Combating necrotic enteritis with l. reuteri vectored nanobodies

Abstract

The invention provides probiotic compositions of live Lactobacillus reuteri as vectors for the in situ delivery of single chain antibodies (e.g. nanobodies) against Clostridium perfringens NetB and a toxins, two key toxins associated with necrotic enteritis, an intestinal disease affecting animal health. The invention provides methods of using and administering such vectors and nanobodies to combat disease, particularly necrotic enteritis, and to improve productivity' and performance, food conversion, and body weight in animals, particularly poultry.

Description

COMBATING NECROTIC ENTERITIS WITH L. REUTERI VECTORED NANOBODIESINCORPORATION OF SEQUENCE LISTING

[0001] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document encoded as XML in UTF-8 text. The electronic document, created on January 23, 2026, is entitled “2950-43_PCT_ST26.xml”, and is 5,021,761 bytes in size.FIELD OF THE INVENTION

[0002] The present invention relates to probiotic compositions of live Lactobacillus reuteri as vectors for the in situ delivery of single chain antibodies (e.g. nanobodies) against Clostridium perfringens NetB and a toxins, two key toxins associated with necrotic enteritis, an intestinal disease affecting animal health and methods of using and administering such vectors and nanobodies to combat that disease, particularly necrotic enteritis, and to improve productivity' and performance, food conversion, and body weight in animals, particularly poultry'.BACKGROUND

[0003] Direct fed microbials (DFMs), often also called probiotics, are microorganisms which colonize, at least temporarily, the gastrointestinal tract of an animal and provide some beneficial effect to that animal. The microorganisms can be bacterial species, for example those from the genera Bacillus, Lactobacillus, Lactococcus, and Enterococcus. The microorganisms can also be yeast or even molds. The microorganisms can be provided to an animal orally or mucosally or, in the case of birds, provided to a fertilized egg, i.e. in ovo.

[0004] The beneficial activity provided by a DFM or probiotics can be the synthesis of vitamins or other nutritional molecules needed for a healthy metabolism of the host animal. A DFM or probiotic can also protect the host animal from disease, disorders, or clinical symptoms caused by other, pathogenic microorganisms. For example, the DFM or probiotic may produce factors having inhibitoiy or cytotoxic activity against certain species of pathogens, such as deleterious or disease-causing bacteria, or immunomodulatory activity, such as modulating immune response or improving immune response to foreign agent(s) or foreign antigen.

[0005] Necrotic enteritis (NE) is a common intestinal disease that causes significant economic losses ( -6 billion dollars annually) to the poultry industry worldwide. NE generally manifests as clinical orsubclinical forms. Clinical NE is acute and characterized by high mortality (30%-60%) and associated symptoms such as ruffled feathers, wet litter, diarrhea, and passage of undigested feed. In some cases, mortality is the only sign with no other premonitory symptoms. Subclinical NE is chronic and associated with damage to the intestinal mucosa, leading to reduced digestion and absorption of nutrients, decreased weight gain (3%-5%), and elevated feed conversion ratio (6-9 points) (Hofacre et al. (2018) Poultry Science 97: 1929-1933). Subclinical NE contributes to the majority of economic losses associated with NE and is the most prevalent form of NE (Emami e al (2021) Poultry Science 100:101055).

[0006] NE is caused primarily by Clostridium perfringens type A strains that infiltrate the mucosa of the small intestine and produce toxins such as NetB and a toxin. C. perfringens is a spore-forming, anae¬ robic, Gram-positive commensal that is ubiquitously found in the gastrointestinal tract of animals and the environment. NE is a complex disease and several factors are known to influence the gut environment of the host and favor the growth of C. perfringens strains. Mucosal damage caused by Eimeria species, nature of the feed, sudden diet change, high-density' bird housing conditions, and extreme environmental temperatures are among the key factors that predispose birds to NE (Fernandes Da Costa et al. (2016) Avian Pathology 54:381-388). The proliferation of C. perfringens leads to dramatic shifts in microbiota, with decreased abundance of beneficial species such as those belonging to Lactobacillus (Yang, Liu, Robinson, et al. (2021) J Animal Science and Pathology 12:107).

[0007] The C. perfringens NetB is a pore-forming toxin and it plays a key role in NE (Keybum et al., (2008) PLoS Pathogens 4, e6). A nontoxic variant of NetB called W262A is commonly used for immunization and has been shown to only partially protect birds from NE (Fernandes Da Costa et al. (2016) Avian Pathology 45, 381-388; Hunter et al. (2019) Peer! 7, e6600). The role of a toxin in NE is not clear; however, immunization with a toxin antigen partially protects birds from NE (Cooper et al. (2009) Veterinary Microbiology 133, 92-97). A combination of NetB and a toxin provides improved protection against NE (Fernandes Da Costa et al. (2016) Avian Pathology 45, 381-388). Nevertheless, vaccine approaches are only partially effective in reducing NE and the efficacy of vaccines depends on several factors such as the host genetics, immune system, and nutrition. To date, the administration of antibiotics has been the only effective treatment for NE; however, there is an increasing demand to reduce the use of antibiotics due to concerns around antimicrobial resistance. In addition, vaccines and antibiotics address only some aspects of NE (C. perfringens and their toxins), necessitating the need for developing safe and effective alternatives that address multiple aspects of NE biology.

[0008] Since their discovery' in the 1990s, nanobodies (Nbs) have emerged as a promising alternative for disease prevention and treatment in both animal and human health. Nbs are single-domain antibodies, derived from heavy chain only (lack light chains and the first constant CH1 domain) antibodies that naturally occur in the serum of camelids (dromedaries, camels, llamas, alpacas, guanacos, vicunas)(Jovcevska & Muyldermans (2020) Bio Drugs 34, 11-26). The variable domain of these heavy-chain only antibodies is the only domain involved in binding of this special class of antibodies (therefore called VHH, Variable domain of the Heavy chain of Heavy chain only antibodies).

[0009] Compared to traditional antibodies, Nbs possess several unique and favorable properties such as small size, high stability', strong antigen-binding affinity, water solubility', and ease of production in bacteria. Despite all these advantages, like any other protein and peptide biotherapeutics or preventatives, oral delivery of Nbs remains unsuccessful due to their degradation in the acidic and enzyme-rich environment ofthe stomach (Gleeson et al. (2021) Adv Drug Delivery Reviews 173, 112-124). Desired efficacy also demands frequent administration at higher concentrations, which is not economically feasible.

[0010] Microbial vectors offer an opportunity for oral delivery of bio-therapeutics and preventatives and include bacteria such as Limosilactobacillus Lactobacillus'), Lactococcus, Salmonella, Bacillus, Listeria, and Escherichia coli, engineered to deliver target molecules directly to the site of action. Delivery using microbial vectors not only protects the target molecules from the harsh gastrointestinal (Gl) environment but also maximizes effectiveness and minimizes off-target effects. Microbial vectors also have the advantage of easy and inexpensive manufacturing with flexible scalability and storage.

[0011] Lactobacilli are Gram-positive bacteria that are mainly characterized by their ability to produce lactic acid from sugar and have been used to produce fermented products for decades. Many Limosilactobacillus species have “Qualified Presumption of Safety' (QPS)” status from the European Food Safety Authority (EFSA) and “Generally Regarded As Safe (GRAS)” status from US Food and Drug Administration (FDA). Lactobacilli are members of the endogenous microbiota of oral, GI, respiratory', and urogenital mucosa and play a key role in regulating local microbiota, restoring barrier function, preventing inflammation associated with GI diseases, improving growth performance, and protecting against infectious diseases. Of particular importance, Lactobacilli have been shown to possess antagonistic activity against C. perjringens, inhibit toxin production, reduce proinflammatory cytokines, improve intestinal integrity and immune response, correct microbial dysbiosis, restore performance deficiencies associated with subclinical NE, and protect chickens from clinical NE (Gong et al. (2020) Frontiers in Immunology 8, 865; Guo et al. (2017) Frontiers in Microbiology 8, 2081). Lactobacilli are also well known for surviving the harsh environment of the GI tract. Furthermore, Lactobacilli are generally associated with mucosa and thus ensure delivery of target molecules directly to the mucosa. The availability of genetic tools for engineering Lactobacillus / Limosilactobacillus further makes them attractive candidates for in situ deliveiy of biomolecules.

[0012] There is a need in the art for approaches and therapeutics to combat NE, and the toxic impact it has on poultry. In particular, there is a need for approaches and therapeutics that are not antibiotic-basedand which reduce mortality and also further provide commercially relevant and important impacts on the overall productivity and performance, conversion of food to body weight and the overall body weight of animals susceptible to the pathogen and disease.SUMMARY OF THE INVENTION

[0013] The present invention provides compositions and methods for improving animal health. The composition and methods improve mortality and productivity in animals. The invention provides compositions and methods for treatment and amelioration of necrotic enteritis (NE) in animals, including poultry.

[0014] In particular aspects, the invention provides compositions and methods for treatment and amelioration of subclinical necrotic enteritis (NE) in animals, including poultry. In particular aspects, the invention provides compositions and methods for treatment and amelioration of chronic subclinical necrotic enteritis (NE) in animals, including poultry. In particular aspects, the invention provides compositions and methods for improvement of performance or productivity in animals, including poultry, with subclinical necrotic enteritis (NE). In aspects, improved performance or productivity includes increased body weight, decreased food conversion rate (FCR).

[0015] The invention provides a composition comprising at least two Lactobacillus strains of bacteria genetically engineered to express and secrete one or more nanobody directed against at least one toxin of Clostridium perfringens, wherein at least one toxin is selected from alpha toxin and NetB, wherein the composition comprises at least 1x106CFU of each of the one or more bacteria, and wherein the Lactobacillus strains are strains that have native probiotic capability.

[0016] In an embodiment, the Lactobacillus strains are Lactobacillus reuteri strains. In a particular embodiment, the strains are L. reuteri strain 3630 and strain 3632, or a variant thereof having at least 80%, 90%, 95%, 97% or 99% to the nucleic acid sequence or the genome sequence of L. reuteri strain 3630 and strain 3632. In a particular embodiment, the strains are L. reuteri strain 3630 and strain 3632.

[0017] In an embodiment, the strains are genetically engineered to express and secrete a nanobody directed against C. perfringens alpha toxin and a nanobody directed against C. perfringens Net B. In an embodiment, the strains are genetically engineered to express and secrete a nanobody directed against C. perfringens alpha toxin and a nanobody directed against C. perfringens Net B, wherein the nanobody directed against C. perfringens alpha toxin is selected from(a) EAT-1F2:EVQLVESGGGLVQAGGSLRLSCAGSGRTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:56);(b) EAT-1F2_R27H:EVQLVESGGGLVQAGGSLRLSCAGSGHTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:57);(c) EAT-1F2_T28P:EVQLVESGGGLVQAGGSLRLSCAGSGRPGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:58);(d) EAT-1G4:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAY DS VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHYGQGTQVTVSS (SEQ IDNO:59); and (e) EAT-1G4_Y103W:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADS VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHWGQGTQVTVSS (SEQ ID NO:60); and wherein the nanobody directed against C. perfringens NetB is selected from(f) ENB-1A4:EVQLVESGGGLVQAGGSLRLSCAASGSIFSIТSVMGWYRQAPGKQREFVAGITIGGTARYPDS VKGRFTTSRDNTQNTVYLQMNNLKPEDTAVYYCNAVLPSDQRRWSWGQGTQVTVSS (SEQ ID NO:61);(g) ENB-1D11:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGRTNYADS VKGRFTISIDNAKNSIHLQMNSLKPDDTARYYCNSPYHALWGQGTQVTVSS (SEQ ID NO:62); and (h) ENB-ID11 R56H:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGHTNYADS VKGRFTIALWGQGTQVTVSS (SEQ ID NO:63).

[0018] The invention provides a composition comprising at least two Lactobacillus strains of bacteria genetically engineered to express and secrete one or more nanobody directed against at least one toxin of Clostridium perfringens, wherein at least one toxin is selected from alpha toxin and NetB, wherein the composition comprises at least 1x106CFU of each of the one or more bacteria, and wherein the Lactobacillus strains are strains that have native probiotic capability. In an embodiment, the Lactobacillus strains are Lactobacillus reuteri strains. In an embodiment, the strains are L. reuteri strain 3630 and strain

[0019] In a particular embodiment, the strains are genetically engineered to express and secrete a nanobody directed against C. perfringens alpha toxin and a nanobody directed against C. perfringens Net B, wherein the nanobody directed against C. perfringens alpha toxin is selected from(a) EAT-1F2: EVQLVESGGGLVQAGGSLRLSCAGSGRTGSLYSMGWFRQAPGKEREFV AAITWRPSSTYYADSVKGTPQAYDYWGQGTQVTVSS (SEQ ID NO:56);(b) EAT-1F2_R27H:EVQLVESGGGLVQAGGSLRLSCAGSGHTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:57);(c) EAT-1F2_T28P:EVQLVESGGGLVQAGGSLRLSCAGSGRPGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:58);(d) EAT-IG4:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHYGQGTQVTVSS (SEQ ID NO:59); and (e) EAT-lG4_Y103W:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHWGQGTQVTVSS (SEQ ID NO:60); and wherein the nanobody directed against C. perfringens NetB is selected from(f) ENB-1A4:EVQLVESGGGLVQAGGSLRLSCAASGSIFSTNVMGWYRQAPGKQREFVAGITIGGTARYPDSVK GRFTISRDNTQNTVYLQMNNLKPEDTAVYYCNAVLPSDQRRWSWGQGTQVTVSS (SEQ ID NO:61);(g) ENB-1D11:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGRTNYADSVKGRFTISIDNAKNSIHLQMNSLKPDDTARYYCNSPYHALWGQGTQVTVSS (SEQ ID NO:62); and (h) ENB-ID11_R56H:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGHTNYADSVK GRFTIALWGQGTQVTVSS (SEQ ID NO:63).

[0020] The invention includes a method of increasing mortality, feed conversion ratio and body weight in an animal at risk of necrotic enteritis comprising administering to the animal at least two doses of the composition provided herein. In an embodiment, a method is provided for increasing mortality, feedconversion ratio and body weight in an animal, including poultry, in the presence or circumstances of subclinical necrotic enteritis, including chronic subclinical NE, including in a commercial animal housing. In an embodiment, the method provides administering to the animal at least one dose, at least two doses, or up to three doses, of the composition provided herein, particularly wherein the first dose is administered at birth or upon or shortly after hatching.

[0021] A method is provided for increasing mortality, feed conversion ratio and body weight in an animal at risk of necrotic enteritis comprising administering to the animal at least two doses of a composition comprising at least two Lactobacillus strains of bacteria genetically engineered to express and secrete one or more nanobody directed against at least one toxin of Clostridium perfringens, wherein at least one toxin is selected from alpha toxin and NetB, wherein the composition comprises at least IxlO5CFU of each of the one or more bacteria, and wherein the Lactobacillus strains are strains that have native probiotic capability. In an embodiment, the Lactobacillus strains are a first and a second Lactobacillus reuteri strain, wherein the first Lactobacillus reuteri strain has a genomic nucleic acid sequence comprising least one of SEQ ID NOs: 49-55, or having at least one nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 49-55 and further having at least 98% sequence identity with one or more of SEQ ID NOs: 49-55; and wherein the second Lactobacillus reuteri strain has a genomic nucleic acid comprising at least one of SEQ ID NOs: 44-48, or having at least one nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 44-48 and further having at least 98% sequence identity with one or more of SEQ ID NOs: 44-48.

[0022] In an embodiment of the method, the animal is housed in a commercial setting or breeding facility or pen and the mortality from necrotic enteritis is subclinical wherein there is less than 10% NE-associated mortality in the absence of the composition.

[0023] In another aspect of the method, the strains are genetically engineered to express and secrete a nanobody directed against C. perfringens alpha toxin and a nanobody directed against C. perfringens Net B, wherein the nanobody directed against C. perfringens alpha toxin is selected from(a) EAT-1F2: EVQLVESGGGLVQAGGSLRLSCAGSGRTGSLYSMGWFRQAPGKEREFV AAITWRPSSTYYADSVKGTPQAYDYWGQGTQVTVSS (SEQ ID NO:56);(b) EAT-1F2_R27H:EVQLVESGGGLVQAGGSLRLSCAGSGHTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:57);(c) EAT-1F2 T28P:EVQLVESGGGLVQAGGSLRLSCAGSGRPGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:58);(d) EAT-1G4:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHYGQGTQVTVSS (SEQ ID NO:59); and (e) EAT-1G4_Y103W:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHWGQGTQVTVSS (SEQ ID NO:60); and wherein the nanobody directed against C. perfringens NetB is selected from(f) ENB-1A4:EVQLVESGGGLVQAGGSLRLSCAASGSIFSTNVMGWYRQAPGKQREFVAGITIGGTARYPDSVK GRFTISRDNTQNTVYLQMNNLKPEDTAVYYCNAVLPSDQRRWSWGQGTQVTVSS (SEQ ID NO:61);(g) ENB-1D11:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGRTNYADSVK GRFTISIDNAKNSIHLQMNSLKPDDTARYYCNSPYHALWGQGTQVTVSS (SEQ ID NO:62); and (h) ENB-ID11_R56H:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGHTNYADSVK GRFTIALWGQGTQVTVSS (SEQ ID NO:63).

[0024] In aspects of the method(s), the animal is not administered antibiotic. In another aspect, the animal is poultry. In an embodiment, the poultry is chicken or turkey. In one embodiment, the chicken or turkey are raised in a commercial setting or hatchery.

