Novel anthelmintic cry proteins and uses thereof

Abstract

Compositions and methods for treating or reducing the severity or likelihood of occurrence of a parasitic worm infection in a human, an animal, or a plant are described. Compositions including isolated, native, bioactive crystals of CryH1 or CryH13 and inactivated or live recombinant bacteria expressing a CryH1 or CryH13 crystal proteins in the cytosol of the bacterium are described. Compositions suitable for pharmaceutical and agricultural applications are provided. Methods of administering a therapeutically effective amount of a composition to a human or animal suffering from a parasitic worm infection are provided. Compositions are provided for use as animal feed, feed supplement, or in waste treatment. Methods of agricultural applications for control of parasitic worm infections are provided.

Description

[0001] NOVEL ANTHELMINTIC CRY PROTEINS AND USES THEREOF

[0002] STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under Grant No, AI056189 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 649,526, filed May 20, 2024, the contents of which are incorporated herein by reference in its entirety for all purposes.

[0006] SEQUENCE LISTING

[0007] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on May 2, 2025, is named 765433-UM9-310PC_Sequence Listing.xml, and is 15 kilobytes in size.

[0008] BACKGROUND

[0009] Helminths are a group of parasitic worms living in resource-limited settings. Helminths can infect a wide variety of hosts including humans, domesticated animals, wild animals, plants, and fish. Helminths are the. most common infectious agents of humans in developing countries and produce a global burden of disease that exceeds better-known conditions, including malaria and tuberculosis. Helminths infect 2.3 billion of the poorest peoples and greater than 400,000,000 of the poorest children worldwide. (Hall, A., et al. Matern Child Nutr 4 Suppl 1, 1 18-236 (2008).) Infected children can exhibit growth stunting, retarded cognitive development, lethargy, malnutrition, increased school absenteeism, and vulnerability to secondary infections. (Bethony, J. et al. Lancet 367, 1521-32 (2006); Hotez, P.J. Forgotten people, Forgotten diseases. (2008)) Pregnant women who are infected are at increased risk for low birth-weight babies and for maternal and infant mortality. (Brooker et al., PLoS Negl Trap Dis 1, e291 (2008)). Infected individuals have lower energy, lower productivity, and immune defects that result in increased virulence of EUV / AIDS and a higher likelihood of contracting malaria and tuberculosis (Stothard et al., Ann Trap Med Parasitol 103, 357-60 (2009); Moran, M. et al., G-finder Report (2009)) Helminths thus trap large populations of the developing world in poverty. The common link of helminth transmission is poor sanitation, which requires a massive investment in infrastructure and public health.

[0010] Parasitic worms also infect animals including livestock and companion animals like cows, sheep, goats, horses, pigs, poultry, dogs, and cats. Parasitic worm infections can lead to devastating economical losses to animal based farm production. Farm animals can suffer from malnutrition,, tissue damage, and blood loss resulting in impaired production traits and reproduction parameters. Moreover, infected animals can transmit parasites to humans. Thus, treatment of animal helminth infection is of great commercial interest.

[0011] Some helminths, viz. nematodes (roundworms), also parasitize: plants. Nematodes feed on all parts of the plant, including roots, stems, leaves, flowers and seeds. Root vegetables like carrots, beets, parsnips, and potatoes are especially prone to nematode infestation. While feeding on plants, nematodes create large lesions on plant tissue making the plants vulnerable to infection by other disease-causing organisms. When the feeding process damages the plant's root system, it reduces the plant's ability to absorb water and nutrients. Damaged plants result in stunted growth and reduced productivity. Parasitic worms that eat root vegetables are particularly difficult to manage because the above ground symptoms of root damage are relatively nondescript and goes unnoticed until the end of the season, when the roots are harvested. Preventive treatments applied to root vegetable crops are thus highly desirable. (Shah, M. M., & Mahamood, M. (Eds.). (2017). Nematology - Concepts, Diagnosis and Control. InTech, doi: 10.5772 / 66851)

[0012] Fish are another host affected by helminth infections, can also infect fish and have a major impact on public health as well as national economies that rely on sustainable fish production in fisheries.

[0013] There are some chemicals, called anthelmintics, that are currently used to treat helminth infestations. These chemicals are toxic to helminths but can be expensive and have serious side effects On the hosts they are meant to protect. Conventional chemotherapy approved by the World Health Organization for helminth infections in humans involves treatment with benzimidazoles (e g., albendazole, mebendazole) or nicotinic acetylcholine receptor (nAChR) agonists (pyrantel, levamisole). (Keiser and Utzinger, JAMA 299, 1937-48 (2008)). These compounds, however, lack full efficacy against most human helminth parasites. Reports in humans of resistance to both classes of drugs are increasing (e.g., Tanzania, 2010 (Stothard et al., Ann Trop Med Parasitol 103, 357-60 (2009)), potentially rendering ineffective current strategies for controlling helminth infections. A notable challenge in this field is that the infected populations are among the poorest in the world, and economic incentives to develop new drugs are low (~$700,000 / year is spent to develop new drugs against human hclminths(Moran, M. et al. G-fmder Report (2009)), The poverty of infected populations demands that helminth therapeutics be safe, effective, and also inexpensive; highly stable even in the absence of a cold chain; transportable through distribution routes to infected populations; and amenable to culturally acceptable delivery systems,

[0014] Ciystal (Cry) proteins made by the soil bacterium Bacillus thuringiensis (Bt) may be candidate agents that provide safe and effective treatment or control of helminths. Cry proteins have been in use for 60+ years as safe, natural, organic insecticides for control of crop pests, mosquitoes, and black flies. (Roh, J.Y., et al. J MICROBIOL BIOTECHNOL 17, 547- 59 (2007)). They are also effective against nematodes, (Wei, J. Z. et al. PROC NATL ACAD SCI 100, 2760-5 (2003)). Cry proteins are non-toxic to vertebrates and are EP A approved for expression in transgenic food (e g., com, potato). (Mohamadzadeh et al. PNAS 106,4331 -6 (2009); Betz F.S., et al. REGUL TOXICOL PHARMACOL 32, 156-73 (2000)). They are stable and cheap to mass-produce. Activity of Cry proteins against nematode plant parasites and against helminths has been described, e.g, in W02007 / 062064; US2010 / 0024075; WO2010 / 053517; and US2011 / 0263489; see also, e,g,, Li, X.-Q. et al., 2008 Biol. Control 47:97-102, which describes activity of a Cry5B protein truncated at amino acid residue 698 against C. elegans and plant parasitic nematodes.

[0015] Cry proteins can be expressed at high levels and safely delivered to humans or animals using the B. thuringiensis in the inactivated bacteria with cytosolic crystals (IBaCC system).

[0016] However, known Cry proteins are not always effective against all parasitic worms and parasitic worms can develop resistance to Cry proteins over time. Thus, there exists a need to identify new Cry proteins effective against parasitic worms that infect humans and animals.

[0017] SUMMARY

[0018] In one aspect, an anthelmintic composition comprising a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein is CryH13 or CryHl, is provided. In certain exemplary embodiments; the Cry protein is in the form of a native, bioactive crystal, wherein the crystal is produced by a host bacterium that is genetically engineered to express the gene encoding the Cry protein.

[0019] In certain exemplary embodiments, the host bacterium is a non-sporulating bacterium.

[0020] In certain exemplary embodiments, the composition is substantially free Of any bacterial spores.

[0021] In another aspect, an anthelmintic composition comprising a killed or inactivated bacterium that is genetically engineered to express a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein is CryH13 or CryHl, is provided.

[0022] In certain exemplary embodiments, the bacterium is genetically engineered to have a genetic mutation that results in a defect in sporulation such that the Cry protein is trapped in the cytosol of the bacterium.

[0023] In certain exemplary embodiments, the expression of the gene encoding the Cry protein is under control of a non-sporulation-specific promoter. In certain exemplary embodiments, the promoter is a Cry 3 A, GerA, GNAT, or Tad A promoter.

[0024] In certain exemplary embodiments; the bacterium is a Gram-positive bacterium. In certain exemplary embodiments, the bacterium is a species of Bacillus. In certain exemplary embodiments, the bacterium is Bacillus thuringiensis (Bt).

[0025] In certain exemplary embodiments, the bacterium has a genetic mutation, wherein the genetic mutation is a deletion or inactivation of one or mote genes resulting in a defect of sporulation, and wherein the one or more genes resulting in a defect in sporulation is selected from the group consisting of kinA, kinB, spoOA, spoOB, spoOE, spoOF, spoO J, spoOM spoIIB, spoil D, spoHE, spoUF, spoIIG, SpoIIL, spoIIM, spoHIA, spoIIIB, spoIIIE, spoIVA, spOlVC, spoTVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, oil, oF, oE, oG, and cK. In certain exemplary embodiments, the genetic mutation is a deletion or inactivation of the spoOA gene.

[0026] In certain exemplary embodiments, the bacterium is a Gram-negative bacterium. In certain exemplary embodiments, the bacterium is E. colt or P.Jluoresccns.

[0027] In certain exemplary embodiments, the composition further comprises a pharmaceutical carrier or excipient. In certain exemplary embodiments, the composition is encapsulated by a pharmaceutical grade capsule in a dry powdered form. In certain exemplary embodiments, the composition is an orally-available composition. In another aspect, a method for producing an anthelmintic composition is provided, the method comprising: (a) growing a non-sporulating form of a host bacterium that is genetically engineered to express a single type of nematicidal crystal (Cry) protein, wherein the single type of nematicidal Cry protein is CryHl or CryH13, wherein the non-sporulating host bacterium produces native, bioactive nematicidal crystals formed from the single type of nematicidal crystal protein, wherein the host bacterium has a genetic mutation such that CryHl or CryHl 3 is trapped in the cytosol of the bacterium, wherein the host bacterium is grown in a growth medium, optionally wherein the non-sporulating host bacterium releases die native, bioactive nematicidal crystals into the growth medium; and (b) isolating and concentrating the native, bioactive nematicidal crystals to form isolated, native, bioactive nematicidal crystals.

[0028] In certain exemplary embodiments, the genetic mutation results in a defect of sporulation.

[0029] In certain exemplary embodiments, the method further comprises formulating the composition in an orally-avallable dosage form. In certain exemplary embodiments, formulating comprises one or both of lyophilizing or spray drying the bacterium, and encapsulating the bacterium in a pharmaceutical-grade capsule.

[0030] In certain exemplary embodiments, the antimicrobial agent is an antimicrobial compound or gamma irradiation. In certain exemplary embodiments, the antimicrobial agent is: (a) a food-grade antibiotic; (b) a beta-lactam antibiotic; or (c) a terpene, iodine or formaldehyde. In certain exemplary embodiments, the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol, and citral, and combinations thereof. In certain exemplary embodiments, the terpene is carvacrol.

[0031] In certain exemplary embodiments, CryH l or CryHl 3. expression Is under control of a non-sporulation specific promoter. In certain exemplary embodiments, the promoter is a Cry3A, GerA, GNAT, or TadA promoter.

[0032] In certain exemplary embodiments, the bacterium is Bacillus sp. In certain exemplary embodiments, the bacterium is Bacillus thurmgiensis (Bt).

[0033] In certain exemplary embodiments, the genetic mutation is a deletion or inactivation of one or more genes resulting in a defect of sporulation, wherein the one or more genes is selected from the group consisting of kinA, khiB, spoOA, spoOB, spoOE, spoOF, spoOJ, spoOM spoIIB, spoIID, spoIIE, spoUF, spoIIG, spoIIL, spoIIM, spoUIA, spoIHB, spoIHE, spoTVA, spolVC, spol VD, spoVG. spoVK, spoVL, spoVM, spoVM, spoVP, spoVQ, spoVID, oH, aF, oE, oG, and uK, In certain exemplary embodiments, the genetic mutation is a deletion or inactivation of the spoOA gene.

[0034] In certain exemplary embodiments, the bacterium is a Gram-negative bacterium. In certain exemplary embodiments, the bacterium is an E. colt or P.fluofescens species.

[0035] In another aspect, a method for producing an anthelmintic composition is provided, the method comprising exposing a non-sporulating bacterium to an antimicrobial agent, thereby killing or inactivating the bacterium, wherein the bacterium is genetically engineered to express CrylHl or CryHl 3, and. wherein the bacterium has a genetic mutation such that CryHl or CryH13 is trapped in the cytosol of the bacterium.

[0036] In certain exemplary embodiments, the genetic mutation results in a defect of sporulation.

[0037] In certain exemplary embodiments, the method further comprises formulating the composition in an orally-ayailable dosage form, In certain exemplary embodiments, formulating comprises one or both of lyophilizing or spray drying the bacterium, and encapsulating the bacterium in a pharmaceutical-grade capsule.

[0038] In certain exemplary embodiments, the antimicrobial agent is an antimicrobial compound or gamma irradiation. In certain exemplary embodiments, the antimicrobial agent is: (a) a food-grade antibiotic; (b) a beta-lactam antibiotic; or (c) a terpene, iodine or formaldehyde. In certain exemplary embodiments, the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol# and citral, and combinations thereof In certain exemplary embodiments, the terpene is carvacrol.

