injection, application, spray, immersion, or spraying.

17. A pharmaceutical composition or biocide composition according to any one of claims 10 to 15, applicable to animals selected from birds, pigs, cattle, horses, cats, dogs, sheep, rabbits, and bees.

18. The pharmaceutical composition or biocide composition according to claim 11, 13, or 14, wherein the control and / or reduction of mite infestation results in a mite mortality rate of at least 25%.

19. The pharmaceutical composition or biocide composition according to claim 18, wherein a mortality rate of at least 25% of the mites occurs within 48 hours from the moment the composition is applied to and / or brought into contact with the mites.

20. A method for controlling and / or reducing mite infestation in a vector, comprising using the composition described in any one of claims 1 to 9.

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