[0025] In embodiments of the method(s), the animal or poultry is administered at least two doses of the composition. In an embodiment, a first dose is administered on the day of birth or hatch. In an embodiment, at least one dose is administered in drinking water or is sprayed on feed or food. In an embodiment the first dose is administered in a gel diluent and gel drops are administered hours after or on the day of birth or hatch.

[0026] In embodiments of the method, subclinical NE mortality is present in the absence of the composition. In an embodiment, NE mortality is less than 10% in the absence of the composition. In an embodiment, mortality is reduced by at least 25% with administration of the composition. In an embodiment, mortality is reduced by at least 40% with administration of the composition. In an embodiment, mortality is reduced by greater than 40% with administration of the composition.

[0027] In an embodiment, feed conversion ratio (FCR) is lowered by at least 4 points. In some embodiments, feed conversion ratio (FCR) is lowered by at least 5 points. In some embodiments, feed conversion ratio (FCR) is lowered by at least 7 points.

[0028] In some embodiments, wherein weight gain is significant. In an embodiment, weight gain is significant in at least two phases of growth of the animal, poultry, or chicken.

[0029] In an embodiment, the animal is not administered an antibacterial agent or antibiotic. In an embodiment, the animal is not administered an antibacterial agent or antibiotic directed against C. perfringens.

[0030] In one embodiment, the animal is selected from poultry, sheep, cows, pigs, goats and horses. In an embodiment, the animal is selected from lambs, calves, pigs and foals. In one embodiment, the animal is poultry. In one embodiment, the animal is turkey or chicken. In one such embodiment, the poultry is chicken. In an embodiment, the chicken is a broiler chicken or a laying hen.

[0031] In an embodiment, the animal or poultry is administered a dose or first dose of the composition on the day of birth or upon or shortly after hatching. In an embodiment, the animal or poultry is administered at least two doses of the composition. In an embodiment, the animal or poultry is administered at least three doses of the composition.

[0032] In an embodiment, a first dose is administered on the day of birth or hatch, or within one to 2 days of birth or hatch. In an embodiment, first dose is administered on the day of birth or hatch, within hours of birth or hatch. In an embodiment, first dose is administered on the day of birth or hatch, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours of birth or hatch.

[0033] In an embodiment, the first dose is administered by spray administration. In an embodiment, the first dose is administered in a water mediated diluent. In an embodiment, the first dose is administered in a gel. In an embodiment, the first dose is administered in a gel and is dropped on the animal(s), such as in gel drops or droplets.

[0034] In an embodiment, at least one dose is administered in drinking w'ater or is sprayed or dropped on feed or food.

[0035] In an embodiment at least one dose is administered in drinking water or is sprayed or dropped on feed or food at least 7 days, 8 days, up to 14 days, up to 15 days, 14 days, 16 days, up to 1 days after birth or hatch. In an embodiment at least one dose is administered in drinking water at least 7 days, 8 days, up to 14 days, up to 15 days, 14 days, 16 days, up to 18 days after birth or hatch. In an embodiment at least one dose is administered in drinking water or is sprayed on feed or food at a dose of at least 1 x 106CFU / animal least 7 days, 8 days, up to 14 days, up to 15 days, 14 days, 16 days, up to 18 days after birth or hatch.

[0036] In an aspect of the method, mortality is reduced and productivity is improved. In an aspect of the method each of mortality and feed conversion ratio are decreased and body weight is increased in the animal, particularly poultry or chicken. In an aspect of the invention, final weight of an animal, particularly mean final weight of production animals (animals grown for food such as poultry or chickens or turkeys) is increased. In an aspect, the animal is exposed to subclinical NE by subclinical infection via C. perfringens, such as wherein the pen, enclosure, cage, etc where it is housed is experiencing a subclinical C. perfringens infection such as whereby the NE mortality across the animals in the pen, enclosure, cage etc is below 10%, particularly below 5%, particularly in the range of 2-5%.

[0037] In one embodiment, mortality is reduced by at least 20%. In one embodiment, mortality is reduced by at least 25%. In one embodiment, mortality is reduced by at least 30%. In an embodiment, mortality is reduced by at least 40%. In one embodiment, mortality is reduced by at least 45%. In one embodiment, mortality is reduced by about 50%. In one embodiment, mortality is reduced by more than 25%. In one embodiment, mortality is reduced by more than 40%. In one embodiment, mortality with a subclinical infection rate and mortality at less than 10% is reduced by more than 25% with administration of the composition. In one embodiment, mortality with a subclinical infection rate and mortality at less than 5% is reduced by more than 25% with administration of the composition. In one embodiment, mortality with a subclinical infection rate and mortality at less than 5% is reduced by more than 40% with administration of the composition.

[0038] In an embodiment, feed conversion ratio (FCR) is lowered significantly. In an embodiment, feed conversion ratio (FCR) is lowered by at least 4 points. In an embodiment, feed conversion ratio (FCR) is lowered by at least 5 points. In an embodiment, feed conversion ratio (FCR) is lowered by at least 6 points. In an embodiment, feed conversion ratio (FCR) is lowered by at least 7 points.

[0039] In an embodiment, weight gain is significant. In an embodiment, weight gain is significant in at least two phases of growth of the animal, poultry, or chicken or turkey.BRIEF DESCRIPTION OF DRAWINGS

[0040] FIG. 1 depicts a graph of total % NE mortality in both the Challenge Control (Group 3) with the standard deviation bar shown as a dashed line and in the LRCVP treatment group (Group 4) shown with standard deviation bar as a solid line.

[0041] FIG. 2 depicts a graph of survival - the number of chick subjects remaining on each study day through to the end of the study day 28 comparing results from birds treated with LRCVP at a dose of I x 106CFU / chick shown in the top line to birds in the Challenge Control group in the bottom line.

[0042] FIG. 3 provides a graph of the Prevented Fraction (PF) of NE mortality in the group of chicks vaccinated with LRCVP.

[0043] FIG.4 provides a plot of the FCR Challenge results on Days 15 (lightest bar on the left of each set of bars), 28 (darker bar in the middle of each set of bars), and 43 (darkest bar on the right of each set of bars). Results are provided for the control, Bacitracin Methylene Dicalicylate (BMD), 2 doses of LRCVP (LRCVPx2), a single dose of LRCVP (LRCVPxl) and the chassis strains (L. reuteri strain 3632 and 3630, not expressing nanobody).

[0044] FIG. 5 A depicts average of adjusted feed conversion ratio (FCR) on day 15. Results are provided for the control, Bacitracin Methylene Dicalicylate (BMD), 2 doses of LRCVP (LRCVPx2), a single dose of LRCVP (LRCVPxl) and the chassis strains (L. reuteri strain 3632 and 3630, not expressing nanobody).

[0045] FIG. 5B depicts average of adjusted feed conversion ratio (FCR) on day 28. Results are provided for the control, Bacitracin Methylene Dicalicylate (BMD), 2 doses of LRCVP (LRCVPx2), a single dose of LRCVP (LRCVPxl) and the chassis strains (L. reuteri strain 3632 and 3630, not expressing nanobody).

[0046] FIG. 5C depicts average of adjusted feed conversion ratio (FCR) on day 43, Results are provided for the control, Bacitracin Methylene Dicalicylate (BMD), 2 doses of LRCVP (LRCVPx2), a single dose of LRCVP (LRCVPxl) and the chassis strains (L. reuteri strain 3632 and 3630, not expressing nanobody).

[0047] FIG. 6 provides a plot of mean weight gain for Test Groups 1 through 4, measured and indicated on day 15, day 28 and day 43. Results are provided for the control, Bacitracin Methylene Dicalicylate (BMD), 2 doses of LRCVP (LRCVPx2), a single dose of LRCVP (LRCVPxl ) and the chassis strains (L. reuteri strain 3632 and 3630, not expressing nanobody).DETAILED DESCRIPTION

[0048] The present disclosure provides live probiotic vectors, compositions of the same, and methods for combatting necrotic enteritis, particularly in poultry.

[0049] In one embodiment, the invention provides a method of using a composition of at least one recombinant L. reuteri vector for in situ delivery of nanobodies against NetB and a toxin to combat necrotic enteritis (NE). In one embodiment, the invention provides a method of using a composition of at least one recombinant L. reuteri vector for in situ delivery of nanobodies against NetB and a toxin to combat necrotic enteritis (NE) and improve performance and productivity. In one embodiment, the invention provides a method of using a composition combining recombinant L reuteri vector strains for in situ delivery of nanobodies against NetB and a toxin to combat necrotic enteritis (NE) and improve performance and productivity, particularly in poultry, particularly in chickens.

[0050] In another embodiment, the invention provides a method of using a composition of a combination of two recombinant L. reuteri vectors (strains) for in situ delivery of nanobodies against NetB and a toxin, wherein one of the two vectors delivers nanobodies against NetB and the other delivers nanobodies against a toxin. In another embodiment, the invention provides a method of using such a composition wherein the two vectors are present in about a one to one ratio in the composition.Lactobacillus reuteri Strains

[0051] One or more strains of Lactobacillus reuteri with probiotic and inherent anti- infective activity have been identified and can be produced using methods known in the art.

[0052] Lactobacillus reuteri strains 3630 and 3632 have been described and detailed as novel strains suitable as DFMs, including in combination, and also as suitable strains for genetic modification and as live delivery or production strains. Lactobacillus reuteri strain 3632 was deposited on 19 June 2020 in the ATCC Patent Depository and assigned ATCC Patent Deposit Number PTA-126788. Lactobacillus reuteri strain 3630 was deposited on 19 June 2020 in the ATCC Patent Depository and assigned ATCC Patent Deposit Number PTA- 126787.

[0053] The L reuteri strains 3630 and 3632 are described as probiotic strains in PCT / US2020 / 016668, published as WO 2020 / 163398. A live delivery system based on L reuteri strain 3630 or 3632 is described and detailed in PCT / US2020 / 016522, published as WO 2020 / 163284. This PCT / US2020 / 016522 application describes native bacterial promoters, signal sequences suitable for expression and vectors and bacterial genome sites / genes for integration to generate stable modified strains.

[0054] In an earlier study, the two particlar Limos ilactobacillus reuteri (L. reuteri) isolates, ATCC PTA- 126787 (Z. reuteri 3630) and ATCC PTA-126788 (Z. reuteri 3632) were found to possess favorable safety properties based on the results from in silico, in vitro and in vivo analyses in chickens and Sprague Dawley rats (Gangaiah et al (2021) PLoSOne 17(1), e0262663).

[0055] In embodiments of the invention, a isolated first Lactobacillus reuteri strain (3632 or an equivalent / variant strain) is utilized and includes at least one of: a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3, and a nucleic acid that encodes for an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.

[0056] In some embodiments, a second isolated second Lactobacillus reuteri strain (3630 or an equivalent / variant strain) includes at least one of: a nucleic acid sequence having at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28, and a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at feast 98%, or at least 99% sequence identity with SEQ ID NO: 29.

[0057] In one embodiment, the isolated first Lactobacillus reuteri strain has a nucleic acid or amino acid sequence including at least one of SEQ ID NOs: 1-24, 26, and 49-55, sequences having at least one nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 1-24, 26, and 49-55, sequences having one or more nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 1 -24, 26, and 49-55, sequences having at least one nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 1-24, 26, and 49-55 and further having at least 99% sequence identity with at least one of SEQ ID NOs: 1-24, 26, and 49-55, sequences having at least one nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 1-24, 26, and 49-55 and further having at least 99% sequence identity with one or more of SEQ ID NOs: 1-24, 26, and 49-55, sequences having at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with at least one of SEQ ID NOs: 1-24, 26, and 49-55. In one embodiment, the isolated first Lactobacillus reuteri strain has a genomic nucleic acid sequence including at least one of SEQ ID NOs: 49-55, sequences having one or more nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 49-55, sequences having at least one nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 49-55 and further having at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with one or more of SEQ ID NOs: 49-55, sequences having at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NOs: 49-55. In an embodiment, the isolated first Lactobacillus reuteri strain is Lactobacillus reuteri strain 3632, which corresponds to ATCC Patent Deposit Number PTA-126788.

[0058] In one embodiment, the isolated first Lactobacillus reuteri strain comprises or has a genomic nucleic acid sequence corresponding to the genomic nucleic acid sequence of ATCC strain PTA-126788, or a variant thereof comprising or having a nucleic acid sequence at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to the genomic nucleic acid sequence of ATCC strain PTA-126788.

[0059] In some embodiments, the isolated second Lactobacillus reuteri strain has a nucleic acid sequence or amino acid sequence including at least one of SEQ ID NOs: 25, 27-43, and 44-48, sequences having at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with at least one of SEQ ID NOs: 25, 27-43, and 44-48. In one embodiment, the isolated second Lactobacillus reuteri has a nucleic acid or amino acid sequence including at least one of SEQ ID NOs: 25, 27-43, and 44-48,sequences having at least one nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 25, 27-43, and 44-48, sequences having one or more nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 25, 27-43, and 44-48, sequences having at least one nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 25, 27-43, and 44-48 and further having at least 99% sequence identity with at least one of SEQ ID NOs: 25, 27-43, and 44-48, sequences having at least one nucleic acid or amino acid sequence difference from the sequence of at least one of SEQ ID NOs: 25, 27-43, and 44-48 and further having at least 99% sequence identity with one or more of SEQ ID NOs: 25, 27-43, and 44-48, sequences having at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with at least one of SEQ ID NOs: 25, 27-43, and 44-48. In one embodiment, the isolated second Lactobacillus reuteri strain has a genomic nucleic acid sequence including at least one of SEQ ID NOs: 44-48, sequences having one or more nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 44-48, sequences having at least one nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 44-48 and further having at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with one or more of SEQ ID NOs: 44-48, sequences having at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NOs: 44-48. In an embodiment, the second Lactobacillus reuteri is Lactobacillus reuteri strain 3630, which corresponds to ATCC Patent Deposit Number PTA- 126787.

[0060] In one embodiment, the isolated second Lactobacillus reuteri strain comprises or has a genomic nucleic acid sequence corresponding to the genomic nucleic acid sequence of ATCC strain PTA- 126787, or a variant thereof comprising or having a nucleic acid sequence at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to the genomic nucleic acid sequence of ATCC strain PTA- 126787.

[0061] Necrotic enteritis (NE) is one of the most economically impactful diseases of poultry with estimated global losses ~$6B USD per year. Financial losses due to NE can occur from high mortality rates (1-40%) and also from poor performance due to subclinical disease (manifested in altered feed conversion to body weight and reduced overall body weight of animals for example). Further, restricted use of antibiotics as growth promoters and the voluntary implementation of ‘drug-free’ broiler production program have further led to increased incidence of NE.

[0062] In high mortality situations NE mortality can be in the range of 10-52%. This can be modeled in NE Mortality models where challenge models are designed and evaluated where C. perfrmges NE-mediated mortality is at least 10% of animals and up to just over 50% of animals. In other models, which resemble subclinical situations and particularly commercial- like environments, C. perfrmges NE- mediated mortality is on the order of 2-5% of animals. Such commercial-like environment models seekto evaluate and determine overall productivity, such as body weight of animals and feed conversion to body weight. In commercial environments, there is often an inherent low / subclinical level of NE and more limited C. perfringens infection across or throughout the animal housing, particularly where animals are introduced periodically where some are infected, even if sub clinically. Modeling such subclinical environments and demonstrating positive net effects on productivity can be particularly relevant and promising for positive outcome and methods in commercial-like settings.

[0063] Necrotic enteritis is a complex disease and requires a multipronged solution. The stages of the disease include inflammation, altered intestinal integrity, dysbiosis (imbalanced gut microbiome), followed by C. perfringens infection. In accordance with the invention, compositions and methods are provided which impact and mitigate multiple stages, particularly suppressing inflammation, improving gut functionality, stabilizing the gut microbiome, and also inhibiting the C. perfringens pathogen. This results in significantly enhanced overall affects and impacts to prevent NE via a multipronged solution.

[0064] In an embodiment of the invention, the L. reuteri vector composition is a combination of a recombinant form of a culture of the isolate ATCC PTA-126787 (Z. reuteri 3630) and of the isolate ATCC PTA-126788 (Z. reuteri 3632), where the strains deliver one or more nanobodies targeted specifically against C. perfringens. In an embodiment, the Z. reuteri vector composition is a combination of a recombinant form of a culture of the isolate ATCC PTA-126787 (Z. reuteri 3630) and of ATCC PTA- 126788 (Z. reuteri 3632), where one of the strains delivers nanobodies against NetB and the other delivers nanobodies against a toxin. These strains of L. reuteri were previously initially evaluated as live vectors for in situ delivery of llama derived Nbs against C. perfringens NetB and a toxin to prevent NE in poultry (Gangaiah et al, 2022, “Recombinant Limosilactobacillus (Lactobacillus) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis,” MicrobiologyOpen, 022; 11:el270, pp 1 -37). A high mortality model was evaluated (greater than 10% mortality in the challenged control) and only mortality was assessed and described. Further, various dosing scenarios, dosing amounts, timing, as well as productivity and performance effects including feed conversion, body weight were not evaluated or described.Antibodies Including Nanobodies Directed Against C. PerfringesI. Clostridium perfringens Toxin Antibodies

[0065] Toxins to be targeted by single chain antibodies include Clostridium perfringens alpha toxin and NetB. Camelid heavy-chain only (VHH) antibodies against C. perfringens alpha toxin and NetB are generated. Briefly, two llama calves each are immunized with either recombinant alpha toxin or NetB variant W262A. Neither of these immunogens are haemolytic. The immunized llamas are boosted twice with toxin peptides. On days 44 and 72 after the primary immunization, blood samples are taken andRNA isolated for phage library construction. Phage libraries are screened for binding activity towards each of the two toxins. The candidate antibodies are sequenced and further screened in bioassays.A. Alpha Toxin Antibodies

[0066] Alpha toxin causes membrane damage to a variety of erythrocytes and cultured cells. It is preferentially active towards phosphatidylcholine (PC or lecithin) and sphingomyelin (SM), two major components of the outer leaflet of eukaryotic cell membranes. The N-terminal domain possesses full activity towards phosphatidylcholine but lacks the sphingomyelinase activity and is not haemolytic or cytotoxic. The C-terminal domain is devoid of enzymatic activity, but interaction between the N- and C- terminal domain is essential to confer sphingomyelinase activity, haemolytic activity and cytotoxicity to the toxin. Although alpha toxin is a potent haemolysin, the lysis of erythrocytes is only seen after intravenous administration of toxin in experimental animals or in cases of clostridial septicaemia.