[0039] In certain exemplary embodiments, CryHl or CryHl 3 expression is under control of a non-sporulation specific promoter. In certain exemplary embodiments, the promoter is a Cty3A, GcrA, GNAT, or TadA promoter.

[0040] In certain exemplary embodiments, the bacterium is Bacillus sp. In certain exemplary embodiments, the bacterium is Bacillus thuringiensis (Bt).

[0041] In certain exemplary embodiments, the genetic mutation is a deletion or inactivation of one or more genes resulting in a defect of sporulation, wherein the one or more genes is selected from the group consisting ofkinA, kinB, spoOA, spoOB, spoOE, spoOF, spoOJ, spoOM spoUB, spoIID, spoIIE, spoUF, spoIIG, sppHL, spoIIM, sppIHA, spolllB, spoIHE, spoIVA, spoIVC, spoFVD, spoVG, spoVK, spoVL. spoVM, spoVN, spoVP, spoVQ, spoVID, CTH, CTF, CTE, OG, and cK. In certain exemplary embodiments, the genetic mutation is a deletion or inactivation of the spoOA gene.

[0042] In certain exemplary embodiments, the bacterium is a Gram-negative bacterium. In certain exemplary embodiments, the bacterium is an E. coli or P. fluorescent species.

[0043] In another aspect, a method of controlling a parasitic worm infection of a plant is provided, said method comprising applying an effective amount of a composition described above to the plant.

[0044] In certain exemplary embodiments, the parasitic worm is resistant to one or more other anthelmintic treatments. In certain exemplary embodiments, the parasitic worm is resistant to Cry5B.

[0045] In certain exemplary embodiments, the parasitic worm infecting the human is selected from the group consisting of roundworm, whipworm, hookworm, flatworm, tapeworm, fluke, and pinworm (threadworm).

[0046] In certain exemplary embodiments, the animal treated is selected from the group consisting of cattle, sheep, goat, equine, pig, poultry, dog, and Cat. In certain exemplary embodiments, the parasitic worm infecting the animal is selected from the group consisting of roundworm, hookworm, whipworm, heartworm, lungworm, and a strongyle (cyalhostomin).

[0047] In certain exemplary embodiments, the animal is a sheep and tire parasitic worm is a roundworm. In certain exemplary embodiments, the roundworm is Haemonchus contorius.

[0048] In certain exemplary embodiments, the animal is an equine and the parasitic worm is a strongyle (cyalhostomin).

[0049] In certain exemplary embodiments, the animal is a pig and the parasitic worm is a roundworm. In certain exemplary embodiments, the roundworm is Ascaris spp. In certain exemplary embodiments, the roundworm is Ascaris suum.

[0050] In certain exemplary embodiments, the parasitic worm infecting the human is Ancylostoma spp or Necator spp. In certain exemplary embodiments, the parasitic worm is Ancylostoma eeylanicum or Ancylostoma duodenale. In certain exemplary embodiments, the parasitic worm is Necator americanus.

[0051] In certain exemplary embodiments, the parasitic worm is Ascaris spp. In certain exemplary embodiments, the parasitic worm is Ascaris lumbricoides.

[0052] In certain exemplary embodiments^ the parasitic worm is Thrichuris spp. In certain exemplary embodiments, the parasitic worm is Thrichuris trichiura. In certain exemplary embodiments; the parasitic worn is Strongyloides stercoralis.

[0053] In another aspect, a method of treating a parasitic worm infection in an animal or a human is provided, comprising orally administering an effective amount of a composition described above to an animal or a human infected with a parasitic worm.

[0054] In certain exemplary embodiments, the parasitic worm is resistant to one or more other anthelmintic treatments. In certain exemplary embodiments, the parasitic worm is resistant to Cry5B.

[0055] In certain exemplary embodiments, the parasitic worm infecting die human is selected from the group consisting of roundworm, whipworm, hookworm, flatworm, tapeworm, fluke, and pinworm (threadworm).

[0056] In certain exemplary embodiments, the animal treated is selected from the group consisting of cattle, sheep, goat, equine, pig, poultry, dog, and cat. In certain exemplary embodiments, the parasitic worm infecting tire animal is selected from the group consisting of roundworm, hookworm, whipworm, heartworm, lungworm, and a strongyle (cyathostomin).

[0057] In certain exemplary embodiments, the animal is a sheep and the parasitic worm is a roundworm. In certain exemplary embodiments, the roundworm is Haemon chits contortus.

[0058] In certain exemplary embodiments, the animal is an equine and the parasitic worm is a strongyle (cyathostomin).

[0059] In certain exemplary embodiments, the animal is a pig and the parasitic worm is a roundworm. In certain exemplary embodiments, the roundworm is Ascaris spp. In certain exemplary embodiments, the roundworm is Ascaris sum.

[0060] In certain exemplary embodiments, the parasitic worm infecting the human is Ancylostoma spp or Necator spp. In certain exemplary embodiments, the parasitic worm is Ancylostoma ceylanicum or Ancylastoma duodenale. In certain exemplary embodiments, the parasitic worm is Necator americanus .

[0061] In certain exemplary embodiments; the parasitic worm is Ascaris spp. In certain exemplary embodiments, the parasitic worm is Ascaris lumbrieoides.

[0062] In certain exemplary embodiments, the parasitic worm is Tkrichuris spp. In certain exemplary embodiments, the parasitic worm is Tkrichuris trichiura.

[0063] In certain exemplary embodiments, the parasitic worm is Strongyloides stercoralis.

[0064] In another aspect, an animal feed composition is provided, comprising: (a) abase animal feed, and (b) a Supplement Comprising a composition described above. In certain exemplary embodiments; a method of controlling or preventing a parasitic worm infection in an animal comprising feeding the animal the feed composition is provided.

[0065] In certain exemplary embodiments, a method of promoting gut health or boosting immunity of an animal comprising feeding the animal the feed composition is provided.

[0066] In certain exemplary embodiments, a composition for human or animal waste treatment, comprising a composition described above is provided. In certain exemplary embodiments, a method of waste treatment, comprising contacting waste with the composition for human or animal waste treatment is provided. In certain exemplary embodiments, waste, litter, or bedding is contacted with the composition.

[0067] In another aspect, an orally-available anthelmintic composition is provided comprising CryHl or CryHl 3 protein and one or more additional nematicidal proteins, wherein the CryHl or CryHl 3 protein is in the form of a native, bioactive crystal.

[0068] In certain exemplary embodiments, the one or more additional nematicidal proteins is Cry5B.

[0069] In another aspect, an orally-available composition comprising a killed, non- sporulating bacterium that expresses CryHl or CryHl 3 and one or more additional nematicidal proteins is provided, wherein the bacterium has a genetic mutation Comprising a deletion or inactivation of one or more genes selected from the group consisting of kinA, kinB, spot) A, spoOB, spoOE, spoOF, spoOJ, spoQM, spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spollL, speHM, spoIIIA, spoIIIB, spot HE. spoIVA, spoIVC, SpoIVD, spOVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, cH, oF, oE, cG, and oK that results in a defect in sporulation, and wherein the CryHl or CryHl 3 and the one or more additional nematicidal proteins are trapped in the cytosol of the bacterium.

[0070] In certain exemplary embodiments, the one or more additional nematicidal proteins is Cty5B.

[0071] In another aspect, a method for producing an anthelmintic composition described above is provided, the method Comprising: (a) growing a non-sporulating form of a host bacterium that is genetically engineered to express nematicidal crystal (Cry) proteins, wherein the Cry proteins are CryHl or CryHl 3 and one or more additional Cry proteins, wherein the non-sporulating host bacterium produces native, bioactive nematicidal crystals formed from the Cry proteins, wherein the bacterium has a genetic mutation that prevents sporulation such that CryHl, CryH12 and tire one or more nematicidal proteins are trapped in tiie cytosol of the bacterium, optionally wherein the npn-sporulating host bacterium releases the native, bioactive nematicidal crystals into the growth medium; (b) isolating and concentrating the native, bioactive nematicidal crystals to form isolated, native, bioactive nematicidal crystals; and (c) formulating the isolated, native, bio active nematicidal crystals in an orally-available dosage form.

[0072] In certain exemplary embodiments, the further method comprises: (a) exposing a non- sporulating bacterium to an antimicrobial agent, thereby killing or inactivating the bacterium, wherein the bacterium is genetically engineered to express CtyHl and CryH13 and one or more additional nematicidal proteins, wherein the bacterium has a genetic mutation that prevents sporulation such that CryHl, CryH12 and the one or more nematicidal proteins are trapped in the cytosol of the bacterium; and optionally, (b) formulating the killed or inactivated bacterium in an orally-available dosage form.

[0073] In another aspect, a method of treating a parasitic worm infection in an animal or a human is provided, comprising orally administering an effective amount of a composition described above to the animal or to the human infected with a parasitic worm.

[0074] In another aspect, an animal feed is provided, comprising: (a) a base animal feed; and (b) a supplement comprising a composition described above.

[0075] In certain exemplary embodiments, a method of Controlling or preventing a parasitic worm infection in an animal comprising feeding the animal the animal feed composition is provided.

[0076] In certain exemplary embodiments, a method of promoting gut health or boosting immunity in an animal comprising administering to the animal feed composition is provided.

[0077] In another aspect, an isolated Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1 ; or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

[0078] In another aspect, an isolated Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the Cry protein is encoded by a nucleic acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2; or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

[0079] In certain exemplary embodiments, a method of Controlling a parasitic worm infection of a plant is provided, said method comprising applying an effective amount of the Bacillus thuringiensis nematicidal Cry protein to the plant.

[0080] In certain exemplary embodiments, a method of controlling a parasitic worm infection in a human is provided, said method comprising administering to the human an effective amount of the Bacillus thuringiensis nematicidal Cry protein.

[0081] In certain exemplary embodiments, a method of controlling a parasitic worm infection in an animal is provided, said method comprising administering to the animal an effective amount of the Bacillus thuringiensis nematicidal Cry protein.

[0082] In certain exemplary embodiments, the animal is a domestic animal, a wild animal or a fish.

[0083] In certain exemplary embodiments, a method of producing a Bacillus thuringiensis nematicidal Cry protein described above is provided.

[0084] In certain exemplary embodiments, a method of purifying a Bacillus thuringiensis nematicidal Cry protein described above is provided.

[0085] In certain exemplary embodiments, a composition comprising a B a cillus thuringiensis nematicidal described above is provided, wherein the composition comprises a pharmaceutical excipient or carrier, or wherein the composition comprises carrier material suitable for agricultural application.

[0086] In another aspect, a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1 9 or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

[0087] In certain exemplary embodiments, said nucleotide sequence Is a synthetic sequence that has been designed for expression in a plant. In certain exemplary embodiments, said nucleotide sequence is operably linked to a promoter capable of directing expression of said nucleotide sequence in a plant cell. In certain exemplary embodiments; a vector comprising a recombinant nucleic acid molecule described above is provided.

[0088] In certain exemplary embodiments, a host cell that contains a recombinant nucleic acid described above is provided. In certain exemplary embodiments, the host cell is a bacterium. In certain exemplary embodiments, the host cell is a plant cell.

[0089] In certain exemplary embodiments; a transgenic plant comprising a host cell described above is provided. In certain exemplary embodiments, said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.

[0090] In certain exemplary embodiments, a transgenic seed comprising a nucleic acid molecule described above is provided.

[0091] In another aspect, a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO; 4,

[0092] In certain exemplary embodiments, said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant. In certain exemplary embodiments, said nucleotide sequence is operably linked to a promoter capable of directing expression of said nucleotide sequence in a plant cell.

[0093] In certain exemplary embodiments, a vector comprising a recombinant nucleic acid molecule described above is provided.

[0094] In certain exemplary embodiments, a host cell that contains a recombinant nucleic acid described above is provided. In certain exemplary embodiments, the host cell is a bacterium. In certain exemplary embodiments, the host cell is a plant cell.

[0095] In certain exemplary embodiments, a transgenic plant comprising a host cell described above is provided. In certain exemplary embodiments, said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0096] In certain exemplary embodiments, a transgenic seed comprising a nucleic acid molecule described above is provided. In another aspect, a plant or plant cell having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: I, or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

[0097] In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.

[0098] In certain exemplary embodiments, a composition comprising the Bacillus thuringiensis nematicidal crystal (Cry) protein or the host cell is provided. In certain exemplary embodiments, the host cell is live, inactivated, or killed. In certain exemplary embodiments, said composition is in a form selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.

[0099] In certain exemplary embodiments, a method for protecting a plant frpm a parasitic worm, comprising applying the composition to a plant, seeds, or soil is provided. In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0100] In another aspect, a plant or plant cell having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

[0101] In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0102] In certain exemplary embodiments, a composition comprising the Bacillus thuringiensis nematicidal crystal (Cry) protein or the host cell is provided. In certain exemplary embodiments, the host cell is live, inactivated, or killed. In certain exemplary embodiments, said composition is in a form selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.