[0067] The inhibitory capacity of the VHH antibodies directed towards alpha toxin on the alpha toxin lecithinase activity is determined by measuring its effect on egg yolk lipoproteins. Fresh egg yolk is centrifuged ( 10,000 x g for 20 min at 4 °C) and diluted 1: 10 in PBS. The ability of the VHHs to neutralize the alpha toxin activity is assessed by pre-incubating a two-fold dilution series of the VHHs (two wells per dilution, 5 pM starting concentration) with a constant amount of alpha toxin (either 5 pg / ml recombinant alpha toxin or 3.33 x 10-4 U / pl alpha toxin from Sigma, P7633) for 30 minutes at 37 °C prior to the addition of 10 % egg yolk emulsion. As a positive control, serum from calves immunized with the recombinant alpha toxin is used, starting from a 1:4 dilution. After incubation at 37 °C for 1 hour, the absorbance at 650 nm (A650) was determined. Alpha toxin activity is indicated by the development of turbidity which results in an increase in absorbance.

[0068] Control serum is able to neutralize the lecithinase activity of both the commercial and the recombinant alpha toxin. An eight- fold dilution of the antiserum (corresponding to 3.12% serum) is able to completely neutralize the alpha toxin lectihinase activity of the recombinant alpha toxin, whereas only the highest dilution of the antiserum (corresponding to 25% serum) is able to completely neutralize the lecithinase activity of the commercial alpha toxin. Difference in inhibitory capacity is observed between five candidate VHH antibodies. VHH EAT-1F3 had no effect on the lecithinase activity of either of the alpha toxins. The neutralizing capacity of EAT- 1 A2 and EAT- 1 C 8 is very similar and is the same for both the recombinant and commercial alpha toxin. The maximal inhibitory capacity is preserved until a 32-fold dilution (0.16 pM VHH) of the VHHs. However, both EAT-1A2 and EAT-1C8 are unable to completely neutralize the lecithinase activity, resulting in 40% to 50% residual lecithinase activity. Two other VHHs, EAT-1F2 and EAT-1G4 show' a difference in neutralizing capacity towards the recombinant and the commercial alpha toxin. EAT-1F2 has a high neutralizing capacity towards the recombinant alpha toxin but is unable to completely neutralize the commercial alpha toxin, resulting in about 25% residuallecithinase activity. In contrast to EAT- 1 F2, EAT- 1 G4 neutralizes 100% of the lecithinase activity of the commercial alpha toxin, but is less capable of neutralizing the recombinant alpha toxin.

[0069] Neutralization of the alpha toxin haemolytic activity by the VHH antibodies directed towards alpha toxin is determined by measuring its effect on sheep erythrocytes. Similar to the inhibition of the alpha toxin lecithinase activity, the ability to neutralize the haemolytic activity is assessed by preincubating a two-fold dilution series of the VHH antibodies (two wells per dilution, 5 pM starting concentration) with a constant amount of alpha toxin (6.25 x 10-5U / µl alpha toxin from Sigma, P7633) for 30 minutes at 37 °C prior to the addition of 1% sheep erythrocytes. As a positive control, serum from calves immunized with the recombinant alpha toxin is used, starting from a 1:4 dilution. After incubation at 37 °C for 1 hour, the plates are centrifuged to pellet intact red blood cells. The supernatant is transferred to a new 96 well plate and the A550 is determined. Alpha toxin activity is indicated by the increase in absorbance due to release of haemoglobin from the erythrocytes.

[0070] The inhibitory capacity of the VHH antibodies towards the alpha toxin haemolytic activity is determined using the commercial alpha toxin only, as the recombinant alpha toxin shows no haemolytic activity. Up to a 16-fold dilution of the control serum (corresponding to 1.56% serum) completely inhibits the alpha toxin haemolysis. To the contrary, none of the candidate VHHs has an effect on the haemolytic activity of alpha toxin. Because the control serum contains polyclonal antibodies, whereas the VHHs are monoclonal, the combined effect of all 5 VHHs towards alpha toxin is determined (1 pM of each VHH in the highest dilution, corresponding to 5 pM VHHs in total). Combining the VHHs has no effect on the alpha toxin haemolysis.

[0071] Based on the above results, VHH antibodies EAT-IF2 and EAT-IG4 are selected for further characterization and expression. The VHH antibody sequences, including a C terminal section of sequence associated with vector expression and including a poly histidine amino acid sequence useful for characterization and isolation (but not altering or influencing nanobody binding to target antigen) (depicted in bold) are set out below:

[0072] The peptide sequence of EAT-1F2 including a C terminal section of sequence associated with vector expression and a poly histidine C -terminal amino acid sequence is:EVQLVESGGGLVQAGGSLRLSCAGSGRTGSLYSMGWFRQAPGKEREFVAAITWRPSSTY YADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGT QVTVSSAAASGSLEQKLISEEDLNGAAHHHHHHGAA (SEQ ID NO:64)

[0073] An R27H mutant of EAT-1F2 including a C terminal section of sequence associated with vector expression and a poly histidine C -terminal amino acid sequence may also be generated to improve protease resistance of the antibody:EVQLVESGGGLVQAGGSLRLSCAGSGHTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYA DSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVT VSSAAASGSLEQKUSEEDLNGAAHHHHHHGAA (SEQ ID NO:65)

[0074] The peptide sequence of EAT-1G4 including a C terminal section of sequence associated with vector expression and a poly histidine C-terminal amino acid sequence is:EVQLVESGGGLVQPGGSLRLSCAASGSIATFNDMGWFRQAPGKQRDWVATIVSDGSTAY DSVK. GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHYGQGTQVTVSSAAASGSL EQKLISEEDLNGAAHHHHHHGAA (SEQ ID NO:66)

[0075] A person of skill in the art would recognize that, because of the redundancy of the genetic code, multiple nucleic acid sequences could encode the above peptides. However, an exemplary nucleic acid sequence encoding EAT-1F2 is: (SEQ ID NO:67)1 GAGGTGCAGC TCGTGGAAAG TGGCGGAGGT CTTGTTCAGG CTGGGGGATC GCTCCGTCTG61 AGCTGTGCGG GGTCTGGCAG AACAGGTAGT CTCTATTCCA TGGGTTGGTT TCGGCAGGCC121 CCGGGTAAGG AGCGGGAGTT CGTTGCAGCG ATTACGTGGA GGCCCAGCTC TACCTACTAC181 GCGGACAGCG TAAAGGGACG ATTCACCATT AGTAGAGACG ACGCAAAGAA TACTGTATAT241 TTGCAGATGA ATTCGTTGAA GCCTGAGGAC ACCGCTGTCT ATTTTTGCGC GGCGCGACCG301 AGGGGCGGTC TCTCCCCGAC ACCTCAAGCA TATGATTACT GGGGACAAGG GACCCAAGTC361 ACTGTATCCA GTGCGGCCGC GAGCGGCAGC CTTGAACAAA AGCTGATAAG CGAGGAGGAT421 CTCAATGGTG CTGCACATCA TCATCACCAT CACGGGGCAG CG

[0076] Exemplary nucleic acid sequence encoding EAT-1G4 is: (SEQ ID NO:68)1 GAAGTTCAGC TTGTAGAGTC CGGTGGGGGT CTTGTACAGC CCGGCGGGAG CTTGCGACTC61 TCATGCGCTG CTTCCGGAAG CATTGCGACA ATAAATGATA TGGGTTGGTT TAGACAAGCC121 CCCGGGAAGC AGCGTGACTG GGTCGCGACT ATTGTGAGTG ACGGCAGCAC GGCTTATGCG181 GACTCAGTGA AAGGGAGATT TACGATTTCG CGAGATAACG CGAAAAACAC TGTATACCTG241 CAGATGAATT CACTCAAGCC GGAAGATACA GCTGTGTATT ATTGTTCTGC CCGACGGCAC301 TACGGACAGG GGACCCAGGT CACAGTCTCG AGCGCTGCCG CCAGTGGGTC ACTCGAGCAG361 AAGCTGATAT CAGAGGAGGA CCTTAACGGT GCGGCGCACC ATCACCACCA TCATGGTGCG421 GCGB. NetB Antibodies

[0077] NetB is a heptameric beta-pore-forming toxin that forms single channels in planar phospholipid bilayers. The NetB activity is influenced by membrane fluidity and by cholesterol, which enhances the oligomerization of NetB and plays an important role in pore formation. NetB has high haemolytic activity towards avian red blood cells.

[0078] Neutralization of the NetB haemolytic activity by camelid VHH antibodies directed towards NetB is determined by measuring NetB-mediated lysis of chicken erythrocytes. The ability to neutralize NetB haemolytic activity is assessed by pre-incubating a two-fold dilution series of the VHH antibodies (two wells per dilution, 5 pM starting concentration) with a constant amount of NetB toxin (20 pg recombinant NetB) for 30 minutes at 37 °C prior to the addition of 1% chicken erythrocytes. The nontoxic NetB variant W262A is included as a negative control as this variant displays no haemolysiticactivity. Positive control serum from rabbits immunized with the recombinant NetB (wild type NetB) is used, starting from a 1:4 dilution. After incubation at 37 °C for 1 hour, the plates are centrifuged to pellet intact red blood cells. The supernatants is transferred to a new 96 well plate and the A550 is determined. NetB activity is indicated by the increase in absorbance due to release of haemoglobin from the erythrocytes.

[0079] The control serum is able to neutralize the haemolytic activity of NetB. VHH antibodies ENB- 1F4 and ENB-1F10 have no effect on the NetB haemolysis. ENB-1B9 has intermediate inhibitory capacity, while ENB-1 DI 1 and ENB-1A4 are able to neutralize the NetB haemolysis up to a 4- to 8-fold dilution (1.25 pM -- 0.625 jiM VHHs).

[0080] Based on the above results, VHH antibodies ENB-1 A4 and ENB-1 DI 1 are selected for further characterization and bacterial expression. The VHH antibody sequences, including a C terminal section of sequence associated with vector expression and including a poly histidine amino acid sequence useful for characterization and isolation (but not altering or influencing nanobody binding to target antigen) (depicted in bold) are set out below:

[0081] The peptide sequence of ENB-1 A4 including a C terminal section of sequence associated with vector expression and a poly histidine amino acid sequence is:EVQLVESGGGLVQAGGSLRLSCAASGSIFSTNVMGWYRQAPGKQREFVAGITIGGTARYP DSVKGRFTISRDNTQNTVYLQMNNLKPEDTAVYYCNAVLPSDQRRWSWGQGTQVTVSS AAASGSLEQKLISEEDLNGAAHHHHHHGAA(SEQ ID NO:69)

[0082] The peptide sequence of ENB- 1 D 11 including a C terminal section of sequence associated with vector expression and a poly histidine amino acid sequence is:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGRTNYA DSVKGRFTISIDNAKNSIHLQMNSLKPDDTARYYCNSPYHALWGQGTQVTVSSAAASGS LEQKLISEEDLNGAAHHHHHHGAA (SEQ ID NO: 70)

[0083] An R56H mutant of ENB-ID11 including a C terminal section of sequence associated with vector expression and a poly histidine amino acid sequence may also be generated to improve protease resistance of the antibody:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGHTNYADS VKGRFTISIDNAKNSIHLQMNSLKPDDTARYYCNSPYHALWGQGTQVTVSSAAASGSLEQK LISEEDLNGAAHHHHHHGAA (SEQ ID NO:7I)

[0084] A person of skill in the art would recognize that, because of the redundancy of the genetic code, multiple nucleic acid sequences could encode the above peptides. However, an exemplary nucleic acid sequence encoding ENB-1A4 is: (SEQ ID NO:72)1 GAGGTACAAC TGGTTGAGAG TGGGGGTGGT TTGGTGCAAG CCGGAGGTTC CTTACGTTTG61 TCTTGCGCGG CTAGTGGGAG CATCTTTTCA ACAAACGTAA TGGGGTGGTA CCGCCAAGCC 121 CCAGGTAAGC AGCGGGAATT TGTGGCCGGG ATACGATCGG AGGAACTGCG AGGTATCCT 181 GATAGTGTGA AAGGGCGTTT CACAATTAGT CGAGATAATA CACAGAATAC TGTCTATCTC 241 CAAATGAATA ATCTCAAGCC CGAAGACACA GCAGTTTATT ATTGTAATGC CGTTCTCCCC 301 TCTGATCAGC GTCGATGGAG CTGGGGACAA GGCACCCAGG TTACGGTTAG CAGCGCGGCA 361 GCGTCTGGTT CGCTCGAGCA AAAGCTCATA TCTGAGGAGG ACCTGAACGG GGCAGCCCAC 421 CATCACCACC ATCACGGAGC AGCT

[0085] An exemplary nucleic acid sequence encoding ENB-1D11 is: (SEQ ID NO:73)1 GAGGTACAGC TGGTGGAGTC CGGCGGTGGT TTGGTGCAAA CCGGGGGTAG TCTGCGGCTT61 AGTTGCACGG CGTCTGGGAC AATAGACATG ACTTATGGTC TCATATGGTA CAGGCAAGCG121 CCTGGGAAAG AGAGGGAACT CGTTGCGAGT ATCAGAAGGG ACGGCCGCAC AAATTACGCT181 GATTCAGTGA AAGGGCGCTT CACTATCTCG ATCGATAATG CGAAAAACAG TATTCACCTT241 CAAATGAACT CCCTTAAGCC CGATGATACC GCCAGGTATT ATGCAACAGC CCATATCAC301 GCACTTTGGG GTCAGGGTAC GCAGGTAACA GTGTCTAGTG CGGCAGCCTC TGGTAGTTTG361 GAGCAAAAGT TGATAAGTGA GGAGGACTTA AATGGGGCGG CACATCACCA CCACCATCAT421 GGGGCGGCT

[0086] In an embodiment of the invention, the nanobody (Nb) against NetB is aNb having an amino acid sequence selected from one of the following:

[0087] ENB-1A4:EVQLVESGGGLVQAGGSLRLSCAASGSIFSTNVMGWYRQAPGKQREFVAGITIGGTARYPDSVK GRFTISRDNTQNTVYLQMNNLKPEDTAVYYCNAVLPSDQRRWSWGQGTQVTVSS (SEQ ID NO:61),

[0088] ENB-ID11:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGRTNYADSVK GRFTISIDNAKNSIHLQMNSLKl’DDTARYYCNSPYI-LALWGQGTQVTVSS (SEQ ID NO:62), and

[0089] ENB-ID 11. _R56H:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGHTNYADSVK GRFTIALWGQGTQVTVSS (SEQ ID NO:63).

[0090] In an embodiment of the invention, the nanobody (Nb) against a toxin is a Nb having an amino acid sequence selected from one of the following:

[0091] EAT-IF2:EVQLVESGGGLVQAGGSLRLSCAGSGRTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:56);

[0092] EAT-IF2 R27H:EVQLVESGGGLVQAGGSLRLSCAGSGHTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:57);

[0093] EAT-1F2JT28P:EVQLVESGGGLVQAGGSLRLSCAGSGRPGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVrVSS (SEQ ID NO:58);

[0094] EAT-IG4:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHYGQGTQVTVSS (SEQ ID NO:59); and

[0095] EAT- 1 G4 Y 103 W:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHWGQGTQVTVSS (SEQ ID NO:60).

[0096] Any one or more of the above nanobodies are / can be delivered by the bacteria, particularly a direct feed microbial, particularly one or more or both of the Lactobacillus strains detailed and described herein.

[0097] In aspects of the invention, the strains, particularly Lactobacillus, particularly Lactobacillus reuteri, in aspects strains L. reuteri 3630, or 3632 or 3630 and 3632, or an equivalent or variant thereof, are genetically engineered via a specific expression vector to express and secrete the one or more nanobodies as provided herein. In an embodiment, the expression vector includes a promoter derived from the strains, particularly in an aspect a promoter derived from L. reuteri 3630 or 3632. In an embodiment, the expression vector includes a promoter derived from the strains, particularly in an aspect a promoter derived from L. reuteri strain 3632.

[0098] The expression vectors include expression cassettes. In one embodiment, the expression casettes are integrated in the Lactobacillus strain genomes for stability' and continued expression and secretion, including through generations and days of growth of the strains, including prior to administration and after administration to the animals, including poultry, including such as chickens.

[0099] In aspects, the expression cassettes include sequences at each end that are homologous to target strain genomic sequence, particularly at the site of integration. In an embodiment, the site of integration is pyrE and the sequences at each end that are homologous to pyrE sequence.[000100] Exemplary expression cassete sequences for expression and secretion are provide here for illustration:[000101] L. reuteri ME 01 Expression Cassette with 1-kb homologous regions on either side[0(10102] GAATAGATGGGAGCAATGCGA CGTAGTGGGACGACCAATCAOCCAGAGCTT GACCCAGTTGCTGCTTATCAAGAAG CCA GACCAACCATTTACCTGGGCAAGCGGTCTAAHATCTCCAJtTCTATACCGATAATCSATTGACGATTTCTTATCCAGAAGTTCGGCAAGCA& TT TTTAATGGGATGGTTGAACAAATTAAACTTCATTTTAGTGAGACCGATGTTATTGCCGGAACAGCAACTGCTGGAATTCCTCA GCGGCATGG GTTGCCCAGAATATGGAATTACCGATGATTTATGTTCGGACTAAGCCGAAAGATCATGGTCAAGGCAAGCAAAT GAAGG& S GCTTAAAGAA GGACA A GTCGTCGTG G G CCT CAACTGGTG T GT T TTA CGCTGTGCGGGCGGT AACA TGC GGC G A A A ATTGGCGTTGTTTCTGTCTTTACTTATGATTTGCCAGCTGC GAACA1AAACTTTATGGCAAATGGACTAAAATACTATTCAGTAACGGATT T ATGACTTTGATTAAGGTCGCAAAGGAAAATGACCAAATTAGTGCGGACCACCrCAAGTCTCTTCAAGAATGGCGGAAAGACCCGTTAAGCTGG AGi^AAGSG^GCATrara^lAATTAAAAGGCTGGATTTTTTCGGCCTTTTTTTAGTGCAAATAATTATTTTTTACGT ATTTATATTATAGGGCTAATCACTAAACTAAT / iATTAGTGGTTGAAGCGCTGAAAATTTTCTGCTATTTTATTA AT AG TTT GATAATAAAATA A T G AT AT T T AAT AT AAAGAGGG A T AAAC GAAA T AJiTGAAATCAACAAGAAAGAAA ATTCTTGCATCGTCGTTAGGGG'rAGCTGGCGCAATGGCAATGGGCACGGTAACTGCA AGGCTGATACGACCGT TACGGTCAATGCTGGCGATAGTTTGA TGGGATTGCTCAAAAGTATAATGTTAGTGCGGATGATATTGCAACCG CTAATCACTTGCAAAATAAAGAGTTGATTTCTGTGGGACAAAAGTTGACAATTCCAACCAAAGATAAAAATGAA ACAGAAcyr CAACTTGTAGAGTCCGGTGGGGGATTAGTTCAACCGGGTGGGTCATTACGATTATCATGCGCTGC TAGCGGATCTATTGCAACAATCAACGACATGGGCTGGTTTCGTCAJGGCTCGAGGTAAGCAACGGGAT’fGGGTAG CCACT TAGTT GTGAC G TCG CAGCTT TGC G CTCAG A GGG CGTTTC CTAT AGTCGGGATAAT GCGAAAAATACGGTCTACCTCCAAATGAATTCTCTAAAACCTGAAGATACCGCCGTTTATTATTGTAGCGCACG C GACACTACGGACAAGGCACCt^GGTTAC GTGAGTAG GCCGCTGCA CTGGATCACTGGAAC AAA'E'gSA TTAaTGMGAAGATGTTAACGGTGCAGCTCAgGACCATQATCATGATGGTGGAGCGTACTTCCCTGGAAATTM:TATCTTGTGATGX^TTAATATTCCCTTTCACCTCACCTrTAaTAATTZ^TTAg'r^TTATCTCeCgCATCaC AA^^GAGTGCTA ATACTATTTCAGATTAGAAAGTTTTATGAGGGAGAC^&&^GTAA TAATTATCAAAAAAGCCTAA GATTGATTCAGAAAAAAGTTAATCTGAATTAATCTTAGGCTT ATCTTA't'TGTTTATCTTCCCAAATCTTTTGGAGCATTGGGTAGCGTAAGT CACCAACTTGTACTACTTGATAATCTGCATTTGCTAATGTTGTAGCATCACCAACGCCAATTGCAATTGCATTTATATGGTGAATATTTTCAA T TCA TGAGC TGTCGTTCCAACTCCAA TGCAA TTAGCTCC'GA TAAGA TTTAA T GA TTAACAGCAGTTAGA TA TGGGCTTTCTTTA TC TTAG TCCTAACGATACTATCGACATAATTATCAAGGCCAATTTGTTTAAGGACATTTTGTACATCTCCATTAGCATCTAACAC& GCAATTTTA& CGT AATGATCATAGAGATTTAGTAAAAGACGC’i'TAATCCCTGGAAGTTGATCCTTTTCACTTAGGC ATCCGCTTCTTCGTTAAGGAATTTTTGCC A TCTGCAATCATTGAAGCCTTTTCGCT GGGGCAGGATTAGCATTAAAATGTGCTAAGACTAGGGAGAGCCCTTGCGAA‘XAGTAAGGTTAT CGAATn^(^(^TAATTT CX’AGGl! AATCCCATCCKAAATTCATACATTACAAAACG rGCCAl^CAGTAAAGGATAAC AT TGTArTA3 CA AA G A TA TTA TTAAACGAAAAAA TCGCACCA TCCA TGA TTAAACA T CCC T TCA T AGA TAA T TA TG T C TA TA T T T TAACGTAATCGAAAATGA AAGGGAATACATTAiATTAAGCATTTACTTTAGCGAGGTTAGCGGCATAATCGATTCCCTTTACATCTAAATACTCAATAAT GATTGATTGT AATAAATGGTACATGAACi'CGGTTAGCAAGGACTGACAGACTATCACGACGAGCCATCGTGCCGTCACGCTTTAACACTTCGATAATAACAGC AACTGGTCZ: CTTTCCTGCTAAATAGGCAAGATCAATAGCTGCTTCGGTGTGACCGATTCGATCTCTTAJiTCCATGAGGTTGGGCGTAAAGAGG TTGAATGTGGCCGGGA GATTAAAGTCAGATCCTTGGGCAGTTGGCT GGCAAT TGCCGAATCGTTGCTGCGCGGTCAAAAGCGGAAACACC AGTAGTAAC CCGGTTGCTTCTAAGGZACCATCAGT GTA CC'ATAAA GGTG TTGGZG (SEQ ID NO:74) Italicized: 5 ' and 3 ’ homologous regions in the L, reuteri 3630 chromosome Regular text: CwlS promoterUnde lined text: CwlS secretion signalBold text: CwlS anchor sequencesBol and Undesri ined text: VHH1 (EAT-1G4, alpha toxin nanobody) Italicized snd under 1 ined: Terminator[000103] 1. reuteri NE06 Expression Cassette with 1-kb homologous regions on either side[080104] GAfi?v; GaTSGtAGC^JSTGCG^rC'G2?iGTGGG / ; CcyiCC^A2,C / iCC'rAGAGCrTr,GAC'CC'Ae7'? GCTSC:"7A7CAAGAaGI'CaA AAAAGCATTCTTAAATGAGGAGGAAGAAAAATGACATATTCACAACGCGTTGCAAAAGCATTATTAGATATTCATGCTGTTACCCTTAGCCCA GACCAACCATTTACCTGGGCAAGCGGTCTAAAATCTCCAATCTATACCGATAATCGATTGACGATTTCTTATCCAGAACTTCGGCAAGCAATT TTTAA TGGGA TGGTTGAACAAA TTAAACTTCA TTTTAGTGAGGCCGA TGTTA TTGCCGGAACAGCAACTGCTSGAA TTCCTCA TGCGGCA TGGGTTGCCCAGAAZAZG AATZACCGAZGATT'tAI'GZTCGGACTAA CCGAAAGAT'CAZGGI'CAAOGOAAGCAAAT'rGAAGGG TGCTlAAAGAA GGACAAAAAGTCGTCGGGATTGATGACCT'lATT CAACTGGTGGTAGTGTATTAAACGCTGTGCGGGCGG TAACAATGCAGCO GAAAAG A ATTGGCQTTGT'r CTGTCTGTACTTATGATTTGCCAGC’rGCTGAACAAAACTTTAAGGCAAATGGAC'AAAAATACTA’rTCAGT& ACGGATTAT AGGACAGTGATTAAGGTCGCAAAGGAAAATGACCAAATTAGGGCGGACCACCTCAAGTCTCT'ACAAGAATGGCGGAAAGACCCGTTAAGCTGG AGTAAAGAGCAAGCATCATAAAAATTAAAAGGC GGATTTTTTCGGCCTTTTTTTAGTGCAAATAATTATTTTTTACG ATTTATATTATAGGGCTAATCACTAA CTAAT ATTAGTGGTTGAAGCGCTGAAA TTTTCTGCTATTTTATTA ATAG I T TGATAAT AAAATAATGAT T I TAAT G AAAGAGGGATAAACGAAATAA3«AAA GAAeAAOAA GAAA ATTCTTGCATCGTCGT GGG AGCTG CGCAATGGCAA GGGC CGGT ACTGC A GGCTGA ACGACCGT TACGGTCAATGCTGGCGATAGTTTGAATGGGATTGCTCAAAAGTATAATGTTAGTGCGGATGATATTGCAACCG CTAATCACTTGCAAAATAAAGAGTTGATTTCTGTGGGACAAAAGTTGACAATTCCAACGAAAGATAAAAATGAA ACAGAAGTACAACTTGTTGAATCCGGTGGCGGATTGGTGCAGACTGGTGGGTCACTTAGATTAAGTTGTACTGC TAGTGGTACGA TGATATGACCTATGGACTGATTI’GGTATCGCCAAGCGCCAGGAaAAG. AGCGTGAAC’.ryG G C TCT TTCG CGGG CGG C AC A T CGCGGATTCTGT A GG TCGG T C ATCTCT AGAC AT GCC ^AAC CAA ’IC. A^TACAAA GAA'EAGrTTAAAACCGGATGATACCGCCCGTTATTACTGCAATTCACC TTATC CGCTCTC'TGGGGCCAAGG^AC^CAGGTC^C G^ TC<1AGCGC G^GCTAGCGGATCA T. GAACAAA ^T^jTTAG^^AA^TaTAA^GGTG^^CAgMCATaACCATaATC^gGT^AgCT’i’ACTTCCCTGGA AATTACTATGTTGTGATGCATTAATATTCCCT^CA CTCACCTTiAAlAATTTAAATTAGTAA'rA TCTCGCg CATCACAAA^GAGTGCTATATACTATTTCAGATTAGAAA^TTTTATGAGGGAGACAfiATTGtRKt't& Kl'tKtCRRKRk AGCCTAAGATTGATTCAGASAAAACTTAATCTGA TTAATCTTAGGCTTTATCTTATTGTTTATCT CCCAAAtCTTTTGGAGCATTGGGTAG CGTAAGTCACCAACTTGTACTACTTGATAATCTGCATTTGCTAATGTTGTAGCATCACCAACGCCSATTGCAATTGCAT TATATGGTGAATA TTTrCAArATCATGAGCTGTCGTTCCAACTCCAATGCRATTAGCTCCGaTAAGATTTAATTGATTAACAGCAGTTAGATATGGGC T CTTTA TC TTAGTCCTAACGATACTATCGACATA 'raATCAAGGCCAATTTGnrrT AGG CATTITGTACATCTCCATTAGCATCT ACACGGCAATT TTAACGTAATGATCATAGAGATTTAGTAAAAGACGCTTAATCCCTGGAAGTTGATCCTTTTCACTTAGGCTATCCGCTTC'fTCGTTAaGGAAT TTTTGCCATTCTGCAATCATTGAAGCCTTTTCGCTTGGGGCAGGATTAGCATTAAAATOTGCTAAGACTAGGGAGAGCCCTTGCGAAGGACTA AGGTTATCGaATTGGGAAGCTAATTTTCCAGCTAATCCCATCCaaAATTCATACATTACAAAACGTTGCCATGCAGTSAAGGATAACTTATTT GTATTAACAAAGATATTATOAAACGAABAAATCGCaCCATCCATGATTAA CATCCCTTCATAGATAATTATGTC ATATTTTAACGTAATCG AAAATGAaAGGGAATACATTAATTAAGCATTTACTTTAGCGAGGTTA&t^GCATAATCGATTCCCTTTACATCTAAATACTCAATAATTTGAT TGaTTGTAATAAATGGTACATGAaCTCGGTTAGCAAGGACTGACAGACTATCACGACGAGCCATCGTGCCGTCACGCTTTAACACT’rCGATAA TAACAGCAACTGGTGCCTTTCCTGCTAAATAGGCAAGA’rCABTftGCTGCTTCGGTGTGACCGATTCGATCTCTTAATCCATGAGGTTGGGCGT AAAGAGGTTGAATGTGGCCGGGATGATTAAAGTCAGATGCTTGGGCACTTGGCTTGGCAATTTGCCGaATCGTTGCTGCGCGGTCAAAAGCGG AAACACCAGTAGTAACACCC-GTTGCTTCTBAGGTACCATCAGTAGTAACCATAAATGGTGTTTGGTG (SEQ ID O:75) Italicized: 5fand 3' homologous regions in the L. renter! 3632 chromosome Regular text: CwlS promoterUnderlined text: CwlS secretion signalBold: CwlS anchor sequencesBold and Underlined text: VHH3 (ENB-1D11 R56H, NetB nanobody)Italicized a n d un der Lined: Terminator[000105] In an embodiment, the strains expressing one or more nanobody against C perfringes each and / or together have activity or capability for one or more of decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, reducing inflammation, and decreasing mortality rate in an animal. In embodiments, the strains expressing one or more nanobody against C perfringes together have activity or capability for decreasing feed conversion ratio, increasing body weight, increasing lean body mass, and decreasing mortality rate in an animal.[000106] In some embodiments, compositions disclosed herein include an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain at a ratio of approximately 0.75-1.5: 1. In a preferred embodiment, the composition includes about equal amounts of the isolated first Lactobacillusreuteri strain and the isolated second Lactobacillus reuteri strain, or approximately 1: 1. In an embodiment, the composition includes about equal amounts, such as equal amounts measured as CFU / kg or CFU / ml of the composition, of the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain, or approximately 1:1.[000107] The compositions disclosed herein can be formulated as animal feed, feed additive, food ingredient, food additive, medicament additive or ingredient, water additive, water-mixed additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. In one embodiment, the composition includes water.[000108] In some embodiments, the compositions disclosed include the isolated first Lactobacillus reuteri strain in an amount of about 102- 108CFU / kg of the composition, about 10s- 108CFU / kg of the composition, about 104- 107CFU / kg of the composition, about 10 05CFU / kg of the composition, about 102CFU / kg of the composition, about 103CFU / kg of the composition, about 106CFU / kg of the composition, about 107CFU / kg of the composition, or about 108CFU / kg of the composition. In some embodiments, the compositions disclosed herein includes the isolated first Lactobacillus reuteri strain in an amount of about I02-108CFU / ml of the composition, about 106- 10sCFU / ml of the composition, about 104- 107CFU / ml of the composition, about 103- 105CFU / ml of the composition, about 103CFU / ml of the composition, about 104CFU / ml of the composition, about 105CFU / ml of the composition, about 106CFU / ml of the composition, about I07CFU / ml of the composition, or about 108CFU / ml of the composition.[000109] In some embodiments, the compositions disclosed herein includes the isolated second Lactobacillus reuteri strain in an amount of about 102- 108CFU / kg of the composition, about 106- 108CFU / kg of the composition, about 104-107CFU / kg of the composition, about 103- 105CFU / kg of the composition, about 102CFU / kg of the composition, about 103CFU / kg of the composition, about 106CFU / kg of the composition, about 107CFU / kg of the composition, or about 108CFU / kg of the composition. In some embodiments, the compositions disclosed herein includes the isolated second Lactobacillus reuteri strain in an amount of about 102- 108CFU / ml of the composition, about 106- 108CFU / ml ofthe composition, about 104- 107CFU / ml of the composition, about 103- 105CFU / ml of the composition, about 103CFU / ml of the composition, about 104CFU / ml ofthe composition, about l(F CFU / ml of the composition, about 106CFU / ml of the composition, about 107CFU / ml of the composition, or about 10sCFU / ml of the composition.[000110] In particular embodiments, the compositions disclosed herein include two isolated Lactobacillus strains each expressing at least one nanobody directed against C. perfringens in an amount of about 102- 10sCFU / animal ofthe composition. In particular embodiments, the compositions disclosed herein include two isolated Lactobacillus strains each expressing at least one nanobody directed againstC. perfringens in an amount of about 102- 108CFU / animal or bird of the composition. In some embodiments, about 102- 108CFU / animal or bird of the composition is administered in at least two doses to an animal. In some embodiments, about 102- 108CFU / animal or bird of the composition is administered in at least two doses to poultry. In some embodiments, about 102- 108CFU / animal or bird of the composition is administered in at least two doses to a chicken,1000111] In particular embodiments, the compositions disclosed herein include two isolated Lactobacillus strains each expressing at least one nanobody directed against C. perfringens in an amount of about 106CFU / animal or bird of the composition. In some embodiments, about 106CFU / animal or bird of the composition is administered in at least two doses to an animal. In some embodiments, about 106CFU / animal or bird of the composition is administered in two doses to an animal. In some embodiments, about 106CFU / animal or bird of the composition is administered in three doses to poultry. In some embodiments, about 106CFU / animal or bird of the composition is administered in two doses to poultry. In some embodiments, about 106CFU / animal or bird of the composition is administered in at least two doses to a chicken.[000112] In particular embodiments, the compositions disclosed herein include two isolated Lactobacillus strains each expressing at least one nanobody directed against C. perfringens in an amount of at least 106CFU / animal or bird of the composition. In some embodiments, at least 106CFU / animal or bird of the composition is administered in at least two doses to an animal. In some embodiments, at least 106CFU / animal or bird of the composition is administered in two doses to an animal. In some embodiments, at least 106CFU / animal or bird of the composition is administered in three doses to poultry. In some embodiments, at least 106CFU / animal or bird of the composition is administered in at least two doses to poultry. In some embodiments, at least 106CFU / animal or bird of the composition is administered in at least two doses to a chicken.[000113] In embodiments, the compositions comprising a combination of Lactobacillus strains expressing a nanobody directed against NetB and a nanobody directed against a toxin are administered in a first administration to animals, particularly poultry, particularly chickens, on the day of or shortly after (hours after or less than 24 hours after) hatch or birth. In embodiments, a first administered composition comprises two isolated Lactobacillus strains each expressing at least one nanobody directed against C. perfringens formulated in a water based diluent for administration, such as by spray administration, in an amount of at least 106CFU / animal or bird. In some particular embodiments, a first administered composition comprises two isolated Lactobacillus strains each expressing at least one nanobody directed against C. perfringens formulated in a gel diluent for administration, such as by spray administration or particularly by gel drop, in an amount of at least 106CFU / animal or bird. In some embodiments, thesuitable gel diluents is selected from Hydrodrop gel (Cleared, Westbrook. ME) and CEVAGEL (Ceva Animal Health, Lanexa, KS).[000114] The present disclosure also provides methods of increasing animal health, wherein the method includes administering an effective amount of the composition to an animal. The present disclosure provides methods of improving animal performance or productivity, wherein the method includes administering an effective am ount of the composition to an animal. In some embodiments, improving animal performance or productivity comprises one or more of decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, reducing inflammation, or decreasing mortality rate. In some embodiments, improving animal performance or productivity comprises decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen- associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, reducing inflammation, and decreasing mortality rate. In some embodiments, improving animal performance or productivity comprises decreasing feed conversion ratio, increasing weight, increasing lean body mass, and decreasing mortality rate. Methods are provided for decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, reducing inflammation, and decreasing mortality rate.[000115] The composition disclosed herein and above increases animal health by providing positive health benefits when administered to an animal, as compared to an animal that has not been administered the composition. As used herein, “animal” includes bird, a human, or a non-human mammal. Specific examples of birds include poultry such as chickens or turkey. Specific examples of animal include chickens, turkey, dogs, cats, cattle, goats, horses and swine. The animal may be poultry', including chickens or turkeys. The chicken may be a broiler chicken or egg-laying or egg-producing chicken. The animal may be a human. The animal may be a non-human mammal.[000116] Positive health benefits include decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, reducing inflammation, and decreasing mortality rate.[000117] The compositions may be administered orally, parentally, nasally, or mucosally. Parental administration includes subcutaneous, intramuscular and intravenous administration.[000118] In some aspects, administration includes feeding the animal, or spraying onto the animal. In other aspects, administration includes on ovo administration or in ovo administration. In an embodiment, administered comprises in ovo administration. In some aspects, administration includes feeding the poultry, or spraying onto the poultry'. In some aspects, feeding includes providing via drinking water,such as adding to drinking water that is consumed in its entirety, or nearly in its entirety. In other aspects, administration includes on ovo administration or in ovo administration. In an embodiment, administered comprises in ovo administration. In an embodiment, administered comprises spray administration. In an embodiment, administered comprises immersion, intranasal, intramammary, topical, or inhalation.[000119] In some aspects the animal is vaccinated in conjunction with administration. The animal may be vaccinated prior to administration of the compositions disclosed herein. The animal may be vaccinated with an coccidiosis vaccine. Coccidiosis vaccines are known in the art, for example, COCCIVAC.[000120] In some embodiments, administration is by way of injection or infusion. In one embodiment, the composition is administered to a cow by way of intra-mammary infusion.[000121] In an embod iment of the method(s), the method does not comprise administration of an antibiotic.[000122] In some embodiments, the compositions or combinations may additionally include one or more prebiotic. In some embodiments, the compositions may be administered along with or may be coadministered with one or more prebiotic. Prebiotics may include organic acids or non-digestible feed ingredients that are fermented in the lower gut and may serve to select for beneficial bacteria. Prebiotics may include mannan-oligosaccharides, fructo- oligosaccharides, galacto- oligosaccharides, chito- oligosaccharides, isomalto- oligosaccharides, pectic- oligosaccharides, xylo- oligosaccharides, and lactose- oligosaccharides.