[0103] In certain exemplary embodiments, a method for protecting a plant from a parasitic worm, comprising applying the composition to a plant, seeds, or soil is provided. In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers,, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0104] In another aspect, a method for protecting a plant from a parasitic worm, comprising expressing in a plant or a cell thereof a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1, or wherein the C ry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% of is 100% identical to SEQ ID NO: 3.

[0105] In certain exemplary embodiments, the plant is selected frpm the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0106] In certain exemplary embodiments, a composition comprising the Bacillus thuringiensis nematicidal crystal (Cry) protein or the host cell is provided. In certain exemplary embodiments, the host cell is live, inactivated, or killed. In certain exemplary embodiments, said composition is in a form selected from the group consisting of a powder, dust, pellet, granule^ spray, emulsion, colloid, and solution.

[0107] In certain exemplary embodiments, a method for protecting a plant from a parasitic worm, comprising applying the composition to a plant, seeds, or soil is provided. In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers,, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0108] In another aspect, a method for protecting a plant from a parasitic worm, comprising expressing in a plant or a cell thereof a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the nucleic acid molecule has a Sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%. about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

[0109] In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0110] In certain exemplary embodiments, a composition comprising the Bacillus thuringiensis nematicidal crystal (Cry) protein or the host cell is provided. In certain exemplary embodiments, the host cell is live, inactivated, or killed. In certain exemplary embodiments, said composition is in a form selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.

[0111] In certain exemplary embodiments, a method for protecting a plant from a parasitic worm, comprising applying the composition to a plant, seeds, or soil is provided. In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0112] In another aspect, a method for increasing yield in a plant comprising growing in a field a plant of or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1, or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

[0113] In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0114] In certain exemplary embodiments, a composition comprising the Bacillus thuringiensis nematicidal crystal (Cry) protein or the host cell is provided. In certain exemplary embodiments, the host cell is live, inactivated, or killed. In certain exemplary embodiments, said composition is in a form selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution. In certain exemplary embodiments, a method for protecting a plant from a parasitic worm, comprising applying the composition to a plant, seeds, or soil is provided. In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0115] In another aspect, a method for increasing yield in a plant comprising growing in a field a plant of or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein is provided, wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

[0116] In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0117] In certain exemplary embodiments, a composition comprising the Bacillus thuruigiensis nematicidal crystal (Cry) protein or the host cell is provided. In certain exemplary embodiments, the host cell is live, inactivated, or killed. In certain exemplary embodiments, said composition is in a form selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.

[0118] In certain exemplary embodiments, a method for protecting a plant from a parasitic worm, comprising applying the composition to a plant, seeds, or soil is provided. In certain exemplary embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

[0119] BRIEF DESCRIPTION OF THE DRAWINGS

[0120] The foregoing and other features and advantages of the present specification will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The file of this patent contains at least one drawing / photograph executed in color. Copies of this patent with color drawing(s) / photograph(s) will be provided by the Office upon request and payment of the necessary fee.

[0121] FIG. 1 depicts the effect of CryHl and CryH13 IBaCC lysate on the viability of threadworm, Strongyloides stercoralis, adults in vitro.

[0122] FIG. 2A — FIG. 2B depict the effect on the relative motility of adult hookworms, Ancylostoma ceylanicum, when exposed to com oil extracted lysates of IBaCC system comprising vector expressing Cry5B (positive control), empty vector (EVC, negative control), and vector expressing candidate Cry proteins. Effect after 24-hour exposure to Ipg lysate (A) and 48-hdur exposure to 10 pg lysate (B).

[0123] FIG. 3A - FIG. 3C depict SDS-P AGE showing expression of CryHl in IBaCC (A), CryHl 3 in IBaCC (B), and efficacy of CryH13 IBaCC against Caenorhabditis elegans L4 stage (B). EVC = empty vector control; CryH13 = CryH13 IBaCC. MW = molecular weight markers. N2 = wild-type C elegans. bre-5(yel 7) = CrySBa-resistant C. elegans. Cry5Ba = CrySBa IBaCC.

[0124] FIG. 4A - FIG. 4C depict the effect of various doses of CryH13 IBaCC on the in vitro motility of Ancylostoma ceylanicum adult worms (A) and Ascaris suum L4 stage lafvae(B) and the effect of various doses of CryHl IBaCC on the in vitro motility of Ascaris suum L4 stage larvae (C). EVC: empty vector control (negative control).

[0125] FIG. 5A — FIG. 5C depict the in vivo effect of treatment with a single dose of CrySB IBaCC (positive control), CryHl IBaCC, or CryH13 IBaCC on f ceylanicum worm burdens (A),fecal egg count (B) and body weight in infected hamsters. Hamsters were treated water (Group A), Cry5B IBaCC (Group B), CryHl IBaCC (Group C) or CryHl 3 IBaCC (Group D).

[0126] FIG. 6A — FIG. 6B depict the in vivo effect of two oral doses of CryHl IBaCC and. CryHl 3 IBaCC on the intestinal worm burdens or eggs per gram (EPG) of Ascaris suum in infected mice.

[0127] FIG. 7 compares the effects of various doses of Cry5B IBaCC and CryHl IBaCC on the in vitro motility of Ancylostoma ceylanicum adult worms.

[0128] FIG. 8 compares the effects of various doses of CryHl IBaCC lysate and CryH13 IBaCC lysate on the in vitro motility of Trichuris muris adult worms, DETAILED DESCRIPTION

[0129] Disclosed are methods of treating or preventing helminth infection by administering to a subject a preparation of killed or inactive bacteria recombinantly expressing a nematicidal protein (e.g., crystal protein from Bacillus thuringiensis) in the cytosol of the bacterium. Such recombinant bacteria are treated with an anti-microbial agent such that the bacteria are killed before or during administration. In these particular methods, because the bacteria are dead when administered, any bacterium, including non-food grade bacteria, can be administered to a subject to treat a helminth infection.

[0130] Microbes

[0131] In certain embodiments, the bacteria of the specification are non-sporulating bacteria. As used herein, the term “non-sporulating bacterium” includes wild-type bacteria that are incapable of producing spores (e.g., certain Gram-negative bacteria) as well as genetic variants of spore-forming bacteria that have been engineered to be defective in sporulation (e.g., certain Gram-positive bacteria). As used herein, unless the context makes clear otherwise, “a mutation resulting in a defect in sporulation” or “a genetic mutation that results in a defect in sporulation” refers to any genetic mutation that results in a defect in a member of the sporulation pathway and / or any genetic mutation that prevents the formation of viable spores.

[0132] In some embodiments, sporulation-deficient bacteria are advantageous. An example of a sporulation deficient bacterium is a spoOA- Bacillus thuringiensis. Any mutation or combination of mutations that confers sporulation deficiency but that does not substantially affect viability or heterologous gene expression can be used. These mutations include but are not limited to mutations in the following genes: kinA, kinB, spo0A, spo0B, spo0E, spo0F, spoOJ, spoOM spoUB, spoIlD, spoHE, spoITF, spoIIG, spoHL, spoHM, spoHIA, spoIIIB, spolllb, spoIVA, spoIVC, spoIVD, spoVG, spoVK, SpoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. (Silvaggi, J., et al. Unmasking novel sporulation genes in Bacillus subtillus. J Bacteriol. 186, 8089-8095, 2004; Sandman, K_, et al. Genetic Analysis of Bacillus subtilis spo Mutations Generated by Tn917-Medialed Insertional Mutagenesis. Genetics. 117, 603-617, 1987; Malvar and Baum, Tn5401 Disruption of the spoOF Gene, Identified by Direct Chromosomal Sequencing, Results in CrylHIA Overproduction in Bacillus thuringiensis. J Bacteriol. 176, 4750-4753, 1994). Bacteria are particularly applicable to the control of helminths because: 1) recombinant bacteria can cheaply express large amounts of Cry proteins prior to administration into the GI tract of a mammalian subject, and Cry proteins so expressed, independent of any Cry proteins that may be secreted by bacteria in the GI tract, have been shown to have a significant impact on helminths; 2) studies using purified Cry protein to treat hookworms, whipworms, and Heligmosomoides bakeri, all in infected rodents, demonstrate that helminths in the mammalian GI tract can ingest and be kllled / intoxicated by Cry proteins-; 3) recombinant bacteria expressing a therapeutic protein, in which the protein is not purified, are cheaper to produce since no purified protein is needed; and 4) recombinant bacteria delivering helminth curing proteins (e,g., CrySB) are more effective that purified proteins (e.g., CrySB) at the same bio-active protein dose (e.g., total CrySB) in curing infections.

[0133] Microbes of the disclosed compositions and methods include killed and inactivated forms of Bacillus sp., including Bacillus subtilis (e.g., Bacillus subtilis natto, and Bacillus subtilis PY79), B. cereus, (e.g., B, cereus var. Toyoi (Toyocerin), B. cercus var. toyoii), B. toyonensis, B. claitsii, B. pumilus and Bacillus thuringiensis . Bacillus subtilis has been extensively characterized as a safely ingested food additive in humans (see Example 14, infra, references 15-27). In certain exemplary embodiments, killed and inactive forms of Bacillus thuringiensis are used.

[0134] Other usefill bacteria include but are not limited to non-sporulating variants of Lactococcus sp., Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Clostridium sp., Sporolactobacilliis sp, Sporosarcina sp., Brevibacillus sp, Leuconostoc sp., Pedicoccus sp., Enterococcus sp. and Escherichia sp. Lactococcus sp. includes but is not limited to L. lactis. Lactobacillus sp. includes but is not limited to L. casei, L. paracasei, L. acidophilus, L. bulgaricus, L. delbrueeHi suhsp. bulgaricus, L. helveticus, L. plantarum, L. salivarius, L. reuteri, L. gasseri, and Z. animalis. Bifidobacterium sp. includes but is not limited to B. animalis, B. bifidum, B. breve, B. infantis, and 5. longum. Streptococcus sp. includes but is not limited to X thermophilus. Clostridium sp. includes but is not limited to Clostridium butyrician. Sporolactobacillus sp. includes but is not limited to Sporo lactobacillus vineae. Sporosarcina sp. includes but is not limited to Sporosarcina pasteurii. Brevibacillus sp. includes but is not limited to Brevibd.cillus laterosporus.

[0135] Still other usefill bacteria useful in connection with the specification include killed and inactivated forms of Gram-negative bacteria. In certain exemplary embodiments, the Gram-negative bacteria include E. coll species (e.g., NISSLE 1917) and Pseudomonas specie? (c.g., Pseudomonas fluor escens). Exemplary Cry-expressing Gram-negative bacteria which can be killed or inactivated by the methods of the specification include the Cryexpfessing E. colt strain of Ge et al. (“Hyperexpression of a Bacillus thuringiensis delta- endotoxin-encpding gene in Escherichia eoli: properties of the product,” Gene, 93 : 49-54 (1990)) and the P. fluor escens strain of Peng et al. (“A Delta-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity,” App, Microbiol Biotech., (2003), 63:300-306).

[0136] Nematicidal Proteins

[0137] As used herein, unless the context makes clear otherwise, “nematicidal protein” refers to any protein that has toxic activity against nematodes or helminths. Exemplary nematicidal proteins include CryHl and CryH13 protein in the bacterium for delivery into a helminth (e.g-., roundworm)-infected vertebrate animal gastrointestinal tract via oral dosing (gavage, drinking, eating, pill, capsule, powder, etc.). The Cry proteins are expressed in the cytosol of the bacterium, allowing access to the anthelmintic protein after the bacterium lyses or opens up either due to digestion within the gastrointestinal tract ingestion and digestion of bacteria by the parasitic helminths (e.g., roundworms such as hookworms, whipworms, Ascaris, Strongylcides, veterinary parasitic roundworms of the intestine), etc.

[0138] In certain embodiments, a bacterium as provided herein may be introduced that expresses an individual Giy protein or that simultaneously expresses multiple Cry proteins. In some embodiments, multiple bacteria may be introduced, each of which expresses either a different individual Cry protein or simultaneously expresses multiple Cry proteins. In these and related embodiments, it is contemplated that the GI tract may be seeded with bacteria that express either one Cry protein or multiple Cry proteins at the same time. For example, due to the laek of cross-resistance between Cry5B-resistant roundworms and Cry21 A-resistant roundworms, simultaneous administration of Cry5B and Cry21A in the gastrointestinal tract may inhibit the development of parasite resistance to the combination therapy.

[0139] In the long run, removing antibiotic selection capability (e.g., genetic selection markers) from the plasmids that are used to introduce heterologous Cry protein-encoding sequences, as well as using bacterial strains that are unable to replicate outside the vertebrate host, may be desirable in order to environmentally contain the genetically modified bacteria. For example, LAB (lactic acid bacteria) have been engineered to be autotrophic in thymidine or thymine synthesis such that they can only grow in the vertebrate intestine where thymidine or thymine is present and not in the environment where thymidine or thymine is not present. See, e.g., Steidler L, et al. “Biological containment of genetically modified Laetococcus lactis for intestinal delivery of human interleukin 10.” Nat Biotechnol 21 : 785-789 (2003).