[000123] The compositions may further include one or more component or additive. The one or more component or additive may be a component or additive to facilitate administration, for example by way of a stabilizer or vehicle, or by way of an additive to enable administration to an animal such as by any suitable administrative means, including in aerosol or spray form, in water, in feed or in an injectable form. Administration to an animal may be by any known or standard technique. These include oral ingestion, gastric intubation, or broncho-nasal spraying. The compositions disclosed herein may be administered by immersion, intranasal, intramammary, topical, mucosally, or inhalation. When the animal is a bird the treatment may be administered in ovo or by spray inhalation.[000124] Compositions may include a carrier in which the bacterium or any such other components is suspended or dissolved. Such carrier(s) may be any solvent or solid or encapsulated in a material that is non- toxic to the inoculated animal and compatible with the organism. Suitable pharmaceutical carriers include liquid carriers, such as norma! saline and other non-toxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose and which can also be incorporated into feed for farm animals. When used for administering via the bronchial tubes, the composition is preferablypresented in the form of an aerosol. A dye may be added to the compositions hereof, including to facilitate checking or confirming whether an animal has ingested or breathed in the composition.[000125] When administering to animals, including humans or farm animals, administration may include orally or by injection. Oral administration can include by bolus, tablet or paste, or as a powder or solution in feed, food, or drinking water. Administration may be by ingestion. The method of administration will often depend on the species being feed or administered, the numbers of animals being fed or administered, and other factors such as the handling facilities available and the risk of stress for the animal.[000126] The dosages required will vary and need be an amount sufficient to induce a response or to effect a biological or phenotypic change or response expected or desired. Routine experimentation will establish the required amount. Increasing amounts or multiple dosages may be implemented and used as needed.[000127] The strains disclosed herein demonstrate certain phenotypic properties. Without wishing to be bound by theory, it is believed that these phenoty pic properties at least contribute to increasing anima! health.[000128] In some embodiments, the isolated strains secrete at least one of cyclic dipeptides (cyclo(his- phe) and cyclo (phe-pro), short chain fatty acids (2-hydroxy-3 -methylvalerate and alphahydroxyisocaproate), betaine, dimethylglycine, essential amino acids (e.g., allo-threonine, phosphothreonine, histidine, lysine, phenylalanine, tryptophan, leucine, isoleucine, and cysteine s-sulfate), nucleotides (e.g., adenosine 5 ’-monophosphate (AMP), uridine 5’-monophosphate (UMP), cytidine 5 ’-monophosphate (5’-CMP), and cytidine 2’3’-cyclicmonophosphate), myo-inositol, and indolin-2-one. Some of the aforementioned molecules pro ide beneficial characteristics to the host, including increased weight, pro-inflammatory' effects, and antibiotic effects.[000129] In some embodiments, the composition including the isolated first Lactobacillus reuteri strain (strain 3632) and the isolated second Lactobacillus reuteri strain (strain 3630) in combination, will secrete certain beneficial molecules in larger quantities than when individually cultured. For example, with combinations and cocultures of strains 3630 and 3632, increased levels of each of the following are provided or secreted (as determined from culture supernatants): dimethylglycine, allo-threonine, 1-methyl-4-imidazoleacetate, 4- imidazoleacetate, lysine, N6-methyllysine, N6, N6-dimethyllysine, 5- aminovalerate, and tyrosine, 4-hydroxyphenylpyruvate, indolacetate, and gamma-glutamylglutamine, glucose 6-phosphate, 4-hydroxyl-2-oxoglutaric acid, and myo-inositol, Uridine 5 ’-monophosphate (UMP), Cytidine 5 ’-monophosphate (5 ’-CMP), 3’-5’-uridylyluridine, O-sulfo-L-tyrosine, indole 3 acetamide, indolin-2-one and daidzein. In particular, when the strains 3630 and 3632 are combined in cultures or are grown together, significant and synergistic amounts (more than just additive) of somebeneficial molecules are present or secreted. In particular embodiments, significant amounts of the molecules 4-hydroxyphenylpyruvate and glucose 6-phosphate are secreted or present with combinations of strains 3630 and 3632, or with compositions including a mix of about equal amounts of strains 3630 and 3632.[000130] In some embodiments, the animal administered the composition exhibits a shift in the microbiome content of the gastrointestinal tract. For example, there may be an increase in the amount of bacteroidaceae bacteria in the gut of an animal that has been administered the composition described herein, as compared to an animal that was not administered the composition.Definitions[000131] As used herein, “isolated” means that the subject isolate has been separated from at least one of the materials with which it is associated in a particular environment, for example, its natural environment.[000132] Thus, an “isolate” does not exist in its naturally occurring environment; rather, it is through the various techniques known in the art that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture in association with an acceptable carrier.[000133] As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can include substantially only one species, or strain, of microorganism.[000134] As used herein, the term “bacterial consortia”, “bacterial consortium”, “microbial consortia”, or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest (e.g. increasing vaccine efficacy). The community may comprise two or more species, or strains of a species (eg., Lactobacillus reuteri strains 3632 and 3630), of microbes. In some instances, the microbes coexist within the community symbiotically.[000135] As used herein, the terms “colonize” and “colonization” include “temporarily colonize” and “temporary colonization”.[000136] As used herein, “probiotic” refers to a substantially pure microbe (i.e., a single isolate) or a mixture of desired microbes, and may also include any additional components (e.g., carrier) that can be administered to an animal to provide a beneficial health effect. Probiotics or microbial compositions ofthe invention may be administered with an agent or carrier to allow the microbes to survive the environment of the gastrointestinal tract, i.e., to resist low pH and to grow in the gastrointestinal environment.[000137] As used herein, “carrier”, “acceptable carrier”, or “pharmaceutical carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, in some embodiments as injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. The choice of carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. See Hardee and Baggo (1998. Development and Formulation of Veterinary Dosage Forms. 2nd Ed. CRC Press. 504 pg.): and E. W. Martin (1970. Remington’s Pharmaceutical Sciences. 17th Ed. Mack Pub. Co.).[009138] As used herein, “delivery” or “administration” means the act of providing a beneficial activity to a host. The delivery may be direct or indirect. An administration could be by an oral, nasal, or mucosal route. For example without limitation, an oral route may be an administration through drinking water, a nasal route of administration may be through a spray or vapor, and a mucosal route of administration may be through direct contact with mucosal tissue. Mucosal tissue is a membrane rich in mucous glands such as those that line the inside surface of the nose, mouth, esophagus, trachea, lungs, stomach, gut, intestines, and anus. In the case of birds, administration may be in ovo, i.e. administration to a fertilized egg. In ovo administration can be via a liquid which is sprayed onto the egg shell surface, or an injected through the shell.[090139] As used herein, the terms “treating”, “to treat”, or “treatment”, include restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. A treatment may also be applied prophylactically to prevent or reduce the incidence, occurrence, risk, or severity of a clinical symptom, disorder, condition, or disease.[000140] As used herein, “subject” includes bird, poultry, a human, or a non-human mammal. Specific examples include chickens, turkey, dogs, cats, cattle, and swine. The chicken may be a broiler chicken, egg-laying or egg-producing chicken. As used herein, the term “poultry” includes domestic fowl, such as chickens, turkeys, ducks, quail, and geese.[000141] As used herein, the term “immunogenic” means than an agent is capable of eliciting an immune response, including an innate, humoral, or cellular immune response, and both. “Immunogenic”includes “immunomodulatory”. An immunogenic composition is a composition that elicits an innate, humoral, or cellular immune response, or both.[000142] As used herein, the term “immune response” includes a response by a subject that involves generation of antibodies that bind to an antigen (i.e., an antibody response). This does not exclude generation of a cell- mediated response.[000143] By “stimulating” is meant directly or indirectly increasing the level and / or functional activity of a target system (e.g., immune system). In certain embodiments, “stimulation” or “stimulating” means that a desired / selected response is more efficient (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), more rapid (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), greater in magnitude (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), and / or more easily induced (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more) than if the vaccine had been used alone.[000144] The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U. S. Patent Nos. 4,816,397 and 4,816,567. The term “antibody(ies)” includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAI, and IgA2). Also included within the meaning of the term “antibody” are any “antibody fragment”.[000145] An “antibody fragment” means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CH I domains; (iv) a variable fragment (Fv), which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a Single Chain Fv Fragment wherein a VII domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94 / 13804; P. Holiiger et al Proc. Natl. Acad, Sci. USA 906444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson, J Immunol. Methods 242: 193-2049 (2000)); (xii) a minibody, which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sei 17(4):315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9): 1126- 1136); and (xiii) other non-full length portions of heavy and / or light chains, or mutants, variants, or derivatives thereof, alone or in any combination. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are included.[000146] As antibodies or nanobodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, w'hether natural or wholly or partially synthetic, including a nanobody. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.[000147] An "antibody combining site" is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.[000148] Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab1, F(ab')z and F(v), or the VH sequence or nanobody, which portions are preferred for use in the therapeutic methods described herein.[000149] Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recru it an effector function or the like. Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anti-cancer or anti-tumor specific antibody. The other binding domain may be an antibody thatrecognizes or targets a particular cell type, as in a neural or glial cell-specific antibody. In the bispecific antibodies of the present invention the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cel! receptors and / or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor. Thus, the MSLN antibod ies of the invention may be utilized to direct or target agents, labels, other molecules or compounds or antibodies in indications such as wound healing, inflammation, cancer or tumors.[000150] The term “antigen binding domain” describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody heavy chain variable region (VH).[000151] Immunoconjugates or antibody fusion proteins of the present invention, wherein the antibodies, antibody molecules, or fragments thereof, of use in the present invention are conjugated or attached to other molecules or agents further include, but are not limited to such antibodies, molecules, or fragments conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, antimicrobial agent or peptide, cell wall and / or cell membrane disrupter, or drug.[000152] The term “adjuvant(s)” describes a substance, compound, agent or material useful for improving an immune response or immune cell or component stimulation, and may in some instances be combined with any particular antigen in an immunological, pharmaceutical or vaccine composition.Adjuvants can be used to increase the amount of antibody and effector T cells produced and to reduce the quantity of antigen or immune stimulant or modulator and the frequency of injection. Although some antigens are administered without an adjuvant, there are many antigens that lack sufficient immunogenicity' to stimulate a useful immune response in the absence of an effective adjuvant. Adjuvants also improve the immune response from "self-sufficient" antigens, in that the immune response obtained may be increased or the amount of antigen administered may be reduced. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin / Cummings:Menlo Park, California, p. 384). In a preferred aspect an adjuvant is physiologically and / or pharmaceutically acceptable in a mammal, particularly a human. The standard adjuvant for use in laboratory animals is Freund's adjuvant. Freund's Complete adjuvant (FCA) is an emulsion containing mineral oil and killed mycobacteria in saline. Freund's incomplete adjuvant (FIA) omits the mycobacteria. Both FIA and FCA induce good humoral (antibody) immunity, and FCA additionally induces high levels of cell-mediated immunity'. However, neither FCA nor FIA are acceptable for clinical use due to the sideeffects. In particular, mineral oil is known to cause granulomas and abscesses, and Mycobacterium tuberculosis is the agent responsible for tuberculosis. Previously known and utilized adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvant such as BCG (bacille Calmette-Guerin) and Corynebacteriu parvum. Mineral salt adjuvants include but are not limited to: aluminum hydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide and calcium hydroxide. Preferably, the adjuvant composition further comprises a lipid of fat emulsion comprising about 10% (by weight) vegetable oil and about 1-2% (by weight) phospholipids. Preferably, the adjuvant composition further optionally comprises an emulsion form having oily particles dispersed in a continuous aqueous phase, having an emulsion forming polyol in an amount of from about 0.2% (by weight) to about 49% (by weight), optionally a metabolizable oil in an emulsion-forming amount of up to 15% (by weight), and optionally a glycol ether-based surfactant in an emulsion-stabilizing amount of up to about 5% (by weight). There have been many substances that have been tried to be used as adjuvants, such as the lipid-A portion of gram negative bacterial endotoxin, and trehalose dimycolate of mycobacteria. The phospholipid lysolecithin exhibited adjuvant activity (Arnold et al., Eur. J Immunol. 9:363-366, 1979). Some synthetic surfactants exhibited adjuvant activity, including dimethyldioctadecyl ammonium bromide (DDA) and certain linear polyoxypropylenepolyoxyethylene (POP-POE) block polymers (Snippe et al., Int. Arch. Allergy Appl. Immunol. 65:390-398, 1981; and Hunter et al., J. Immunol. 127:1244-1250, 1981).[000153] The term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partners). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.[000154] It should be appreciated that also within the scope of the present invention are sequences for or encoding specific binding members (antibodies, nanobodies etc) of the invention which code for e.g. an antibody or nanobody having amino acid sequence as provided herein, but which are degenerate thereto. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.[000155] Mutations can be made in the sequences encoding the amino acids, antibody fragments, VHHs or nanobodies, CDR region sequences thereof, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an aminoacid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention includes sequences containing amino acid changes and substitutions, including conservative changes, which do not significantly alter the activity or binding characteristics of the resulting protein.[000156] Two amino acid sequences are "highly homologous” or "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90% or 95% or 98% or 99%) are identical, or represent conservative substitutions. The sequences, CDR regions or VH or heavy chains of two antibodies or nanobodies are substantially homologous when one or more amino acids, or one or a few, or one to three, or one or two are substituted with a similar or conservative amino acid substitution, and wherein the antibody / antibodies / nanobodies have the profile of binding and activities of one or more of the antibodies / nanobodies.[000157] Performance in commercial poultry / chicken settings is defined by high-efficiency production metrics ---primarily fast growth for broilers and high egg output for layers — achieved through optimized, data-driven environments. Key Performance Metrics include feed conversion ratio, European production efficiency factor, mortality, uniformity, and body weight. Feed Conversion Ratio (FCR): The primary measure of efficiency. Modem broilers can achieve an FCR of 1.5, meaning 1.5 kg of feed produces 1 kg of weight, allowing a 2 kg bird to reach market weight in roughly 29 days. European Production Efficiency Factor (EPEF): A common formula,Liveability(%)xLive Body Weight (kg)xl00 / Age (days)xFCR, is used to assess overall flock success. Mortality: In well-managed systems, mortality is kept minimal, though high-density, fast-growth environments can increase risks from heat stress (above 2.5°C from optimum) or, in the case of free-range, environmental factors. Uniformity: Carcass weight uniformity is crucial for processing, with beter results often seen in summer months or smaller flock sizes (11,000-30,000 birds).[000158] Any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such nonlimiting examples andillustrations includes, but is not limited to: “for example,” “for instance,” “e.g.,” and “in one embodiment.” In this specification, groups of various parameters containing multiple members are described. Within a group of parameters, each member may be combined with any one or more of the other members to make additional sub-groups. For example, if the members of a group are a, b, c, d, and e, additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.[000159] Throughout this specification, quantities are defined by ranges, and by lower and upper boundaries of ranges. Each lower boundary can be combined with each upper boundary to define a range. The lower and upper boundaries should each be taken as a separate element. Two lower boundaries or two upper boundaries may be combined to define a range.[000160] The following embodiments and aspects illustrate and are not intended to limit scope of the present invention. Instead, these embodiments and aspects provide guidance to any skilled artisan on how' to prepare and use compositions and methods taught by the present invention, w'here such skilled artisans will appreciate that modifications may be made without departing from the spirit and scope of the invention. The present disclosure is exemplified by specific embodiments below.[000161] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLES[000162] The abbreviations and acronyms in Table I, below, are used in the Examples below':[000163] Table 1i Abbreviation Definition! AE Adverse EventBAR Bright, alert, and responsiveBMD Bacitracin Methylene DicalicylateBSL Bio-Safety LevelCFR Code of Federal RegulationsCFU Colony Forming UnitsCp Clostridium perfringensCOA Certificate of Analysiscs Coarse SprayDOA Day(s) of AgeDOH Day-of-hatchDW Drinking WaterEMA Eimeria maximaFSR Final Study ReportID IdentificationLR NEO I Limosilactobacillus reureri vectored nanobodies against C. perfiringens alpha toxin LRNE06 Limosilactobacillus reureri vectored nanobodies against C. perfringens NetB toxin LRCVP Limosilactobacillus reureri combination vector product comprising LR NE01 and LRNE06MRS De Man, Rogosa and Sharpe (agar)Nb NanoantibodyNE Necrotic EnteritisPCT Polymerase Chain ReactionRPM Revolutions Per MinuteSD Study DaySMF Study Master FileSOP Standard Operating ProcedureFSM Veterinary Service MemorandumEXAMPLE 1Production of L. reuteri Combination Vector Product[000164] L. reuteri 3630 and 3632 were isolated from chicken cecum. Morphology of these strains include opaque, circular colonies with slight whitish center. The LR 3630 colonies have whitish pigmentation and the LR 3632 colonies include dull orange pigmentation. These strains are non-spore forming.f 000165] A Limosilactobacillus reureri combination vector product (LRCVP) was prepared from strains of LR NE01 and LR NE06 produced independently using the same production process, as follows, and formulated with cryoprotectant before mixing together. The LR NE01 strain used was a L. reuteri strain 3630 with intact pyrE delivering EAT-1G4 (Nb against alpha toxin) while LR NE06 used was a L. reuteri strain 3632 with intact pyrE delivering ENB-1D11 R56H (Nb against NetB). (See above descriptions of exemplary expression sequences; Gangaiah, Dharanesh et al MicrobiologyOpen, 16 February 2022, pages 1-37, incorporated herein by reference). Descriptions of L. reuteri strains 3630 and 3632 used to produce the LR NE01 and LR NE06 strains can be found in PCT publications WO 2020 / 163284 Al and WO 2023 / 076353.[000166] Seed vials of LR NE01 were inoculated into one single flask containing 400 mL of Limosilactobacillus reuter / -(LR-4) media (Table 2) while seed vials of LR NE06 were inoculated intoanother single flask containing 400 mi of the same media, both under microaerophilic conditions at 37°C for 16 hours. After that about 350 mL of each resulting seed culture was transferred to a separate production fermenter containing 7L of LR-4 media. The production fermentors used microaerophilic operations at 37°C, pH 5.5, with an agitation rate of 100 RPM. Fermentation continued until cells reached stationary phase (about 6-9h) as indicated by osmolality measurements. Bacteria were then harvested by centrifugation and cell pellets resuspended in a 10% lactose cryoprotectant solution. To combine strains, the formulated solutions of LR-NE01 and LR-NE06 were mixed at approximately 1-1 ratio based on osmolality measurements. This solution was then transferred into 20 mL sterile serum vials (7 mL fill volume), frozen at -80o C for at least 12 hours and lyophilized to yield the final LRCVP. The LRCVP was stored at 2 - 8°C.[000167] The batch of LRCVP used in the studies described in the next two Examples below was prepared using X+23 passages of LR NE01 and LR NE06 from master seeds, while the batch of LRCVP used in the study described in the third Example below was prepared using X-r3 passages of LR NE01 and LR NE06 from the same master seeds.[000168] Table 2 - Media Recipe for LR-4 Media, per LiterIngredient Amount (g / L)Yeast Extract 20K2IIPO4 2MgSO4*7H2O 0.1MnSO4*H3O 0.05Ammonium Citrate 2.Sucrose 50Tween 80 1Purified Water 800 plus amount added forfinal volume of 1 LiterEXAMPLE 2Efficacy of LRCVP in Broiler Chickens Against a Virulent Necrotic Enteritis Challenge [000169] In initial studies, a coarse spray of LRCVP at 1 x 108CFU / chick was administered at day 0, followed by LRCVP at 1 x 108CFU / chick added to daily water / drinking water at Day 13. Challenge with C. perfringens was given at day 19 and again at day 20, and the study was terminated at day 28-29. In a first evaluation, mortality was reduced 51% and in a second similar evaluation, mortality was reduced 25%. Meta analysis confirmed significant efficacy.[000170] Additional studies to further evaluate were conducted as follows:[000171] I. Objective:[000172] The objective of this study was the demonstration of reduction in mortality after primary' coarse spray (CS) vaccination of broiier chicks with LRCVP on day of hatch (DOH) followed by booster vaccination with LRCVP via drinking water (DW) at 13 days of age (DOA) at a target dose of 1 x 106CFU / chick against NE challenge. Mortality was evaluated for each chicken after receiving a predisposing dose of Eimeria maxima (Ema) on SD 14 and a virulent challenge of C. perfringens (Cp) on SD 19, SD 20, and SD 21 (if needed). The chickens were observed daily through the termination of the study on day 28 (SD 28).[000173] 2. Study Design:[000174] 2.1 Treatment Groups:Table 3Group Treatment Group Size / I DOH CS SD 13 DW Challenge Study Number of Vaccination Vaccination Termination Cages | Target Dose Target Dose| CFU / Bird# CFU / Bird#1 Negative 11 / 1: Distilled Water Distilled Water NoneControl2 Eimeria 11 / 1 i Distilled Water Distilled Water SD 14 EmaControl3 Challenge 484 / 44 I Distilled Water Distilled Water SD 14 Ema SD 28 Control SD 19 CP4 Vaccinate 484 / 44 | I x lO61 x IO6SD 20 CP **SD 21 CP**[000175] Where the following additional points are noted in Table 3, above: * Chicks were placed 11 per cage after enrollment; ** If needed to achieve 10% NE Mortality'; and # Final dose.[000176] 2.2 Study Description:[000177] Broiler chicks were used for this study as they were the intended target population. Nine hundred ninety' (990) DOH mixed sex chicks were enrolled into the study. Each chick was provided a unique identification number and enrolled into a treatment group and cage placement was done based on a randomized assignment by tag number.[000178] Treatment Group 1 (Negative Control, n=l 1, one cage) received distilled water and served as a negative control for vaccination and NE challenge. Treatment Group 2 (E. maxima Control, n=l 1, one cage) received distilled water and served as a control for Eimeria challenge only. Treatment Groups 3 and 4 each consisted of 484 chicks, 11 chicks per cage and 44 cages per group. Treatment Group 3 was administered distilled water on DOH and SD 13 and served as a challenged control group to demonstrate test subject susceptibility to NE challenge. Treatment Group 4 received the IVP at target dose of 1. OE+06 CFU / chick via the routes of administration per study design described in Table 3, above.[000179] Coarse Spray vaccination was performed using a sprayer calibrated to deliver a known amount of liquid per unit of time. Unvaccinated birds (groups 1, 2, and 3) were coarse sprayed with distilled water. All spray applications included green color dye. The target CS volume for placebo or vaccine was 0.25 rnL per bird.[000180] On SD 13, birds were water deprived for a period between 30 and 60 minutes. Unvaccinated birds (groups 1, 2, and 3) received distilled water and treatment group 4 received a target dose of the vaccine via drinking water. The drinking water from the metal troughs of each cage were emptied and the drinker lid from a 1 -liter chick drinker containing the proper amount of water or prepared vaccine solution was issued to the appropriate cage. Once all the treated water was consumed, the water troughs were replaced and filled with fresh non-medicated water.[000181] Treatment Groups 2 -4 received a dose of E. maxima on SD 14 and treatment groups 3 and 4 received a dose of C. perfringens strain CP6 on SD 19, SD 20, and SD 21, as needed to achieve target NE mortality of 20%. Following the first challenge with C. perfringens on SD 19, al! chickens were monitored twice daily (except once daily on weekends and holidays) for clinical signs of NE, morbidity, and mortality until SD 28. All dead or euthanized birds were necropsied to determine a probable cause of disease or death. Lesion scoring was done for the birds that die ofNE (Hofacre et al., 1998) and pathognomonic lesions associated with NE were photographed and scored as follows:Lesion score 1 = Slight mucus covering small intestineLesion score 2 = Necrotic small intestine mucosaLesion score 3 = Sloughed and blood small intestine mucosa and contents[000182] The birds confirmed to have succumbed to NE were included in the final statistical analysis. All live birds observed through SD 28 were included in the analysis as not affected by the NE challenge. All surviving birds were euthanized at the end of the study, necropsied, and lesion scored for NE.[000183] 2.3 Body and Feed Weight:[000184] ll birds and the feed were weighed by cage on SD 0, 14 and 28. Feed added to each cage feeder was weighed on SD 0, 14, and 28. Any additional bags of feed were weighed (and documented) for each cage during each feeding period. Feed was distributed as needed to feeders from pre-w'eighed bags (assigned to each cage) throughout each period. Feed remaining in feeders (and feed bags if applicable) was be weighed and disposed of on SD 28. The trial was terminated on SD 28.[000185] 2.4 Test Subjects and Enrollment Criteria:[000186] Broiler (Ross X Ross) Chickens {Gallus gallus) of mixed sexes were used for the study. The study of each subject began on its day of hatch (DOH). Only healthy birds were selected for the study. Chicks enrolled in the study were observed to be bright, alert, and responsive (BAR). Any chicksdemonstrating physical deformities or not exhibiting BAR behavior were excluded from enrollment in the study. Animals were identified with individual neck tags.6. Group assignment, Randomization, and Blinding:[000187] The experimental unit was an individual cage. There were 90 total experimental units, with 1 per Group I, I per Group 2, and 44 each per groups 3 and 4, as indicated in Table 3. Assignment of birds to treatments and cages was randomized. No birds were replaced in the course of the study.[000188] Partial blinding was achieved by functional segregation. Personnel responsible for treatment were not blinded to bird housing for biosecurity to prevent cross contamination between treatment groups. However, individuals performing post-challenge observations and necropsy were blinded ot the treatment assignment and group membership to prevent observational bias.[000189] 2.5 Husbandry:[000190] Feed and water were provided ad libitum throughout the study except for transient water deprivation prior to the second dose of the vaccine via the SW route on SD 13. Feed met or exceeded the minimal nutritional requirements[000191] Any birds that became ill or injured during the study for reasons not related to the vaccine or challenge were removed to avoid unnecessary distress and euthanized. Any bird unable to eat or drink due to injury or illness for a period greater than 24 hours were to be identified for removal from the study for compassionate purposes. No concomitant treatments were administered to the experimental subjects during the study.[000192] 3.0 Materials[000193] 3.1 Placebo:[000194] Chicks enrolled in a treatment group receiving a placebo (groups 1, 2, and 3) had distilled water administered using the same equipment as the vaccine.[000195] 3.2 Investigational Vaccine Preparation (IVP)[000196] LRCVP prepared as described in Example 1, above was used to vaccinate Group 4. Detailse of suspension of the LRCVP to the target dose, as defined in Table 3, above, was finalized based on the titer of the LRCVP.3.3. Challenge Materials:[000197] The per os challenge model for NE consists of a preliminaiy application of Eimeria maxima, followed by the Clostridium perjringens as previously published (Hofacre et al., 1998). E. maxima isolate MX8 was originally isolated from a single sporulated oocyst from a North Carolina field strain and has been maintained by passage through naive birds as previously described (Schneiders et al., 2019). Fecal recovered oocysts will be confirmed to be sporulated as previously described (You, 2014). The purity of this E. maxima isolate has been verified via polymerase chain reaction (PCR) as previously published(Jenkins et at., 2006). The C. perfringens strain CP6 was originally isolated from field case of NE and produces NetB and alpha-toxin as confirmed by PCR analysis performed on 29 July 2021.[000198] E. maxima oocysts were delivered at a dose of 500 to 7,500 sporulated oocysts per bird by oral gavage on SD 14. Preparation and administration, and dose confirmation of the challenge material was recorded.[000199] C. perfringens were administered by oral gavage at a target dose of about 1.0E+08 CFU per bird on SD 19, SD 20, and SD 21 targeting a mortality of about 20% in the challenged control group (Group 3). If on SD 19 or SD 20, NE mortality was approaching 15% in Group 3, then day 20 and or day 21 it was planned that challenge would not be given.[000200] Dose confirmation was conducted with five separate titrations to determine a geometric mean titer of the challenge dose for each application and recorded.[000201] 3.4 Bacterial Dose Confirmation:[000202] Following investigational vaccine preparation / use or C. perfringens challenge material preparation / application, a 6.0 mL sample was retained for dose confirmation titration and back-up reference.[000203] Dose confirmation was conducted and documented, replicating the confirmation procedure five separate times to determine a geometric mean titer of the vaccine dose as well as the Cp challenge dose.[000204] One (1.0) mL of the sample was combined with 1.0 mL sterile 50% glycerol, mixed, and divided into two 1.0 mL labeled vials for storage ≤ -50o C. The remaining 5 mL was used to perform 10- fold serial dilutions. 1 mL was removed from the undiluted aliquot and added to 9 mL sterile diluent (PBS) and mixed briefly. A ten-fold serial dilution was conducted and 100 pL from each of the serial dilutions (use 3 dilutions - target dilution, one above, and one below) was used to plate onto separate De Man, Rogosa and Sharpe agar (MRS) plates in triplicate. The plates were incubated under anaerobic conditions at 39° C ± 1° C for 18—36 hours. Following incubation, the plates were removed from the incubator, the number of CFU / mL determined from plates containing between 25-250 total colonies, and the resulting orange (LR NE06) and white (LR NE01) colonies were recorded separately.[000205] 4.0 Sequence of Events:[000206] 21 Days before Study Start Date: Eggs were placed in the incubator[000207] 3 Days before Start Date: Eggs candled to remove infertile and dead embryos.[000208] SD 0: Chicks hatched and counted, randomized ID tags applied, chicks from all groups course spray (CS) vaccinated according to treatments in Table 3, using dose of 2.68 x 106CFU / chick for LRCVP administration, allocated birds to cages, and weighed and fed by cages.[000209] SD 13: Booster vaccination of group 4 via drinking water (DW), administering LRCVP at a dose of 2.15E+06 CFU / chick.[000210] SD 14: E. maxima challenge performed by administering 2000 oocysts of E. maxima, via oral gavage, in accordance with Table 3. Birds also fed and weighed by cages.[000211] SD 19, 20, 21 - Gavaged birds with 1.0 ml of solution containing I x 10sCFU / mL of C. perfringens, and only repeated on days 20 and 21 if needed to achieve 20% NE mortality. Total of 2 x 108to 4 x 108CFU / chick of C. perfringens administered by oral gavage.[000212] SD 19-28: Observed all birds, weighed dead or euthanized birds, did necropsy of dead or euthanized birds, and did NE Lesion Scoring.[000213] SD 28: Birds were counted and weighed by cages, study was terminated, birds were euthanized, necropsies were performed, and NE Lesion Scoring was conducted.[000214] 5.0 Conclusion Criteria Used:[000215] 5.1 Validity' criteria: The study was to be considered valid if the negative control group (Treatment Group 1) and the Eimeria control group (Group 2) remained negative for mortality associated with NE.[000216] 5.2 Satisfactory criteria: The study was to be considered satisfactory if NE mortality in the challenged control group 3 is between 15%-25% given the variability of theNE challenge model.[000217] 5.3 Efficacy criteria: The I VP would be considered efficacious if any of the vaccinate groups demonstrated a stastistically significant reduction in mortality due to NE based on a prevented fraction effect size of at least 25% and a significance level (Type 1 Error rate) of 0.10.