[0140] Cry-transformed bacteria such as Bacilli or LAB may be cultured and expression of intracellular, membrane-anchored, or secreted Cry protein by such bacteria may be confirmed using antibodies raised against each Cry protein and standard Western blotting or ELISA techniques.

[0141] To assess the bioactivity of all constructs, recombinant expressing Cry protein (full length, truncated, or variants) may be fed to the free-living nematode, C. elegans. Cry protein toxicity on C. elegans using LC50, brood-size, developmental inhibition assays on solid media and in liquid wells may then be quantitated. C. elegans can access the Cry proteins either via protein secreted onto the solid media / into the liquid well or by their ability to grinds open and digest bacteria. Confirmation that the recombinant bacteria are making bioactive Cry proteins may be obtained. Furthermore, the bioactivity (eg., LCso in pg / mL) may be quantified and the constructs giving the highest activity determined.

[0142] A comparative genomics analysis of public database revealed novel Bacillus thuringiensis Cry proteins, CryHl and Cryll 13. the cloning, expression and nematicidal activity of these two novel proteins is described in this disclosure.

[0143] Truncations, Variants, and Sub-variants

[0144] The crystal proteins may be truncated to enhance their effectiveness. The usefulness Of Bt toxins (e.g., crystal proteins) for controlling helminths may be limited by the protein size that helminths can ingest, Some parasitic roundworms poorly ingest proteins larger than about 40 kD. Thus, the effectiveness of any particular Bt toxin may be limited by size exclusion of proteins that helminths take in and so should be small enough to be readily absorbed by the helminth gut while retaining toxic activity. A truncated toxin may be easier to express in bacteria. Producing a truncated toxin also alleviates the requirement that the target helminth has the proper proteases present to correctly process full Length protoxin (which is inactive) to a truncated, active toxin form. Thus, a truncated toxin is immediately available for intoxication independent of whether the proper protease processing enzymes are present in the helminth target. Truncated toxin may also express at a higher level in microbes because truncated toxins are soluble and less likely to form insoluble inclusions in the cell expressing them, which could be toxic to the cell or which could make the toxin fold incorrectly. Accordingly, it is desirable to produce truncated Bt toxin fragments (c.g., crystal protein fragments). Moreover, fragments of certain Bt toxins have been tested and shown to retain toxic activity and have improved biological properties. By "truncated / * when referring to a Bt toxin protein (crystal protein) is meant a Bt toxin protein that is not full-length but retains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the toxic activity of a corresponding full-length Bt toxin protein.

[0145] "Variants" or “subvariants” of Cry proteins include polypeptides with one or more substitutions, e.g., no more than 20 substitutions, alternatively no more than 10 substitutions, or substitutions at 10% or fewer of the residues, relative to a corresponding wild-type polypeptide or truncated version thereof. The variant, subvariant, or truncated polypeptide has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity, e.g„ toxic activity, of the corresponding wild-type polypeptide or truncated version. Conservative substitutions include substitutions within the following groups: glycine, alanine, threonine, valine, iso leucine, leucine; asp artic acid, glutamic acid, asparagine, glutamine; serine, cysteine; lysine, arginine; aspartic acid, glutamic acid; serine, threonine; asparagine, glutamine; phenylalanine, tyrosine,

[0146] The crystal proteins may be full length, truncated, variants, or subvariants. The truncated crystal protein may include any truncation of the N- and C-termini that still retains toxin activity. The truncated form is not full-length but retains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the toxic activity of a corresponding full- length Bt toxin protein. For example, the truncated portion may be truncated between the end of conserved block 5 mid the C-terminus of the full-length protein.

[0147] Nucleic acid molecules encoding amino acid sequence variants, truncated versions, or both, of a Cry protein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by, for example, oligonucleotide- mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of protein. Moreover, the specification includes synthetic nucleic acid molecules where nucleotides are modified to include codons preferred in a particular organism, remove codons rarely tised in a particular organism, or remove sequences that may inhibit transcription or RNA processing and the like.

[0148] CryHl and CryH13 In some embodiments, the CryHl protein comprises an amino acid identical to the sequence listed in SEQ ID NO: 1. In some embodiments, the CryHl protein comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 1. In some embodiments, the CryH13 protein comprises an amino acid identical to the sequence listed in SEQ ID NO: 3. In some embodiments, the CryHl protein comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 3.

[0149] In some embodiments, the CryHl 3 protein is encoded by a gene comprising a nucleic acid sequence identical to the sequence listed in SEQ ID NO: 2. In some embodiments, the Cry II 13 protein is encoded by a gene comprising a nucleic acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 2. In some embodiments, the CryHl 3 protein is encoded by a gene comprising a nucleic acid sequence identical to the sequence listed in SEQ ID NO: 4. In some embodiments, the CryH13 protein is encoded by a gene comprising a nucleic acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 4.

[0150] Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences or of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides or amino acid residues at corresponding nucleotide or amino acid positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e,, % homolpgy=number of identical positions / total number of positions* 100), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0151] The comparison of s equences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). An exemplary, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sei. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0152] In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e,, a global alignment). An exemplary, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CAB IOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software paekage. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0153] Comparative Genomics Tools

[0154] Although Bacillus thuringiensis is the major source of biopesticides, the number of bacterial species synthesizing proteins with biopesticidal potential is much higher. The Bacterial Pesticidal Protein Resource Center (BPPRC) offers a database of sequences for the control of pests, grouped in structural classes. As described by Diaz-Valerio arid colleagues, IDOPS, is a tool that detects novel biopesticidal sequences and analyzes them within their genetic environment (Diaz-Valerio et al. Front Microbiol. 2021 Jun 28;12:664476). The backbone of the IDOPS detection unit is a curated collection of high-quality hidden Markov models that is in accordance with the BPPRC nomenclature. IDOPS is positively benchmarked with Bt Toxin Digger and Cry Processor. A scan of the UniProtKB database using the IDOPS models returns an abundance of new pesticidal protein candidates distributed across all of the s tructural groups. Gene expression depends on the genomic environment, therefore, IDOPS provides a comparative genomics module to investigate the genetic regions surrounding pesticidal genes. This feature enables the investigation of accessory elements and evolutionary traits relevant for optimal toxin expression and functional diversification. IDOPS contributes and expands the current arsenal of biopesticides.

[0155] Anthelmintic Experiments

[0156] Once heterologous Cry protein expression and bioactivity are confirmed in a desired bacterium, the modified bacteria may be used for curative-type and preventative-type anthelmintic experiments.

[0157] Antibody production: Antibodies against recombinant Cry proteins (e.g., CryHl, CryH13, Ciy5B, Cry21A, Cryl4A, Cry 14 Ab, Cryl3A, and Cry6A, full Length and truncated proteins) may be produced and purified according to standard methodologies (e.g., Cun-ent Protocols in Immunology , John Wiley & Sons, New York, N.Y.(2009).

[0158] Bioacliviiv tests: To assess the bioactivity of all constructs, recombinant bacilli or other bacteria expressing heterologous Cry proteins are fed to the free-living nematode, C. elegans. C. elegans can access the Cry proteins either via protein secreted onto the solid media / into the liquid well, by protein naturally released as bacteria break open, or by their ability to grind and digest bacteria to open the bacterial cells.

[0159] Rodent and parasite tests: Three intestinal parasitic nematodes — Heligmosomoides bakeri, H. poly gyrus (small intestine nematode parasites) in mice, Ascaris suum in mice, Trichuris muns (whipworm) in mice, and A. ceylanicum (hookworm) in hamsters are tested. The tests address: 1) wherein the GI tract do heterologous Cry-expressing bacteria reside and for how long; and 2) how do these bacteria affect the acquisition and progression of intestinal nematode parasites .

[0160] Parasite tests: Naive (uninfected) mice are gavaged with the best heterologous Cryprotein expressing recombinant bacterial strain(s) based on expression and bioactivity.

[0161] Protect against progression test: Mice are infected with H. bakeri. Two weeks later, infected mice are treated with heterologous Cry-protein expressing or control bacteria, respectively. Intestinal worm burdens and fecal egg counts are used to determine if the recombinant bacteria provide anthelmintic therapy in mice with pre-existing nematode infections.

[0162] Exemplary Parasites

[0163] The disclosed methods relate to the control of parasitic worms, e.g., nematodes and platyhelminths, using crystal proteins from Bacillus and their derivatives. Parasitic worms within the scope of the specification include, but are not limited to, those in Class Adenophorea, e.g,, Order Mononchida, Family Plectidae, and Order Stichosomida, Family Mermithidae and Tetradonematidae; Class Secementea, e.g., Order Rhabditida, Family Carabonematidae, Cephalobidae, ChambersielUdae, Heterorhabditidae, Oxyuridae, Panagrolaimidae, Rhabditidae, Steinemematidae, Syrphonematidae, Syrphonematidae, or Thelastomatidae; Order Spirurida, Family Filariidae, Onchocercidae, Physalopteridae, Syngamidae, Spiruridae, Subuluridae, or Thelaziidae; Order Diplogasterida, Family DiplogaSteridae; and Order Tylenchida, Family Allantonematidae, Aphelenchidae, Aphelenchoididae, Entaphelenchidae, Fergusobiidae, Phaenopsitylenchidae, Sphaerulariidae, Anguinidae, Dolichodoridae, Belonolaimidae, Pratylenchidae, Hoplolamidae, Heteroderidae, Criconcmatidac, Tylenchulidae or Tylenehidae. In one embodiment, the parasite is from Class Secementea, Order Ascaridida, Family Ascarididae; Class Adenophorea, Order Trichurida, Family Trichuridae; Class Secementea, Order Strongylida, Family Ancylostomatidae (ancylostomidae) or Trichostrongylidae; or Class Secementea, Order Spirurida, Family Dracunculidae, Filariidae, or Onchocercidae.

[0164] The parasite may be a helminth. Helminths within the scope of the specification include, but are not limited to, those from Phylum Annelida, Class Polychaetae, Class Myzostomida, Class Clitellata, Subclass Hirudinea, Order Cinathobdellidac, Order Rhynchpbdellidae; Phylum Platyhehninth.es (Flatworms), Class Turbellaria, Class Monogenea, Order Monopisthocotylea, Order Polyopisthocotylca, Class Trcmatoda, Subclass Aspidogasrea, Subclass Digenea; Super Order Anepitheliocystida, Order Strigeatida, Family Schistosomatidae, Subfamily Schistosomatinac, Genus Schistosoma, Order Echinostomatida, Family Fasciolidae, Family Paramphistomatidae, Family Echinostomatidae; Super Order Epitheliocystida, Order Plagiorchiida, Family Dicrocoeliidae, Family Troglotrematidae, Order Opisthorchiida, Family Heterophyidae, Family Opisthorchiidae, Class Cestoda, Subclass Cestodaria, Subclass Eucestoda, Order Pseudophyllidea, Family Diphyllobothriidae, Order Cyclophyllidea, Family Taeniidac, Family Hymenolepididae, Family Dilepididae, Family Mesocestoididae, Order Tetraphy llidea, Order Proteocephalata, or Order Spatheobothridea. For example, Cry proteins with the scope of the specification may be employed to prevent, inhibit, or treat Roundworm, Whipworm, Hookworm, Schistosome, or Trematodes.

[0165] The parasite may also be gastrointestinal tract parasitic roundwonns / nematodes. The gastrointestinal tract parasitic roundwonns / nematodes may include but are not limited to the following species: Haemonockus, Cooperia, Ostertagia, Trichostrongylus, Teladorsagia, Nematodirus, Aneylostoma, Cyqthostominea / Cyathpstomjn / Cyaihostome, Strongylus, Parascaris, Ascaris, Trichuris, Oesophagostornum / Oesophagustomum, Trichiuris, Bunosiomum, Oxyuris, Chabertia, Habronema, Draschia, Triodontophorus, Toxocara, Toxascaris, and Uncinaria. Haemonochus species includes but is not limited to Haemonchu.s contortus ahd Haemonchus plaeei, Cooperia species includes but is not limited to Cooperia oncopkora, Cooperia pectinata, and Cooperia curticei. Ostertagia species includes but is not limited, to Ostertagia ostertagi, Ostertagia (Teladorsagia) eircumcincta, and Ostertagia trijurcate. Trichostrongylus species includes but is not limited to Trichostrongylus axei, Trichostrongylus colubyiformis, and T. eircumcincta. Teladorsagia species includes but is not limited to Teladorsagia (Ostertagia) eircumcincta, Nematodirus species includes but is not limited to Nematodirus spathiger. Ancylostoma species includes but is not limited to Ancylostoma caninum, Ancylostoma braziliense, and Ancylostoma tuhaeformc.

[0166] Cyathostominea / Cyathostomin / Cyathostome nematodes are also included. Strongylus species (small and large) includes but is not limited to Strongylus vulgaris, Strongylus equinus, and Strongylus edentatus. Parascaris species includes but is not limited to Parascaris equorum. Strongyloides species includes but is not limited to Strongyloides westeri. Ascaris species includes but is not limited to Ascaris sunm. Trie hurts species includes but is not limited to Trichuris glpbulosq, Trichuris suis, Trichuris campanula, and Trichuris vtdpis.