[000218] 5.4 Statistical methods:[000219] The primary variable was prevented fraction (PF) of NE mortality, measured based on NE mortality counts from the population of birds post-challenge at risk of NE. Summary statistics (n, mean, standard deviation, minimum, 25th percentile, median, 75th percentile, maximum) was presented for all Treatment Groups. The confirmatory analysis below was conducted for Treatment Groups 3 and 4, where PF was with respect to Treatment Group 3. All statistical tests were evaluated at the two-sided 0.10 significance level.[000220] NE mortality counts were modelled using a generalized linear mixed model with fixed effect Treatment Group, random effect Block and the following specifications:Distribution: y,j \bohbn ~Linear Predictor: β₀ + b0i + (β₁ + b1ᵢ)Xᵢⱼ, with (b0ᵢ, b1ᵢ) ~ Bivariate NormalLink function: Logit ηij = log( π_ij / ( 1- π_ij)[000221] A dispersion parameter was to be estimated and, if significant, a Quasi-Binomial error distribution used. The primary variable was evaluated based on 90% confidence intervals estimated via the logistic regression fit and the delta method.6.0 Results and Discussion:[000222] The study results are summarized in the following Table:[000223] Table 4Treatment No of Total Birds Challenge Vaccination Total Total NE Percent Birds Challenged on DOH & Mortality Mortality NE Enrolled SD 13 Mortality Unchallenged 11 0 None Distilled 0 0 o.Control WaterEimeria 11 11 Eimeria Distilled 0 0 0 Control WaterChallenge 484 481 Eimeria Distilled 72 63 13.1 Control + Cperf WaterLRCVP ”484 481 Eimeria LRCVP 46 32 6.65.. + C perf _ _ _..[000224] This study demonstrated a significant reduction in mortality. The NE mortality for chicks vaccinated was reduced by 48.7% with LRCVP.[000225] Figure 1 is a graph of total % NE mortality in both the Challenge Control (Group 3) with the standard deviation bar shown as a dashed line and in the LRCVP treatment group (Group 4) shown with standard deviation bar as a solid line. One can see from the graph that the difference in % NE mortality is statistically significant. There is 13.1% NE mortality in the Challenge Control (Treatment Group 3) and 6.65% NE mortality in the group vaccinated with LRCVP (Treatment Group 4).[000226] Figure 2 is a graph of survival - the number of chick subjects remaining on each study day through to the end of the study day 28 comparing results from birds treated with LRCVP at a dose of I x 106CFU / chick shown in the top line to birds in the Challenge Control group in the bottom line. There are many more chicks surviving in the LRCVP treatment group compared to the challenge control.[000227] Figure 3 is a graph of the Prevented Fraction (PF) of NE mortality in the group of chicks vaccinated with LRCVP. It shows a PF of 48.7%. The p value is 0.0023, demonstrating that the results are statistically significant.EXAMPLE 3Efficacy of LRCVP in Broiler Chickens for Affecting Performance and Mortality in a Mild NE, Commercial-Like Environment[000228] The above examples demonstrate reduction of NE mortality by 20%-48% in severe to moderate NE challenge in batery cage studies. Studies and models were then evaluated to assessproductivity in a commercial-like environment and particularly with a mild challenge and low inherent NE mortality, thus a subclinical infection situation which mimics commercial scenarios. In the commercial setting a lower portion or percentage of animals demonstrate NE mortality as the number infected and ill are a smaller portion or percentage overall. In these situations, the overall performance and productivity is important, including particularly body weight, feed intake, feed efficiency, as well as the mortality percentage.1. Objective[000229] The primary objective of this study was to quantify the effect of LRC VP on overall performance and particularly body weight, feed intake, feed efficiency, and mortality of broiler chickens housed in commercial-like conditions and subjected to a mild NE challenge. This was a full pen study. Birds were housed in dirt floor pens with new pine shavings and stocked at 26 birds per pen. Secondary objectives were to compare a 2-dose application to a single dose on day of hatch (DOH), to an antibiotic labeled for control of NE and shown to effect enhanced feed efficiency (Bacitracin Methylene Dicalicylate (BMD)), and to the application of backbone L reuteri strains which are not expressing nanobody. Tissue and excreta samples were also collected to decipher the mechanism of action of LRC VP. Subclinical NE was induced through Coccivac on day of hatch (DOH) and C. perfringens on DI 5, 16, 17 and 18. Commercially applicable diet formulations were used.2. Study Design[000230] Table 5 outlines the design of this study.Treatment Treatment Chicks per pen' NE Coarse Spray Drinking Group Description xNo. of Challenge2Vaccination Water replicates CFU / Bird3Vaccination CFU / bird31 Challenge Control 26 Chicks x Yes Water Water 20 replicates2 BMD (50 g / US ton 26 chicks x Yes Water Water feed) 20 replicates3 LRCVP 2-dose 26 chicks x Yes 1 x 1041 x 10620 replicates4 LRC VP 1-dose 26 chicks x Yes 2 x 106Water 20 replicates5 Backbone Lr3632 + 26 chicks x Yes 1 x 1061 x 104Lr3630 20 replicates[000231] The following notes apply to Table 5, where indicated:'Chickens were housed in floor pens for 43 days of live phase duration.2Necrotic enteritis challenged protocol of coccidiosis vaccine on DOH, followed by C. perfringens (strain CP6) application of 1.0E+08 CFU / bird on SD 15, 16, 17, and 28.3Final dose.3. Study Description[000232] Broiler chicks were used for this study. Twenty six hundred (2,600) chicks were assigned to five (5) treatment groups, with twenty (20) replicate blocks, and allocated into groups of twenty-six (26) birds per pen. Pens all had new litter. All treatment groups were assigned to pens using a randomized complete block design. Randomization and assignment of treatment groups to pens was completed using Random Permutation Tables (Cochran and Cox, 1992).[000233] Treatment Group 1 was administered water and served as challenge control group. Treatment Group 2 received BMD at 50 g / US ton through the feed for the entirety of the experiment. Treatment Groups 3 & 4 received the vaccine at target doses and route of administration per study design shown in Table 5 above. Treatment Group 5 received backbone L. reuteri Strains 3632 and 3630 at target doses and routes of administration similar to treatment of Group 3. The objectives for each treatment group were as follows:[000234] Group 1: Determine the baseline for untreated FCR weight gain.[000235] Group 2: Determine the effect of daily in feed antibiotic as an “active control.”[000236] Group 3: Establish FCR, weight gain effects of administration of LRCVP.[000237] Group 4: Explore / determine sufficiency of single dose administration.[000238] Group 5: Explore / determine contribution of chassis strains.[000239] Spray vaccination was conducted in the same way as in the preceding Example, above. Sprayers were calibrated to deliver a known amount of liquid per unit of time. Unvaccinated birds (Treatment Groups 1 and 2) were coarse sprayed with water. All spray applications included green color dye similar to the dyes used for coarse spray (CS) application of commercial coccidiosis vaccine at day-of-hatch. The target CS volume for placebo or vaccine was 0.25 mL per bird.[000240] On SD 0, Treatment Groups 1 and 2 were spray vaccinated with distilled water, Treatment Groups 3 and 4 were coarse spray vaccinated with a targeted dose of BE- 101 and Treatment Group 5 received the backbone strains by coarse spray. After the application of vaccine and birds were confirmed to be dry, all birds were vaccinated for coccidiosis (COCCIVAC®-B52, Merck through a commercial spray cabinet. COCCIVAC-B52 is a live oocysts vaccine isolated from chickens, prepared from anticoccidial-sensitive strains of Eimerie acervuline, E maxima, E. maxima MFP, E mivati, and E tenella. (COCCIVAC-B52 Product Bulletin).[000241] On SD 10, one chick / pen was removed and euthanized for sample collection for metagenomic, metatranscriptom ic, and histologic analyses.[000242] On SD 12 or 13, the water consumption of 2 or 3 representative pens was measured to plan for vaccination on SD 14. On SD 14, unvaccinated birds (Treatment Groups 1 and 2), and Treatment Group 4 received distilled water and Treatment Groups 3 & 5 received a targeted dose of the vaccine and backbone strains, respectively, via drinking water. Approximately 2-3 hours prior to administration, water to drinkers was turned off and drinkers were emptied. Vaccine or backbone strain-containing water was added to the volume of water the bird consumed in 30 minutes (eg 0,1 mL vaccine / bird into volume of water to be consumed). Once all the treated water was consumed, the water was turned back on.[000243] To induce subclinical NE, chicks from all the groups received a dose of C. perfringens strain CP6 on SD 15, SD 16, SD 17 and SD 28 to achieve NE mortality of < 5%. Following the first challenge with C. perfringens on SD 15, all chickens were monitored twice daily (except once daily on weekends and holidays) for clinical signs of NE, morbidity, and mortality until SD 43. All dead or euthanized birds were necropsied to determine a probable cause of disease or death. Lesion scoring was done for the birds that die of NE.[000244] On SD 18 and 31, one chick / pen was removed and euthanized for NE lesion scoring and sample collection for metagenomic, metatranscriptomic, and histologic analyses. The birds confirmed to have succumbed to NE were included in the final statistical analysis for efficacy against NE mortality. All live birds observed through SD 43 were included in the analysis as not affected by the NE challenge. On SD 43, one chick / pen was removed and euthanized for NE lesion scoring and sample collection for metagenomic, metatranscriptomic, and histologic analyses. All surviving birds were euthanized at the end of the study.[000245] Body weight and feed intake were monitored throughout the study to compare growth performance between the groups.4 Test Subjects and Enrollment Criteria:[000246] Only male Broiler (Ross X Ross) Chickens {Gallus gallus) of mixed sexes were used for the study. The study of each subject began on its day of hatch (DOH). The source of birds for the study was Aviagen Hatcher}', Blairsville, GA. HVT / SB1 vaccinations were done at the hatchery.[000247] Only healthy birds were selected for the study. Chicks enrolled in the study were observed to be bright, alert, and responsive (BAR). Any chicks demonstrating physical deformities or not exhibiting BAR behavior were excluded from enrollment in the study. The chickens were not individually identified for this study. The pen was the unit of measure, and pen security was used to prevent bird migration. 6. Group assignment, Randomization, and Blinding:[000248] The experimental unit was a pen. There were a total of 100 pens, with 20 per treatment group. Treatment groups were randomized to pens without any treatment blocking using Random Permutation Tables to do so (Cockran and Cox, 1992).[000249] Partial blinding was achieved by functional segregation using the same methods described in the preceding Example.5. Husbandry:[000250] Feed and water were provided ad libitum throughout the study except for transient water deprivation prior to the second dose of the vaccine via the DW route on SD 14 and transient feed deprived prior to C. perfringens application on SD 15, 16, 17, and 28.[000251] Rations were fed as follows: starter SD 0 through SD 15, grower SD 15 through SD 28 and finisher SD 28 to SD 43. Diets were fed as crumbles (starter feed) or pellets (grower and finisher).[000252] Feed formulations for this study consisted of un-medicated commercial-type broiler starter and grower diets compounded with commonly used United States feedstuffs representative of local formulations, calculated analyses to meet or exceed NRC standards. No antibiotics were added to any feed unless specifically stated as a treatment protocol component.[000253]6. Removal Criteria and Humane Endpoint Criteria[000254] Any birds that became ill or injured during the study for reasons not related to the vaccine or challenge were removed to avoid unnecessary distress and euthanized. The reason for removal will be noted and the Attending Veterinarian and / or Study Investigator notified. Body weight and removal day were recorded for calculation of growth metrics.[000255] Any birds that were not able to eat or drink due to injury or illness for a period greater than 24 hours were identified for removal from the study for compassionate purposes. The reason for removal was noted and the Attending Veterinarian and / or Study Investigator notified.7.0 Materials7.1 Placebo[000256] Chicks enrolled in a treatment group receiving a placebo (groups 1 & 2) had distilled water administered using the same equipment as the vaccine.7.2 Investigational Vaccine Preparation (I VP)[000257] LRCVP produced as described in Example 1 above was used to vaccinate Groups 3 & 4.[000258] A second vaccine composition containing backbone L. reuteri strains 3630 and 3632 was used to vaccinate Group 5. It was produced in a similar process to LR NE01 and LRL NE06, as described in Example 1, above. Strains were fermented in LR-media, concentrated through centrifugation, formulated with stabilizer, mixed in a 1-1 ratio and lyophilized in 20 mL vials.7.3 Challenge Materials[000259] The challenge model consisted of coccidia from the SD 0 vaccine (COCCIVAC®‘B52, Merck) and Clostridium perfringens strain CP6 on SD 15, 16, 17, and 28 [Hofacre, et al., 1998] to achieve the targeted < 5% NE mortality in Group 1. Challenge of C. perfringens on SD 16 and SD 17 will be determined based on cumulative NE mortality from the previous day’s challenge. The C. perfringens strain CP6 was originally isolated from field case of NE and produces NetB and alpha-toxin as confirmed by PCR analysis.8.0 Study Procedures:8.1 C. perfringens Cha! lenge[000260] C. perfringens challenge on feed was done by administering through top-dressing of feed at a target dose of about 108 CFU per bird to chicks in all groups. Feed and water were withdrawn for two (2) to three (3) hours prior to administration of C. perfringens. A measured amount of water (100 mL for 25 birds / pen) that would be consumed within 30 minutes was used for each pen to be challenged. The C. perfringens CP6 culture at ~1x108CFU / mL was added to the measured water, thoroughly mixed, and poured onto feed pan of each pen. The mixture was distributed uniformly throughout the entire surface of the feed pan by slowly spinning feeder while pouring mixture.8.2 Bacterial Dose Confirmation[000261] Following investigational vaccine preparation / use or C. perfringens challenge material preparation / application, a 3.0 mL sample was retained for dose confirmation titration and back-up reference.[000262] Dose confirmation was conducted, replicating the confirmation procedure two separate times to determine a geometric mean titer of the vaccine dose as well as the Cp challenge dose.[000263] One ( 1.0) mL of that sample was combined with 1.0 mL sterile 50% glycerol, mixed, and divided into two 1.0 mL labeled vials for storage < -50o C. The remaining 2 mL was used to perform 10- fold serial dilutions. Specifically, 1 mL was removed from the undiluted aliquot and added to 9 mL sterile diluent (PBS) and mixed briefly. A ten-fold serial dilution was conducted and 100 pL from each of the serial dilutions (used 3 dilutions - target dilution, one above, and one below) was used to plate onto separate De Man, Rogosa and Sharpe agar (MRS) plates in triplicate. Plates were incubated under anaerobic conditions at 39° C ± 1° C for 18—36 hours. Following incubation, plates were removed from incubator, number of CFU / mL determined from plates containing between 25-250 colonies, and results were recorded, recording the orange (LR NE06) and white (LR NE01) colonies separately.8.3 Body Weights and Feed Disappearance[000264] All birds were weighed by pen on SD 0, 15, 28, and 43. Birds that died or were euthanized were weighed. Feed was weighed upon addition to each feeder and feed- in-feeder weights were measuredon SD 15, 28. and 43 for calculation of feed disappearance for each phase and the overall experiment. Weight was noted in grams up to at least two decimal places using calibrated scales8.4 Lesion Scoring[000265] Lesion Scoring on SD 18, 31, and 43: one (1) bird per pen was selected, weighed, humanely euthanized and necropsied for lesion scoring and for collection of tissue samples for further analyses. Lesions were scored by the attending veterinarian according to the following scale (Hofacre, 1998):* Lesion score 0 = Normal* Lesion score 1 Slight mucus covering small intestine* Lesion score 2 = Necrotic small intestine mucosa« Lesion mucosa and contents8.5 Tissue Sampling[000266] Tissue Sampling on SD 10, 18, 31, and 43: One ( I ) bird / pen was removed from each pen, weighed, and humanely euthanized. Samples were collected as described in Table 6, below'. Tissue samples were collected only from birds in Treatments 1, 2, 3, and 5.