[0167] Oesophagostomum / Oesophagustomum species includes but is not limited to Oesophagustomum dentatum, Oesophagustomum quadrispinulatum, Oesophagpstomum columbianum, and Oesophagostomum venulosum. Trichiuris species includes but is not limited, to Trichiuris ovis. Bunostomnm species includes but is not limited to Bunostomum trigonocephalum. Oxyuris species includes but is not limited to Oxyuris equi (pin worms). Chabertia species includes but is not limited to Chabertia ovina. Habronema species includes but is not limited to Habronema microstoma and Habronema muscae. Draschia species includes but is not limited to Draschia megastoma. Triodontophorus species includes but is not limited to Triodontophorus minor and Triodontophorus serrdtes. Toxocara species includes but is not limited to Toxocara cants and Toxocara cati. Toxascaris species includes but is not limited to Toxascaris leonine. Uneinaria species includes but is not limited to Uneinaria stenocephala. Human parasitic roundworms of the gastrointestinal tract include but are not limited to the hookworms Ancylostoma dupdenale and Neeator americanus, the whipworm Trichuris trichium, the roundworm Ascpris lumbricoides, the threadworm Strongyloides stercoralis, and the pinworm Enterobius vermiculari.

[0168] Plant parasitic nematodes

[0169] In certain embodiments, the parasite is a plant parasitic nematode. Plant-parasitic nematodes (PPNs) are economically important pests for numerous agricultural and forestry crops, representing a significant constraint on global food security and forestry health. Most plant parasitic nematodes are soilbome root pathogens, but a few species feed primarily upon shoot tissues. Examples of PPNs include, but are not limited to, Xiphinema (dagger nematode), Helicotylenchus spp., Rotylenchuhis reniformis (the reniform (kidney-shaped) nematode), Tylenchulus semjpenetrans (the citrus nematode), Meloidogyne spp. (root knot nematodes) , Pratylenchus (lesion nematode), Radopholus (burrowing nematode), Hirschmanniella (rice root nematode), Globodera spp., Globodera rostoehiensis and Globodera pallida (potato cyst nematodes), Heterodera schachtii (beet cySt nematode), Heterodera avenae (cereal cyst nematode), Heterodera spp. (cyst nematode), Heterodera glycines (soybean cyst nematode), Pratylenchus spp. (root lesion nematodes), Dityletichus spp. (stem and bulb nematodes), Bursaphelenchus xylophilus (pine wood nematode; a nematode that infects pine trees), Anguina spp. (seed gall nematodes), Aphelenchoides spp. (foliar nematodes) and the like, which are some examples of PPNs that are ranked at the top in the list of the most economically and scientifically important species. See Lambert, K. and Bekal S. Introduction to Plant-Parasitic Nematodes. The Plant Health Instructor, Vol. 2 (2002). DOI: 10.1094 / PHI-I-2002- 1218-01, incorporated herein by reference in its entirety.

[0170] Parasitic Nematodes of Fish and Marine Mammals

[0171] Helminth infections have a major impact on the fish industry due to the pathogenic effects of several species affecting productivity, as well as because of the zoonotic potential of many species . Nematodes can affect a wide range of species including eels, sturgeons, bream, pike, cod, trout, carp, bass, rays, skate, bleaks, arapaima, perches, piranha, lingcod, viviparous bleny. (European eelpout) and aquarium species such as guppy (millionfish) and discus fish. Nematodes can also infect marine mammals such as Cetacea (whales, dolphins and porpoises) and Phocidae (seals). Humans acquire fish-borne helminth zoonoses via the consumption of raw or undercooked fish containing infective parasite larvae, The most important fish-borne helminth zoonoses are caused by trematodes of the families Opisthorchiidae and Heterophyidae, nematodes of the families Ahisakidae, Gnathostomatidae and Capillariidae and cestodes of the family Diphyllobothriidae. Nematodes Anisalds spp. and Pseudoterranova spp. of the family Anisakidae, Gnathostoma spp. and Capillaria spp. are the most commonly reported fish-borne nematodes in humans globally. Fish cestodes of the genera Dtphyllobothrtum, Dibothriocephalus and Adenocephalus cause intestinal infections ih humans in Europe, North and South America and Asia, Finally, some acanthocephalan species of the genera Bolbosoma, Corynpsoma and Acanthocephaius may induce abdominal pain, ileus, ulceration and bleeding. Both in wild and cultured fish, parasites may also have an impact on the function, growth, reproduction and survival of the hosts. In cultured fish, however, parasitic diseases are generally more severe, and may cause important economic losses due to stock mortality, declined productivity and reduced marketability. A wide variety of parasite species are known to cause morbidity and mortality in fish. For instance, a number of digenean trematodes, belonging to the families Aporocotylidae, Bolbophoridae, Clinostomidae, Diplostomidae and Heterophyidae, can cause loss of vision, necrosis, hemorrhage, obstructed blood flow and mortality. Monogenean ectoparasites (e.g., of the families Capsalidae, Diplectanidae, Anoplo-discidae) commonly infect external surfaces of fish (gills, fins, skin, etc.) and cause irritation, reduced growth, respiratory distress, gills / skin / tissue damage and mortality (Reed et al., 2009). Furthermore, nematodes, e.g., of the genera Cap i Hana, Camallanus, Rhabdochona are of economic importance, due to their pathological effects in fish, as well as their impact on fish product marketability due to consumer aversion caused by the presence of macroscopic parasites in food. Cestodes are another great concern for global fish populations. For instance, Schyzocoty leacheilognathi of the family Bothriocephaliduf, is known to damage the intestinal tract and cause significant mortality. Acanthocephalans, e.g., of the families Echinorhynchidae, Nepechinorhynchidae, Pomphprhynchidae, may also cause irreversible intestinal damage and impaired nutrient absorption.

[0172] Anti-microbial agents

[0173] In the disclosed methods, the recombinant bacteria expressing a crystal protein can be treated with an anti-microbial agent. Anti-microbial agents can be used on the recombinant bacteria before administration to a subject, or concomitant with administration to the subject. An advantage of killing the recombinant bacteria is that otherwise non-food safe bacteria can be used in the disclosed methods. Such non-food safe bacteria, such as Bacillus thuringiensis which is closely related to Bacillus cereus that can cause food poisoning, express very high levels of Cry proteins such as Cry5B and improve the efficacy of the protein when coadministered versus when the protein is administered in a pure form without the bacterium

[0174] Suitable anti-microbial agents are those that: (I) sufficiently kill the recombinant bacteria; and (2) do not substantially affect the activity and / or levels of the crystal protein. Examples of Suitable anti-microbial agents include, but are not limited to, antib iotics (such aS a beta-lactam antibiotic), bactericidal agents, iodine, terpenes, formaldehyde, and irradiation. Examples of terpenes include, but are not limited to, thymol, eugenol, geraniol, carvacrol, and citral, or a combination thereof. Carvacrol is a particularly exemplary anti-microbial agent.

[0175] Additional therapeutic agents

[0176] In certain embodiments the crystal protein-recombinant bacteria are administered in combination with at least one additional therapeutic agent. This additional therapeutic agent can be, for example, a bacterium expressing or capable of expressing, a crystal protein, a small molecule, or a polypeptide (including antibodies and fragments thereof). In a further embodiment, the additional therapeutic is a nicotinic acetylcholine receptor agonist. In certain embodiments, the additional therapeutic agent is administered simultaneously with recombinant bacteria. In certain embodiments the additional therapeutic agent is administered sequentially (and in either order) with the recombinant bacterium. In certain embodiments, the nicotinic acetylcholine receptor agonist is from the levamisole family of nicotinic acetylcholine receptor agonists. In certain embodiments, the nicotinic acetylcholine receptor agonist is levamisole. In certain embodiments, the levamisole is administered in an amount of about 0.1 mg-'kg to about 5.0 mg / kg. In certain embodiments the nicotinic acetylcholine receptor agonist is pyrantel or tribendimidine. In certain embodiments, the pyrantel is administered in an amount of about 1.0 mg / kg to about 15.0 mg / kg, In certain embodiments, the tribendimidine is administered in an amount of about 0.25 mg / kg to about 10 mg / kg.

[0177] Administration, dosage forms, pharmaceutical compositions

[0178] The present specification describes compositions and methods for administration of killed or inactivated bacterial cells to the gastrointestinal tract of a subject. The methods include administering the bacteria in food or as a food supplement. Oral administration is typically in an aqueous suspension, emulsion, powder Or solid. The composition may be formulated into a food or added to food by the user prior to consumption. Administration to the gastrointestinal tract may also be in the form of an anal suppository (e.g.. in a gel or semisolid formulation). All such formulations are made using standard methodologies.

[0179] The method is typically practiced on any animal where inhibiting pathogen or parasites is desired. In certain embodiment, the animal is a human. However, the animal can be any livestock or zoological specimen where such inhibition of parasites / pathogens provides economic and health benefits. Any animal can benefit by the claimed methods, including birds, reptiles, mammals such as horses, cows, sheep, goats, pigs, and the like domesticated animals, or any of a variety of animals of zoological interest. Other purposes are readily apparent to one skilled in the arts of nutrient absorption, feed utilization and bioavailability.

[0180] The present specification further contemplates a therapeutic system for treating, reducing and / or controlling parasitic infections. Typically, the system is in the form of a package containing a therapeutic composition of the present specification , or in combination with packaging material. The packaging material includes a label or instructions for use of the components of the package. The instructions indicate the contemplated use of the packaged component as described herein for the methods or compositions of the specification . By way of example, and not of limitation, a system can comprise one or more unit dosages of a therapeutic composition according to the present specification . Alternatively, the system can alternately contain bulk quantities of a therapeutic composition. The label contains instructions for using the therapeutic composition in either unit dose or in bulk forms as appropriate and may also include information regarding storage of the composition, disease indications, dosages, routes and modes of administration and the like information.

[0181] Furthermore, depending upon the particular contemplated use, the system may optionally contain either combined or in separate packages one or more of the following components: bifidogenic oligosaccharides, flavorings, carriers, and the like components. One particularly exemplary embodiment comprises unit dose packages of bacterial cells for use in combination with a conventional liquid product, together with instructions for combining the bacteria with the formula for use in a therapeutic method.

[0182] Different dosage regimens may be used in the disclosed methods. In some embodiments, a daily dosage is administered once, twice, three times, or four times a day for one, two, three, four, five, six, seven, eight, nine, or ten days, hi some embodiments, a once- or twice-daily dosage is administered every other day.

[0183] Administration of the compositions containing the active ingredients effective in inhibiting parasite growth in the intestine and in feces generally consist of one to ten unit dosages of 10 mg to 10 g per dosage of the composition for one day up to one month for a human of approximately 100 kg body weight. Unit dosages are generally given once every twelve hours and up to once every four hours. In certain exemplary embodiments, two to four dosages of the composition per day, each comprising about 0.1 g to 50 g per dosage, for one to seven days are sufficient to achieve the desired result,

[0184] An exemplary method involves the administration into the digestive tract of from Ix102to 1 x1010of bacterium per day, in some embodiments from 1 Ix103to Ix106, in other embodiments from Ix106to Ix109, and in some embodiments from 5xl08to Ix109bacterium per day. Exemplary dosages range from about Ix103to Ix106bacterium per day, or alternatively range from about Ix106to Ix109bacterium per day.

[0185] In various specific embodiments, an effective dose of a composition of the present disclosure can be in a range of from 1.0 gm to 15.0 gm for an adult patient, or between about 2.0 gm and about 10.0 gm of the composition. Effective doses can be administered to a subject at any suitable frequency, e.g., at least once a week, e.g., once a day. Pediatric dosages may be in the range of 15% to 90% of adult dosages .

[0186] In other embodiments, a constant dosage of the composition can be administered over time, for example about 2 gm to about 4 gm per day, up to about 6 g to about 10 g per day, depending on the severity of the physiological condition. Once the infection has been effectively ameliorated, the subject can in many instances decrease the dosage to about 2 gm to about 4 gm per day for maintenance purposes. The desired dose may be presented in multiple (e.g., two, three, four, five, six, or more) sub-doses administered at appropriate intervals throughout the day,

[0187] The pharmaceutical compositions comprising the crystal protein-expressing recombinant bacteria can be administered via any of the accepted modes of administration or agents known in the art. However, oral administration is typically used because this route of delivery delivers the recombinant bacteria to the G1 tract. The dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, and can be in unit dosage forms suitable for simple administration of precise dosages. One exemplary embodiment of the dose form is a capsule containing the composition of the disclosure including the bacterial species in a dried form, blended with pharmaceutical carrier. The capsule for such dose form can be of any suitable type, e.g., a gelatin capsule of a conventional variety.

[0188] The physiologically compatible carrier medium with which the bacterial species are employed, can be of any simple type, e.g., a pharmaceutically acceptable carrier such as fructo-ol igo-saccharidc (FOS) medium, or other soluble fiber, sugar, nutrient or base material for the composition, with which the bacterial species can be formulated, e.g,, in an orally administrable form. Other carrier media include mannitol, inulin (a polysaccharide), polydextrose, arabinogalactan, polyolslactulose, lactitol, etc. A wide variety of materials can be used as carrier material in the practice of the present disclosure, as will be apparent to those of ordinary skill in the art, based on the description herein.

[0189] The carrier medium, when present, can be blended with the bacterial species in any suitable amounts,, such as an amount of from 5% to 95% by weight of carrier medium, based on the total weight Of the bacterial species and the carrier medium, in various embodiments. In other embodiments, the amount of carrier medium may be in a range having a lower limit of any of 5%, 10%, 12%, 15%, 20%, 25%, 28%, 30%, 40%, 50%, 60%. 70% or 75%, and an upper limit, higher than the lower limit, of any of 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 95%. The amount of carrier medium in a specific embodiment may be determined based on considerations of the specific dose form, relative amounts of the bacterial species, the total weight of the composition including the carrier medium and the bacterial species, and the physical and chemical properties of the carrier medium, and other factors, as known to those of ordinary skill in the probiotic formulation art.

[0190] In certain embodiments, the bacterial cells are formulated in a composition that protects the cells and / or Cry proteins from the acid environment of the stomach. Accordingly, the specification includes a composition containing a bacterium and a pharmaceutically acceptable acid-resistant ("enteric") carrier. By acid-resistant is meant that the carrier or coating does not dissolve in an acidic environment. An acidic environment is characterized by a pH of less than 7. The acid-resistant carrier is resistant to acids at pH less than about 4.0, in certain exemplary embodiments, the carrier does not dissolve in pH 2-3. In other exemplary embodiments, it does not dissolve in pH of less than 2. To protect bacterial cells from stomach acids, the cells are coated or encapsulated with the acid-resistant carrier.

[0191] In certain embodiments, the coating is pH sensitive. For example, the coating may dissolve after the pH is greater than 4.0. For example, the coating dissolves in a neutral environment as is encountered in the small intestine and does not dissolve in an acidic environment as is encountered in the stomach. Alternatively, the enteric coating dissolves when exposed to specific metabolic event such as an encounter with a digestive enzyme that is found in the small intestine. For example, the coating is digested by a pancreatic enzyme such as: trypsin, chymotrypsin, or a pancreatic lipase. The formulation is hydrated in the small intestine. Digestion or dissolution of the coating allows liberation of bacterial cells, e.g., Bacillus cells, into the intestine.

[0192] In other embodiments, bacterial cells are stabilized in a gel or paste such as an anhydrous carbohydrate paste. In alternate formulations, the cells are lyophilized and / or suspended in a gel or paste. Enteric coating materials are known in the art, e.g., malic acid- propane 1,2-diol. Cellulose derivatives, e.g., cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate (HPMCP), are also useful in enteric acid-resistant coatings. Other suitable enteric coatings include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate and anionic polymers of methacrylic acid and methyl methacrylate. Another suitable enteric coating is a water emulsion of ethylacrylate methylacrylic acid copolymer, or hydroxypropyl methyl cellulose acetate succinate (HPMAS). (See, e.g., U.S. Pat. No. 5,591,433). An enteric coating is designed to resist solution in the stomach and to dissolve in the neutral or alkaline intestinal fluid.

[0193] In certain embodiments, the bacterial cells are formed into dry powders. Suitable drying methods include a natural drying, a forced-air drying, a spray drying, freeze drying, and the like. Of those, a spray drying, drum drying or a forced-air drying are used in exemplary embodiments. A protective agent such as skim milk, sodium glutamate, and saccharides may be used in a time of drying. Saccharides, glucose and trehalose may be used.

[0194] Auxiliary and adjuvant agents may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of contaminating microorganisms, ;if desired, can be accomplished using various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, may also be included.

[0195] Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0196] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the active agent (such as the recombinant bacteria), and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers., as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3- butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polycthylcncglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.

[0197] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. Reference is made, for example, to Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, Easton, Pa., 1990).

[0198] Methods

[0199] Methods are provided for treating a parasitic worm or helminth infection in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-microbial agent treated recombinant bacterium that is engineered to express a crystal protein.

[0200] Furthermore, methods are provided for reducing the severity of a parasitic worm or helminth infection comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-microbial agent treated recombinant bacterium that is engineered to express a crystal protein.

[0201] Methods for the expression of crystal proteins in the IBaCC (inactivated bacteria with crystal proteins) are described in detail in US Patent No. 11,484,568 and in Li et al.

[0202] (Anthnicrob Agents Cheinother. 2021 Feb 17 ;65 (3); cO 1469-20,). The disclosures of both of these references are incorporated herein in their entirety.

[0203] Selected definitions

[0204] As used herein, unless the context makes clear otherwise? “treatment” and similar words such as “treated,” “treating” etc. , indicate an approach for obtaining beneficial or desired results, including clinically desirable results. Treatment can involve optionally either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition.

[0205] As used herein, unless the context makes clear otherwise, “subject” means a vertebrate, such as a mammal, The mammal can be a feline, a rodent, a canine, a bovine, an equine, a swine, a caprine, an ovine, or a primate. In some embodiments, the subject is a human.

[0206] As used herein, unless the context makes clear otherwise, “reducing the likelihood of occurrence," “prevention,” and similar words such as “prevented," “preventing” etc., include approaches for preventing, inhibiting, or decreasing the likelihood of the onset or recurrence of a disease or condition, in a manner that exhibits statistical significance, for example, when compared to the results obtained when the indicated method steps are omitted. Similarly, also included are preventing, inhibiting, or decreasing the likelihood of the occurrence or recurrence of the symptoms of a disease or condition, or optionally delaying the onset or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also include reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition. Methods according to these and related embodiments may be practiced using an effective amount pr a therapeutically effective amount of an agent that subs tantially eradicates, reduces the severity of, or reduces the likelihood of occurrence of a helminth infection, e.g., a soil-transmitted helminth infection. As used herein, an “effective amount” or a ‘‘therapeutically effective amount” of a composition, agent Or substance is that amount sufficient to obtain a desired biological effect, such as beneficial results, including clinical results.

[0207] As used herein “ZBaCC” means inactivated bacteria with cytosolic crystals rendered inviable with food-grade essential oils.

[0208] In certain exemplary embodiments, the herein described compositions for treating or reducing the severity or likelihood of occurrence of a helminth infection are formulated as pharmaceutical compositions, which can be formulated for oral delivery. Pharmaceutical compositions are formulated so as to allow the agent(s) contained therein to be bioavailable upon administration of the composition to a human.

[0209] It will be appreciated that the practice of the several embodiments of the present specification will use, unless indicated specifically to the contrary, conventional methods in virology, immunology, microbiology, molecular biology, and recombinant DNA techniques that are within the skill of the art, and many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology , John Wiley & Sons, New York, N,Y,(20D9); Ausubel et al., Short Protocols in Molecular Biology, 3ldcd., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridisation (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984);^4nz’zwnZ Cell Culture (R, Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

[0210] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein, These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0211] As used in this specification and the appended claims, the singular forms “a,' “an” and "the” include plural references unless the content clearly dictates otherwise. Throughout this specification, unless the context requires otherwise^ the word “comprise,” or variations such as "comprises” or “comprising,” will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0212] Examples

[0213] The following Examples are presented by way of illustration and not limitation.

[0214] Example 1 : Identification and cloning of novel Cry proteins of Bacillus thuringiensis

[0215] Novel, nematicidal Cry proteins of B, thuringiensis were identified within publicly available sequence databases using the Identification Of Pesticidal Sequences (IDOPS) tool. (Diaz-Valerio S. et al., IDOPS, a profile HMM-based tool to detect pesticidal sequences and compare their genetic context. Front Microbiol, 2021'12: 664476).

[0216] The IDOPS tool has at its core a collection of profile hidden Markov models (HMMs). Briefly, HMMs represent the probability for each amino acid to occur in a given protein sequence, as well as the probability for insertion or deletion states. In this way, IDOPS contrasts a candidate sequence against a given model to assign a score which can aid in determining whether a candidate sequence is likely a member of the group represented by the HMM.

[0217] Each HMM within IDOPS was created by iterative rounds of modelling, curation and refinement, this with the goal to achieve maximum sensitivity and specificity regarding the target protein group. The 3-domain Cry toxin group is one of the most studied pesticidal protein classes from B. thuringiensis. Even though all Cry proteins present the characteristic 3 domains, there are variations and alternative blocks within those domains. Therefore, when developing IDOPS, the Cry group was divided accordingly into subgroups, resulting in models called CryMl to CryM6.

[0218] It was discovered that known Cry proteins with nematicidal activity were matched by the HMM called CtyM5, for example the known CrySB. Therefore, biological databases with CryMS were screened and new candidate sequences were selected to synthesize and test for toxic activity.

[0219] The analysis gathered all proteins matched by CryMS from public databases (UniProt, GenBank). Redundant, duplicated, and fragment sequences were filtered. The results were narrowed to sixteen candidate crystal (Cry) protein sequences after further comparative analysis.

[0220] Artificially synthesized gene sequences corresponding to the newly identified, novel crystal proteins sequences were acquired from Genscript. The artificially synthesized genes were cloned in the Bacillus thuringiensis expression system, the inactivated bacteria with cytosolic crystals (IBaCC expression system), described in US Patent No. 11,484,568, the disclosure of which is incorporated herein in its entirety.

[0221] Novel crystal proteins synthesized by expression of artificially synthesized gene sequences in the Bacillus thuringiensis IBaCC expression system can be purified as described in US Patent Nos. 11,844,815 and 11,826,389, the disclosure of which is incorporated herein in its entirety,

[0222] Two novel proteins CryHl and CryH13 were identified by the procedure described above. The amino acid sequences of CryHl and CryH13 are shown in Table 1.

[0223] Table 1. Amino acid sequence of Cry Hl and CryH13 and nucleic acid sequence of genes encoding the proteins.

[0224] The resultant bacterial strain expressing CryHl or CryH13 was called CryHl IBaCC system or CryHl 3 IBaCC system. The IBaCC systems successfully expressed CryHl and CryHl 3 at sufficient levels for bioactivity tests. Cell lysate of the IBaCC systems were tested in vitro against Caenorhabditis elegans, Ancylostoma ceylanicum (human hookworm adults), Ascaris suum (L4 larvae), and Strongyloides stercoralis (human threadworm adults). Anthelmintic efficacy of the cell lysate of the IBaCC systems were also tested in vivo in hamsters against Ancylostoma ceylcmicum (human hookworm adults), Ascaris suum (L4 larvae), and Strongyloides stercoralis (human threadworm adults).

[0225] Example 2 : Effect of CryHl and CryH13 on viability of Strongyloides stercoralis (adult threadworms) and relative motility of Ancylostoma ceylanicum (adult hookworms) in vitro The effect of IBaCC systems expressing the newly identified Cry proteins was tested jo 'vitro on the viability of adult threadworms (Strongyloides stercoralis) and relative motility of adult hookworms (Ancylostoma eeylanieum\

[0226] Viability was tested in vitro using methods described by Charunchaibovorn et al (Charunchaibovorn et aL, Am. J. Trap Med. Hyg 101(5): 1177-1182 (2019) and Chicca et al., Chicca et at, Microbiol. Speetr. 10(4):c0235622 (2022)). For S'. stercdralis, adult worms (20- 40 / well) were added to wells of a 48-well plate. IBaCC was added to test wells at a Cry protein concentration ranging from 0,01 pg / ml to 10 jxg / ml in IX PBS with pen / strep / amphotericin B and £. coll food source. 20 mM Hepes pH 8.0 buffer was added in place of lysates to control wells. The assay was scored at 48 hours. Larvae that moved in the absence or presence of gentle touch with an eyelash were scored as alive. Larvae that did not move were scored as dead. The data show are the averages of three independent trials.

[0227] FIG. 1 shows that, at concentrations as low as 0.1 pg / nil, CryHl and Cry H13 IBaCC significantly reduced viability of adult S'. stercoralis worms, and at concentrations of 10 ng / ml killed all worms (WT = CrySBa control).

[0228] Effect on motility of A. eeylanieum was tested in vitro using methods described by Hu et al. (PLOS ONE 8(7) (2013)) and Chicca et al., (Chicca et al., Microbiol. Speetr. 10(4):e0235622 (2022)). Assays were carried out in a 100 iiL volume in 96-well plates using one adult per well (equal gender per condition) in RPMI plus 50% fetal bovine serum plus pen / strep / amphotericin B plus 25 mM Hepes pH 7.2 (8 hookwonns / condition x 3 independent trials). Plates were put at 37°C in a 5% COi in air incubator. Cells expressing no Cry protein (IBa empty vector control, EVC; negative control) or cells expressing Cry5B IBaCC (positive control),. CryHl IBaCC, or CryH13 IBaCC (at a Cry protein dose of 1 jig / ml or 10 pg / ml was added to the wells). Motility of each parasitic nematode in each well was scored using our Worminator assay system (Chicca et al., above) at 24 hr (A) and 48 hr (B). Data from (A) three and (B) two experiments (n=8 / experiment) was averaged. Motility was normalized to movement of hookworms in the absence of Cry protein using the Worminator, EVC = empty vector control (IBa).

[0229] FIG. 2A shows that at a low dose of about 1 pg / mL, CryHl IBaCC lysate significantly reduced motility of adult A. eeylanieum in vitro and this reduction was slightly better than that seen with IBaCC lysate expressing wild type Cry5B. FIG. 2B shows that though CryHl 3 IBaCC lysate was not as effective as Cry5B in inhibiting motility of A. eeylanieum, a 10 pg / mL dose of CryHl 3 IBaCC lysate reduced motility significantly. Example 3 : In vitro bioactivity of CryH13 IBaCC against Caenorhabditis elegans

[0230] Efficacy of CryH13 IBaCC was tested against Caenorhabditis elegans nematodes. A gene encoding CryHl or CryHl 3 was well-expressed in B. thuringiensis CryHl 3 IBaCC system as seen by SDS-PAGE analysis of the CryHl -IBaCC (FIG. 3A) and the CryH13- IBaCC (FIG. 3B).

[0231] The assays were set up using L4 worms on a48-well format. Specifically, 150 pl S- media + 20 pl OP50 (OD600 - 3) + 20 pl Cry proteins as IBaCC or EVCZIBa (10X) + 10 pl worms in S-media to 20-40 worms in each well. The plates were incubated at 25°C for 6 days wrapped with wetted Kimwipes- C. elegans (fourth larval stage (L4)) were grown for 6 days at 25°C in the presence of bacteria (i.e., feeding on the bacteria) containing empty vector control (EVC; negative control), vector expressing CryHl 3 (CryHl 3 IBaCC), or vector expressing CrySBa (CrySBa IBaCC (positive control). The anti-nematode activity was tested against both wild-type C. elegans (N2) and mutant C. elegans (bre-5(yel7)) that are resistant to CrySBa because they lack the receptor to CrySBa. C elegans were photographed. All images were taken at the same magnification and are depicted in FIG. 3C. Wild-type (N2) C. elegans were healthy (large, motile, and well-fed or dark) when fed negative control (EVC) but are severely intoxicated (stunted, immotile, pale) when fed CrySBa Or CryHl 3 IBaCC. The CrySBa resistant mutant C. elegans (bre-5(yel7)) were healthy when fed or when fed CrySBa (resistant). The data showed that CryHl 3 is highly active against C. elegans and that, unlike CrySBa was able to qualitatively severely intoxicate the bre-5(yel7) resistance mutant (FIG. 3C). Thus CryHl 3 is able to overcome CrySBa resistance. This result was unexpected as bre-5(yel7) mutants are somewhat resistant to Cryl4Aa, which is closely related to CryHl 3 (Griffitts et al., Science, 2001).

[0232] Example 4 : Additional data on the Effect of CryHl on in vitro motility of Ancylostoma ceylanicutn (adult hookworms) and data on the Effect of CryHl and CryII 13 on in vitro motility of Ascaris suum (larvae)

[0233] Bioactivity of CryHl against and A. ceylanicum was tested in vitro using the exact same methods described above. A. ceylanicutn adult worms were added to wells of a 96-well plate to which CryHl 3 IbaCC lysate was added at different concentrations ranging from 0.01 to 10Q pg / ml. For A. suum L4 assays (carried out according to Urban et al. One Health 12:100241 (2021)), A. suum L4 parasites (day 12 harvested from the intestines of B6 / STAT6KO mice) were exposed to no protein, 50 pg / ml, and 100 pg / 'mL Cry proteins for 5 days (37 °C, 5% CO2) in RPMI 1640 with (all final concentrations) 25 mM HEPES (pH 7.2), 5% fetal bovine serum, anti-microbials (100 U / mL penicillin, 100 mg / mL streptomycin;

[0234] 2.5 pg / mL amphotericin B). One adult parasite per well were placed in 100 p.1 medium in a 96-well format with the designated treatment using 5 wells / condition (mixed gender) and then set up 2 independent times for CryH13 (67 or 200 pg / ml for CryH13 IBaCC) and 3 independent times for CryHl (0, 20, 66.7, and 200 pg / ml for CryHl IBaCC). A. suum L4s were scored on a 0 to 3 scale (0, nonmotile even when touched; 1, nonmotile unless touched;

[0235] 2, slowly motile; 3, fully motile) as described previously. In both sets of experiments, wells with 20 mM Hepes pH 8.0 buffer added in place of CryHl or CryH 13 served as negative control as did EVC in the CryH 13 experiment. Plates were put at 37°C in a 5% CO2 in air incubator. Motility of each parasitic nematode in each well was scored over time using the touch 3-0 scale (3 = fully motile; 2 = inhibited motility; 1 = immotile until touched; 0= immotile even with touch).

[0236] FIG. 4A shows that CryH13 IBaCC lysate at doses higher than 0.1 pg / mL significantly reduced motility of A. ceylanicum adult worms in vitro. FIG. 7 shows that QyHl IBaCC lysate at doses higher than 0.1 pg / ml. is more effective than Cry5B for reducing motility of A. ceylanicum adult worms in vitro. The two experiments were carried out the same, (n— 8 parasites / replicate; average of 3 replicates).

[0237] FIG. 4B shows that both the 67 and 200 pg / mL doses of CryH 13 IBaCC lysate significantly reduced motility of adults A. suum worms in vitro.

[0238] FIG.4C shows that 66.7 and 200 pg / mL doses of CryHl IBaCC lysate significantly reduced motility of A. suum L4 stage worms in vitro.

[0239] Example 5: In vivo efficacy of CryHl and CryH13 for reduction of worm burden and fecal egg count of Ancylostoma celyanicum in hamsters

[0240] In vivo efficacy Of CryH I and CryHl 3 IBaCC lysate for reducing fecal eggs counts and worm burdens of A. celyanicum in hamsters was tested using methods described by Hu, Y., et al. PLoS NEGL TROP DIS 4, e6506 (2018) and Li et al., Antimicrob Agents and Chemotherapy 65(3):e01469-20.

[0241] Sixteen (16) 4-week old male Syrian hamsters are infected per os with approximately 130 infectious staged L3i A. ceylanicum hookworm larvae. Eighteen (18) days post-inoculation, the hamsters were grouped together based on equivalent fecal egg counts and then treated per os with water (negative control) or a single dose 10 mg CryHl IBaCC lysate, CryHl 3 IBaCC lysate, or Cry5B IBaCC lysate (positive control) per kilogram of body weight (IDmg / kg). Body weight, hemoglobin levels, and fecal egg counts were taken before treatment and then day 23 post-inoculation, at which point the animals are euthanized and worm burdens in the small intestine are determined. On day 17 post-infection, an overnight collection of stool was taken for determination of fecal egg counts. The hamsters were assigned to groups based on FEC so to ensure roughly the same level of infection (same average egg per gram of feces or EPG) in all groups. On day 18 post-infection, the hamsters were weighed for dosing purposes and treated with IBaCC lysates or control treatments through a blunt-ended gavage needle. On day 22 post-infection, another overnight collection of stool was taken for measurement of FECs. On day 23 post-infection, the animals were euthanized. Total hookworms in the intestine were counted and EPGs calculated.

[0242] As compared to the negative control (treatment with water), treatment with a single dose of either CryHl or CryHl 3 IBaCC lysate significantly reduced both the A. celyanieum worm burden.(FIG, 5A) and EPG (FIG. 5B) in infected hamsters. FIG. 5C shows that infected hamsters treated with water experienced significant parasitic worm infection related weight loss whereas infected hamsters that were treated with Cry5B (positive control), CryHl or CryHl 3 IBaCC lysates suffered no significant weight loss and / or weight gain by the end of the treatment period.

[0243] Example 6: In vivo efficacy of CryHl and CryH13 for reduction otAscaris suum worm burden in infected mice

[0244] In vivo efficacy of CryHl or CryH13 IBaCC lysate for reducing worm burdens of A. suum in mice was tested using methods described by Urban et al. One Health. 2021;12: 100241, and Chicca et al. Microbiol Spectr. 2022; 10: e0235622.

[0245] On day 0, B6 / STAT6KQ mice were infected per os with a suspension of 5000 A. suum L3 eggs. A. suum were allowed to progress to the intestinal phase. Mice wete treated with two oral doses of 20 mg per kilogram of body weight (20 mg / kg) of CryHl IBaCC lysate, CryH13 IBaCC lysate or water (control). First dose was given on day 12 after infection and second dose was given on day 13. All mice were killed on day 18 by exposure to CO5 on day 20 P.I. Total Ascaris counts (worm burden) were measured.

[0246] FIG. 6A shows that treatment with two oral doses of 20 mg / kg of CryHl IBaCC lysate significantly reduced the worm burden. FIG. 6B shows that treatment with two oral doses of 20 tng / kg of CryH13 IBaCC lysate reduced the EPG of A. suum in infected mice by 80%.

[0247] Example 7: Effect of CryHl and CryH13 IBaCC lysates on in vitro motility of Trichuris muris (whipworm)

[0248] The effects of CryHl Or CryHl 3 IBaCC lysate On in vitro motility of whipworm, T. muris was tested using methods described by Hu et al. (PLOS ONE 8(7) (2013)) and Hoang et al., (Hoang et al., PLoS Negl Trop Dis- 2024 Oct 25;18(10):e0012611). Assays were carried out in a 300 p.L volume in 48- well plates using one adult per well (equal gender per condition) in RPMI plus 5% fetal bovine serum plus pen / strcp / amphotcricin B plus 25 mM Hepes pH 72. Plates were put at 37°C in a 5% COa in air incubator. Cells expressing no Cry protein (IBa empty vector control, E VC; negative control) or cells expressing Cry5B IBaCC (positive control), CryHl IBaCC, or CryH13 IBaCC (at a Cry protein dose of 0-500 pg / nil was added to the wells) were incubated. The motility of each parasitic nematode in each well was hand scored.

[0249] FIG. 8 shows that T. muris was intoxicated by CryHl in a dose-dependent manner, resulting in over 50% reduction in nematode motility.

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[0366] 125. Hoang D, Flanagan K, Ding Q, Cazeault NR, Li H, Diaz- Valerio S, Rus F, Darfour EA, Kass E, Petersscn KH, Nielsen MK, Liesegang H, Ostroff GR, Aroian RV. Bacillus thuringiensis Cryl4A family proteins as novel anthelmintics against gastrointestinal nematode parasites. PLoS Negl Trop Dis. 2024 Oct 25;18(10):e0012611. doi: 10.1371 / joumal.pntd.0012611. PMID: 39453964; PMCID: PMC 11540219. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing specification has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this specification that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0367] While particular steps, elements, embodiments and applications of the present specification have been shown and described herein for purposes of illustration, it will be understood, of course, that the specification is not limited thereto since modifications may be made by persons skilled in the art, particularly in light of the foregoing teachings, without deviating from the spirit and scope of the specification . Accordingly, the specification is not limited except as by the appended claims.

[0368] The various embodiments described above can be combined to provide further embodiments. All of the U.S . patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.

[0369] These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited. by the disclosure.

Claims

WHAT IS CLAIMED IS:

1. An anthelmintic composition comprising a Bacillus thuringicnsis nematicidal crystal (Cry) protein, wherein the Cry protein is CryHl 3 or CryHl.

2. The composition of claim 1. wherein the Cry protein is in the form of a native, bioactive crystal, wherein the crystal is produced by a host bacterium that is genetically engineered to express the gene encoding the Cry protein.

3. The composition of claim 2, wherein the host bacterium is a non-sporulating bacterium.

4. The composition of any one of claims 1 to 3, wherein the composition is substantially free of any bacterial spores.

5. An anthelmintic composition comprising a killed or inactivated bacterium that is genetically engineered to express a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein is CryH13 or CryHl .

6. The composition of any one of claims 2 to 5, wherein the bacterium is genetically engineered to have a genetic mutation that results in a defect in Sporulation such that the Cry protein is trapped in the cytosol of the bacterium.

7. The composition of any one of claims 2 to 6, wherein the expression of the gene encoding the Cry protein is under control of a non-sporulation-specific promoter.

8. The composition of claim 7, wherein the promoter is a Cry3A, GerA, GNAT, or TadA promoter.

9. The composition of any one of claims 2 to 8, wherein the bacterium is a Grampositive bacterium.

10. The Composition of any one of claims 2 to 9, wherein the bacterium is a species of Bacillus.

11. The composition of any one of claims 2 to 9, wherein the bacterium is Bacillus thuringiensis (Bt).12 The composition of any one of claims 2 to 11, wherein the bacterium has a genetic mutation, wherein the genetic mutation is a deletion or inactivation of one or more genes resulting in a defect of sporulation, and wherein the one or more genes resulting in a defect in sporulation is selected from the group consisting of kin A, kinB, spoOA, spoOB, spoOE, spoOF, spoOJ, spoOM spoIlB, spoUD, spolIE, spoHF, spoIIG. spoIIL, spoIIM, spoIIIA, spoil IB, spoHIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, SpoVP, spoVQ, spoVID, oH, oF, crE, oG, and aK.

13. The composition of claim 12, wherein the genetic mutation is a deletion or inactivation of the spoOA gene.

14. The composition of any one of claims 2 to 8, wherein the bacterium is a Gramnegative bacterium.

15. The composition of claim 14, wherein the bacterium is K coll or P. fluorescetis ,16. The composition of any one of claims 1 to 15, wherein the composition further comprises a pharmaceutical carrier or excipient.

17. The composition of any one of claims 1 to 16, wherein the composition is encapsulated by a pharmaceutical grade capsule in a dry powdered form.

18. The composition of any one of claims 1 to 17, wherein the composition is an orally-availablc composition.

19. A method for producing an anthelmintic composition, the method comprising:(a) growing a non-sporulating form of a host bacterium that is genetically engineered to express a single type of nematicidal crystal (Cry) protein, wherein the single type of nematicidal Cry protein is CryHl or CryHl 3, wherein the non-sporulating host bacterium produces native, bioactive nematicidal crystals formed from the single type of nematicidal crystal protein, wherein the host bacterium has a genetic mutation such that CryHl or CryH13 is trapped in the cytosol of the bacterium, wherein the host bacterium is grown in a growth medium, optionally wherein the non-sporulating host bacterium releases the native, bioactive nematicidal crystals into the growth medium; and(b) isolating and concentrating the native, bioactive nematicidal crystals to form isolated, native, bioactive nematicidal crystals.

20. A method for producing an anthelmintic composition, the method comprising exposing a non-sporulating bacterium to an antimicrobial agent, thereby killing or inactivating the bacterium, wherein the bacterium is genetically engineered to express CrylHl or CryHl 3, and wherein the bacterium has a genetic mutation such that CryHl or CryHl 3 is trapped in the cytosol of the bacterium.

21. The method of claim 19 or 20, wherein the genetic mutation results in a defect of sporulation.

22. The method of any one of claims 19 to 21 , further comprising formulating the composition in an orally-available dosage form.

23. The method of claim 22, wherein formulating comprises one or both of Lyophilizing or spray drying the bacterium, and encapsulating the bacterium in a pharmaceutical-grade capsule.

24. The method of any one of claims 20 to 23, wherein the antimicrobial agent is an antimicrobial compound or gamma irradiation.

25. The method Of any one of claims 20 to 23, wherein the antimicrobial agent is:(a) a food-grade antibiotic;(b) a beta-lactam antibiotic; or(c) a terpene, iodine or formaldehyde.

26. The method of claim 25, wherein the terpene is selected from the group consisting pf thymol, eugenol, geraniol, carvacrol, and citral, and combinations thereof.

27. The method of claim 26, wherein the terpene is carvacrol.

28. The method of any one of claims 19 to 27, wherein CryHl or CryHl 3 expression is under control of a non-sporulation specific promoter.

29. The method of claim 28, wherein the promoter is a Cry3A, GerA, GNAT, or TadA promoter.

30. The method of any one of claims 19 to 29, wherein the bacterium is Bacillus sp.

31. The method of claim 30, wherein the bacterium is Bacillus thuringiensis (Bt).

32. The method of any one of claims 19 to 31 , wherein the genetic mutation is a deletion or inactivation of one Or more genes resulting in a defect of sporulation, wherein the one or more genes is selected from the group consisting of kinA, kinB. spoOA, spoOB, spoOE, spoOF, sppOj, spoOM spoDB, sppUD, spoHE, spoIIF, spoIIG, spoHL, spoHM, spoIIIA, spoIIIB, spoIHE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, all, aF, crE, oG, and aK.

33. The method of claim 32, wherein the genetic mutation is a deletion or inactivation of the spoOA gene.

34. The method of any one of claims 19 to 29, wherein the bacterium is a Gramnegative bacterium.

35. The method of claim 34, wherein the bacterium is an E. colt or P. jluorescens species.

36. A method of controlling a parasitic worm infection of a plant, said method comprising applying an effective amount of the composition of any one of claims 1 to 15 to the plant.

37. A method of treating a parasitic worm infection in an animal or a human Comprising orally administering an effective amount of the composition of any one of claims 1 to 18 to an animal or a human infected with a parasitic worm.

38. The method of claim.36 or 37, wherein the parasitic worm is resistant to one or more other anthelmintic treatments.39, The method of any of claims 36 to 38, wherein the parasitic worm is resistant to Cry5B.

40. The method of any one of claims 37 to 39, wherein the parasitic worm infecting the human is selected from the group consisting of roundworm, whipworm, hookworm, flatworm, tapeworm, fluke, and pinworm (threadworm).

41. The method any one of claims 37 to 39, wherein the animal treated is selected from the group consisting of cattle, sheep, goat, equine, pig, poultry, dog, and cat.

42. The method of any one of claims 37 to 39 or 41 , wherein the parasitic worm infecting the animal is selected from the group consisting of roundworm, hookworm, whipworm, heartworm, lungworm, and a strongyle (cyathostomin).

43. The method of claim 41 or 42 , wherein the animal is a sheep and the parasitic worm is a roundworm.

44. The method Of claim 43, wherein the roundworm is Haemonchus contortus.

45. The method of claim 41 or 42, wherein the animal is an equine and the parasitic worm is a strongyle (cyathostomin).

46. The method of claim 41 or 42, wherein the animal is a pig and the parasitic worm is a roundworm.

47. The method of claim 46, wherein the roundworm is Ascaris spp.

48. The method of claim 46, wherein the roundworm is Ascaris suum.

49. The method of any one of claims 37 to 40, wherein the parasitic worm infecting the human is Ancyiostoma spp or Necator spp.50, The method of claim 49, wherein the parasitic worm is Ancylostoma ceylanicum or Ancyiostoma duodenale.

51. The method of claim 49, wherein the parasitic worm is Necator americanus .

52. The method Of any one of claims 37 to 40, wherein the parasitic worm isAscaris spp.

53. The method Of claim 52, wherein the parasitic worm is Ascaris lumbricoides.

54. The method Of any one of claims 37 to 40, wherein the parasitic worm is Thrichuris spp.

55. The method of claim 54, wherein the parasitic worm is Thrichuris trichiirra.

56. The method of any one of claims 37 to 40, wherein the parasitic worm is Strongyloides stercoralis.

57. An animal feed composition, comprising:(a) a base animal feed; and(b) a supplement comprising the composition of any one of claims 1 to 15.

58. A method of controlling or preventing a parasitic worm infection in an animal comprising feeding the animal the feed composition of claim 57.

59. A method of promoting gut health or boosting immunity of an animal comprising feeding the animal the feed composition of claim 57.

60. A composition for human or animal waste treatment, comprising the composition of any one of claims 1 to 15.

61. A method of waste treatment, comprising contacting waste with the compositionOf claim 60.

62. The method of claim 61, wherein waste, litter, or bedding is contacted with the composition.

63. An orally-ayailable anthelmintic composition comprising CryHl or CryHl 3 protein and one or more additional nematicidal proteins, wherein the CryHl or CryHl 3 protein is in the form of a native, bioactive crystal.

64. An orally-available composition comprising a killed, non-sporulating bacterium that expresses CryHl or CryHl 3 and one or more additional nematicidal proteins, wherein the bacterium has a genetic mutation comprising a deletion or inactivation of one or more genes selected from the group consisting of kin A, kinB, spoOA, spoOB, spoOE, spoOF, spoOJ, spoOM, spoil B, spoHD, spoIIE, spoIIF, spoIIG, spoIIL, spoHM, spoil I A, spoUIB, spoil IB, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK that results in a defect in sporulation, andwherein the CryHl or CryHl 3 and the one or more additional nematicidal proteins are trapped in the cytosol of the bacterium.

65. The composition of claim 63 or 64, wherein the one or more additional nematicidal proteins is CrySB.

66. A method for producing the anthelmintic composition of claim 63, the method comprising:(a) growing a non-sporulating form of a host bacterium that is genetically engineered to express nematicidal crystal (Cry) proteins, wherein the Cry proteins are CryHl or CryHl 3 and one or more additional Cry proteins, wherein the non-sporulating host bacterium produces native, bioactive nematicidal crystals formed from the Cry proteins, wherein the bacterium has a genetic mutation that prevents sporulation such that CryHl, CryHl 2 and the one or more nematicidal proteins are trapped in the Cytosol of the bacterium, optionally wherein the non-sporulating host bacterium releases the native, bioactive nematicidal crystals into the growth medium;(b) isolating and concentrating the native, bioactive nematicidal crystals to form isolated, native, bioactive nematicidal crystals; and(q) formulating the isolated, native, bioactive nematicidal crystals in an orally- available dosage form.

67. A method for producing the composition of claim 64, the method comprising:(a) exposing a non-sporulating bacterium to an antimicrobial agent, thereby killing or inactivating the bacterium, wherein the bacterium is genetically engineered to express CryHl and CiyH13 and one or more additional nematicidal proteins, wherein the bacterium has a genetic mutation that prevents sporulation such that CryHl, CryH12 and the one or more nematicidal proteins are trapped in the cytosol of the bacterium; and optionally,(b) formulating the killed or inactivated bacterium in an prally-availablc dosage form.

68. A method of treating a parasitic worm infection in an animal or ahuman comprising orally administering an effective amount of the composition of any one of claims 63 to 65 to the animal or the human infected with a parasitic worm.

69. An animal feed composition, comprising:(a) a base animal feed; and(b) a supplement comprising the composition of any one of claims 63 to 65.

70. A method of controlling or preventing a parasitic worm infection in an animal comprising feeding the animal the animal feed composition of claim 69.

71. A method of promoting gut health or boosting immunity in an animal comprising administering to the animal feed composition of claim 69,72. An isolated Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1, or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3,73. An isolated Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein is encoded by a nucleic acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

73. A method of controlling a parasitic worm infection of a plant, said method comprising applying an effective amount of the Bacillus thuringiensis nematicidal Cry protein of claim 72 or claim 73 to the plant.

74. A method of controlling a parasitic worm infection in a human, said method comprising administering to the human an effective amount of the Bacillus thuringiensis nematicidal Cry protein of claim 72 or claim 73.

75. A method of controlling a parasitic worm infection in an animal, said method comprising administering to the animal an effective amount of the Bacillus thuringiensis nematicidal Cry protein of claim 72 or claim 73.

76. The method of claim 75, wherein the animal is a domestic animal, a wild animal ora fish.

77. A method of producing the Bacillus thuringiensis nematicidal Cry protein of one of claim 72 or claim 73.78, A method of purifying the Bacillus thuringiensis nematicidal Cry protein of one of claim 72 or claim 73.

79. A composition comprising a Bacillus thuringiensis nematicidal Cry protein of claim 72 or claim 73, wherein the composition comprises a pharmaceutical excipient or carrier, or wherein the composition comprises carrier material suitable for agricultural application.

80. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1, orwherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

81. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a Bacillus thurtngiensis nematicidal crystal (Cry) protein, wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%. about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

82. The recombinant nucleic acid of claim 80 or 81, wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.

83. The recombinant nucleic acid molecule of claim 82, wherein said nucleotide sequence is operably linked to a promoter capable of directing expression of said nucleotide sequence in a plant cell.

84. A vector comprising the recombinant nucleic acid molecule of claim 80 or 81.

85. A host cell that contains the recombinant nucleic acid of claim 80 or 81.

86. The host cell of claim 85, wherein the host cell is a bacterium.

87. The host cell of claim 85, wherein the host cell is a plant cell.

88. A transgenic plant comprising the host cell of claim 87.

89. The transgenic plant of claim 88, wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.

90. A transgenic seed comprising the nucleic acid molecule of claim 80 or 81.

91. A plant or plant cell having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein comprises ari amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1, or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

92. A plant or plant cell having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

93. A method for protecting a plant from a parasitic worm, comprising expressing in a plant or a cell thereof a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO; 1, or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

94. A method for protecting a plant from a parasitic worm, comprising expressing in a plant or a cell thereof a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.95 , A method for increasing yield in a plant comprising growing in a field a plant of or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 1, or wherein the Cry protein comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 3.

96. A method for increasing yield in a plant comprising growing in a field a plant of or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a Bacillus thuringiensis nematicidal crystal (Cry) protein, wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 2, or wherein the nucleic acid molecule has a sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or is 100% identical to SEQ ID NO: 4.

97. The method of any one of claims 93 to 96, wherein the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

98. A composition comprising the Bacillus thuringicn.si.s nematicidal crystal (Cry) protein of claim 72 or the host cell of claim 86.

99. The composition of claim 98, wherein the host cell is live, inactivated, or killed.

100. The composition of claim 98 or 99, wherein said composition is in a form selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.

101. A method for protecting a plant from a parasitic worm, comprising applying the composition of any one of claims 98 to 100, to a plant, seeds, or soil.

102. The method of claim 101, wherein the plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

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