[000267] Table 6 - Summary of Tissues Sampled and AnalyzedTable 6i SD # birds! Serum2NE Jejunal contents2in Liver, Jejunal & Jejunum i Cecal Excreta1i sampled | (BiomE Lesion RNALater Cecal tissue2tissue2| contents2 / pen <iit) Scoring1Metagenomics & Metatranscripto Histology (Ancera)Metatranscriptom ics mics (Vet 4(BiomEdit) (BioFractal) diagnostic)i 10 ~~ i j 7 y ■ / ..i 18 7 y i [‘24. §______; 31 —i 38 ' 1 1 43 i T 71Samples will be collected only from Treatment Groups 1,2,3,4,52Samples will be collected only from Treatment Groups 1,2, 3, 58.6 Excreta Sampling:[000268] Excreta samples were collected to measure C. perfringens load, Eimeria oocysts per gram (OPG), and LRNE01 & LR NE06 strain quantification. Three composite excreta samples were collected from 3 pens per treatment on SD 10, 18, 24, 31, 38, and 43 from all treatment groups. Briefly, 3 distinct droppings per pen were collected into a single container and were considered one composite sample. Same 3 pens per treatment group were also sampled for excreta throughout the study. Each technicianwore disposable boot covers and gloves when entering a pen. Gloves and boot covers were changed between pens to maintain biosecurity and minimize cross-contamination between pens and treatments.8.7 Serum[000269] Serum samples were collected from the same 1 bird / pen euthanized for sampling on SD 10, 18, 31, and 43 in section 23.5 (see table above) from all treatment groups. Blood specimens collected into plain serum tubes were centrifuged at 4°C and 2,000×g for 10 min ensuring serum separation from blood cells. Resulting serum will be transferred and stored in individually labeled cryo-vials (2 aliquots for each sample) at -80°C.9,0 Sequence of Events During Study:[000270] SD 0 - Male chicks were picked up from the hatchery, weighed, issued starter feed, and spray vaccinated with IVPs as described in Table 3 above, followed by spray vaccinating all chicks with Merck COCCIVAC-B52 once they were dry from IVP application. Chicks were grouped chicks into blocks of 5 'ith 20 replicates per treatment group, weighed birds by pen and placed into pens.[000271] SD 10 - Randomly selected and euthanized one bird / pen, and collected tissue samples from treatment groups 1, 2, 3, and 5 and composite excreta samples from 3 pens / treatment from all treatment groups, all in accordance with the process shown in Table, above.[000272] SD 14 - Booster vaccinations in DW were administered to groups 3 and 5, per Table.[000273] SD 15 - C. perfringens challenge: Birds were weighed by pens, and starter feed was replaced with grower feed. C. perfringens strain CP6 dose of 1.0E+08 CFU / bird was administered, as described above.[000274] SD 16 and 17 - C. perfringens challenged with same dose of strain CP6 as on SD 15.[000275] SD 18 - NE lesion score was determined for one (1 ) bird / pen, collect tissue samples only from treatment groups 1, 2, 3, 5. Excreta samples were collected from all treatment groups.[000276] SD 24 - Excreta samples were collected from all treatment groups.[000277] SD 28 — C. perfringens challenge: Birds weighed by pen, non-consumed grower feed was discarded and replaced with finisher feed, birds were weighed by pens, and birds were dosed with C. perfringens as described in Table using the same dose and process as on SD 15, above.[000278] SD 31 - One (1) bird / pen was randomly selected and euthanized, scored for NE Lesion, and tissue / content / sera samples taken from animals in Groups 1, 2, 5, and 5 as per Table, above. Excreta samples were collected from all treatment groups.[000279] SD 38 - Excreta samples were collected from 3 pens / treatment from all treatment groups.[000280] SD 43 — Birds were weighed by pen. One bird per pen was randomly selected and euthanized, its score for NE lesion determined, and tissue / content / sera samples were taken from treatment groups 1, 3, and 5, as described above. Non-consumed finisher feed was weighed. 3 composite excretasamples were collected from 3 pens / treatment from all treatment groups. Study was then terminated, and all chickens euthanized and disposed of.10.0 Conclusion Criteria[000281] 10.1 Satisfactory criteria: The study was to be considered satisfactory ifNE mortality in the challenge control group was approximately 5% given the variability of the NE challenge model, and if FCR was reduced by BMD in Treatment 2. Both of those criteria were met.[000282] 10.2 Efficacy criteria: The study was to be considered efficacious if any of the vaccinated groups effected a statistically significant reduction in feed conversion rate and / or mortality due to NE at a significance level (Type 1 Error rate) of 0.1. That criterion was met.[000283] 10.3: Statistical methods:[000284] Primary variables included average daily weight gain, average daily feed intake, feed efficiency, mortality (total & NE-specific), and average lesion scores. These variables were modelled using a generalized linear mixed model with fixed effect Treatment Group, random effect Block, and appropriate link function and error distribution. Least square means and confidence intervals were used to evaluate pairwise differences of the levels of fixed effect. All statistical tests were evaluated at the two- sided 0.10 significance level.11. Results[000285] NE mortality rate findings from the study above are summarized in Table 7, below.[000286] Table 7Treatment Treatment Birds / pen # Pens CS Vaccination DW NE group Description CFU / Bird Vaccination Mortality CFU / Bird (absolute number)..1. Challenge Control 26 20 Water Water 102 BMD (50 g / US 26 20 Water Water 7ton feed)3 LRCVP, 2 dose, 1 26 20 2 x 1061 x 1067x 106CFU / Chick4 LRCVP, 1 dose, 1 26 20 1 x 106Waterx 106CFU / Chick 1 5 Backbone Strains 26 20 1 x 1061E+0Lr3632 + Lr3630.1[000287] Results of the FCR measurements are provided in Table 8, below. FCR is feed conversion ratio, determined as the amount of feed ingested / body weight.[000288] Table 8Treatment Treatment Mortality Mortality Mortalitygroup Description Adjusted FCR Adjusted AdjustedDay 15 FCR Day 28 FCR Day 431 Challenge Control 1.364 1.624 1.6722 BMD (50 g / US 1.324 1.608 1.669ton feed)3 LRCVP, 2 dose, 1 1.303 1.551 1.632x 106CFU / Chick4 LRCVP, 1 dose, 1 1.302 1.571 1.659x 106CFU / Chick5 Backbone Strains 1.358 1.631 1.686Lr3632 + Lr3630[000289] Mortality adjusted FCR was reduced at both day 28 and day 43 in the LRCVP, 2 dose treatment group. The FCR results are illustrated graphically in FIG 4, which is a plot of the FCR Challenge results on Days 15 (lightest bar on the left of each set of bars), 28 (darker bar in the middle of each set of bars), and 43 (darkest bar on the right of each set of bars). The results show that on Day 15 there was no difference in adjusted FCR (adjFCR) for 1-dose and 2-dose groups, and both groups were 2 points better than BMD. On Day 28, for the 2-dose group the adjFCR value was about 7 points lower than control, about 5 points lower than BMD, and about 2 points lower than the 1-dose group. On Day 43, the final day of the study, adjFCR for the 2-dose group was about 4 points lower than the control. There was no effect of backbone strains on FCR at day 28 and 43 and only slight improvement at day 15, presumably due to a probiotic effect. BMD had an impact on FCR at day 15, but the effect diminished thereafter.[000290] The FCR results are also illustrated in FIGs 5 A, 5B, and 5C, which are plots of adjFCR for each of the five treatment groups on days 15, 28, and 43, respectively. These A, B and C figures include calculations of p values for each set of bars comparisons, showing that the differences in results were statistically significant.[000291] Average body weight results from this study are provided in Table 8, below:[000292] Table 8Treatment Treatment Average Body Average Body Average Bodygroup Description Weight Day 15 Weight Day 28 Weight Day 431 Challenge Control 0.4145 1.1833 2.63042 BMD (50 g / US ton 0.4230 1.1999 2.6162feed)3 LRCVP, 2 dose, 0.4178 1.2178 2.69311E+06 CFU / Chick _.....4 LRCVP, 1 dose, 0.4183 1.1808 2.60961E+06 CFU / Chick ij 5: Backbone Strains 0.4142 1.1787 2.61041 Lr3632 + Lr3630[000293] FIG. 6 is a plot of mean weight gain for Test Groups 1 through 4. The same bar shading contrast as FIG. 4 was used to indicate the day of study results depicted in each set of bars, specifically, results on Days 15 (lightest bar on the left of each set of bars), 28 (darker bar in the middle of each set of bars), and 43 (darkest bar on the right of each set of bars). FIG. 6 clearly shows that weight gain relative to control increased in all three phases of growth only in the 2 dose group. The effect on weight gain was similar to the test group with just backbone strains (Group 5) at days 28 and 43 for the 1 dose group. That could be due to diminished colonization and resulting lack of nanobody effect.EXAMPLE 4Quantification of Effect of LRCVP on Body Weight, Feed Intake and Feed Efficiencyon Broiler Chickens Housed in Commercial-Like Environment in Absence of Purposeful NE Challenge1. Objective[000294] The objective of this experiment was to quantify the effect of LRCVP on body weight, feed intake, and feed efficiency of broiler chickens housed in commercial- like conditions (floor pens with a dirty liter blend) in the absence of any purposeful NE challenge. This study was performed without NE challenge. Also, fresh and old litters of birds were mixed versus prior study(ies) described above where animals were housed in groups in separate pens. This was done to mimic more commercial-like conditions.2. Study Design2.1 Treatment GroupsTable 8, below, shows the treatment groups that were used in this study:Treatment Treatment Replicates x CS i DW DW Groups Description number of Vaccination I vaccination Supplementation chicks / pen on SDl S on SD 14 from SD 1 to SD... CFU / Bird j CFU / bird 20 CFU / bird / day 1 Negative control 24 x 17 Distilled water 1 RO water RO water 2 LRCVP 2-dose 24 x 17 1E+06 | IE+06 RO water 3 LRCVP lx 10624 x 17 Distilled water i RO water lx 105total over 20 days _........ L........4 LRCVP 4x 10624 x 17 Distilled water | RO water 2x 105total over 20 days5 Lr3632+Lr3630 24 x 17 Distilled water j RO water lx 105j 2x 106total over 20 I 1[ _ I days ] j...2.2 Study Description:[000295] Broiler chicks were used for this study as they were the intended target population. Two thousand forty (2,040) chicks were assigned to five (5) treatment groups, with twenty- four (24) replicate blocks, and allocated into groups of seventeen (17) birds per. Each pen contained a dirty litter blend. All treatment groups were assigned to pens using a randomized complete block design.[000296] Treatment Group 1 was administered with well water and served as the Negative control group. Treatment Group 2 received the LRCVP at target doses and route of administration per study design in section 2.1 above. Treatment Groups 3 and 4 received the LRCVP at target doses delivered through the drinking water from SD 1-20 of the experiment per study design in section 2.1. Treatment Group 5 received the backbone strains at the target dose delivered through the drinking water from SD 1 - 20 of the experiment per study design in section 2.1.[000297] Spray vaccination was conducted in the same way as in the preceding two Examples, above. Sprayers were calibrated to deliver a known amount of liquid per unit of time. All spray applications included green or red color dye similar to the dyes used for CS application of commercial coccidiosis vaccine at day-of-hatch. The target CS volume for placebo or vaccine was 0.25 ml., per bird.[000298] On SD I, Treatment Groups 1 and 3-5 were coarse spray vaccinated with distilled water, Treatment Group 2 was coarse spray vaccinated with a targeted dose of LRCVP resuspended in distilled water. After the application of IVP and birds were confirmed to be dry all birds were vaccinated for coccidiosis (COCCIVAC-B52, Merck).[000299] On SD 14, Treatment Group 2 received a targeted dose of the IVP, LRCVP, via drinking water. Approximately 1 hour prior to administration, nipple lines for water were raised to a height that was inaccessible by the birds, turned off, or a partition temporarily inserted in the pen to deprive of water. Vaccine-containing water held in ajar drinker was then be placed into each pen as the sole source of water. Once all the treated water is consumed, the nipple line waterers were lowered back to their accessible height, turned back on, or the partition removed from the pen.[000300] On SD 1-20, Treatment Groups 3-5 were given free access to water containing the target dose of LRCVP (Groups 3-4) or the backbone strains (Group 5). Briefly, each day a stock solution was made of each material by diluting it into fresh water. That concentrated material will then be further diluted into a pre-determined volume of water to deliver the targeted dose of the LRCVP (cfu / bird) to be consumed entirely within each pen. Water sources delivering the LRCVP or backbone strains will be removed from the pen once fully consumed that day.2.3 Body Weight and Feed Disappearance:[000301] All birds will be weighed by pen on SD 1, 15, 29, and 41. Feed will be weighed upon addition to each feeder and feed- in- feeder weights will be measured on SD 15, 29, and 41 for calculation of feed disappearance for each phase and the overall experiment. Weight will be noted in kilograms up to at least two decimal places for feed, and at least three decimal places for bodyweights, using calibrated scales.2.4 Test Subjects and Enrollment Criteria[000302] The test subjects were all male Broiler (Ross X Ross) Chickens. Age at the start of treatment was day of hatch (DOH)l[000303] Only healthy birds were selected for inclusion in the study, and all were observed to be bright, alert and responsive (BAR). Any chicks demonstrating physical deformities or not exhibiting BAR behavior were excluded from enrollment in the study.[000304] The chickens were not individually identified. However, a set of 17 animal ID’s were assigned to each pen on a pen card for assignment of ID’s for dead / removed birds. Pen security prevented bird migration.2.5 Group Assignment, Randomization & Blinding[000305] There were 120 total pens, with 24 per treatment group. All treatment groups were assigned to pens according to randomized block design, with randomization and assignment of treatment groups to pens being managed using Random function in Excel.[000306] The study was unblinded.2.6 Housing[000307] All birds were housed in concrete floor pens in one room. Pens measuring 4 feet x 4 feet (providing 1.0 ft2 / bird) contained approximately four (4) inches of a dirty / fresh fitter blend, and were capped with approximately one (1) inch of fresh pine shavings as litter. Litter was not replaced during the course of the study.[000308] Each pen contained one ( 1 ) tube feeder and one ( 1 ) bell drinker resulting in a sixteen ( 16) bird / feeder and drinker ratio.[000309] The facility was fan-cooled. Thermostatically controlled gas heaters were the primary heat source. Birds were raised under ambient humidity and were provide[d a lighting program as per the primary breeder recommendations.2.7 Feed and Water[000310] Feed and water will be provided ad libitum throughout the study except for transient water deprivation prior to the second dose of the IVP via the DW route on SD 14.[000311] Table 9, below, shows the composition of feeds provided throughout this study.TABLE 9Ingredient Starter (%) Grower (%) Finisher (%);Com, yellow dent 52.89 57.68 62.74Soybean meal, 47.5% CP 35.71 31.60 27.00Com DDGS 5.00 5.00 5.00Soybean oil 2.06 2.42 2.18 Monocalcium phosphate 21% P 1.13 0.71 0.49Limestone 1.51. 1.11 1.07L-lysine HCI 0.29 0.23 0.27L-threonine 0.11 0.07 0.07DL-methiodine 0.38 0.32 0.31Choline Chloride 60% 0.10 0.05 0.05Salt 0.27 0.29 0.29Sodium bicarbonate 0.16 0.13 0.14Promote phytase 2500 0.04 0.04 0.04Broiler VTM-BRRS 0.35 0.35 0.35[000312] Common rations were fed as follows: starter SD 0 through SD 15, grower SD 15 through SD 29 and finisher SD 29 to SD 41. Diets were fed as mash.[000313] Feed formulations for this study consisted of un-medicated commercial-type broiler starter and grower diets compounded with commonly used United States feedstuffs representative of local formulations, calculated analyses to meet or exceed NRC standards. No antibiotics were added to any feed unless specifically stated as a treatment protocol component.[000314] Rations were changed from starter to grower on SD 15 and from grower to finisher on SD 29. At that time all previous feed was removed from each pen, individually weighed, and replaced with grower feed. On SD 29 all non-consumed grower feed was removed from pens, individually weighed, and discarded. Finisher feed was then fed until study termination.2.8 Removal Citeria and Humane Endpoint Criteria[000315] The same removal criteria and endpoint criteria used with the preceding to studies described above were also observed with this study.3. Materials3.1 Placebo[000316] Chicks enrolled in a treatment group receiving placebo (groups 1 and 3-5) had distilled water administered using the same equipment as the vaccine.3.2 IPV[000317] The lot of LRCPV used in this study for Treatment Groups 2-4 was prepared using X+3 passages of the same master seed lots of NEO 1 and NE06 as were used to produce the LRCPV lot used in the studies described in the preceding two Examples. As in those Examples, the cultures were grown separately in LCM medium, followed by centrifugation, stabilizer addition, mixing, and lyophilization in 20 ml vials.[000318] The same vaccine composition containing backbone L. reuteri strains 3630 and 3632 used in the study in the preceding Example was used for treatment of Group 5.3.3 Bacterial Dose Confirmation[000319] Following IVP resuspension on SD 1 and 14, a 3.0 mL sample was retained for dose confirmation titration and back-up reference. For Treatment Groups 3, 4, and 5, doseconfirmation was carried out on SD 1, 14, and 20.[000320] Dose confirmation was conducted and documented, replicating the confirmation procedure two separate times to determine a geometric mean titer of the vaccine dose.[000321] One (1.0) mL was combined with 1.0 mL sterile 50% glycerol, mixed, and divided into two 1.0 mL labeled vials for storage < -50o C. The remaining 2 mL was used to perform 10-fold serial dilutions in duplication. 1 mL was removed from the undiluted aliquot and added to 9 mL sterile diluent (PBS) and mixed briefly. A ten-fold serial dilution was conducted and 100 pL from each of the serial dilutions (use 3 dilutions - target dilution, one above, and one below) was used to plate onto separate De Man, Rogosa and Sharpe agar (MRS) plates in triplicate. Plates were incubated under anaerobic conditions at 39° C ± 1° C for 18-36 hours. Following incubation, plates were removed from the incubator, the number of CFU / mL was determined from plates containing between 25-250 colonies, recording the orange (LRNE06) and white (LR NEO 1) colonies separately, and the results were recorded. Unused test vaccines were disposed of according to standard laboratory procedures and site procedures.4. Sequence of Events[000322] SD 0 - Chicks were picked up from the hatchery, and gel packs were added to chick boxes. No feed was offered.[000323] SD 1 — Group 2 was spray vaccinated with IPV (LRCVP) and all other groups were sprayed with distilled water. All chicks were spay-vaccinated with COCCIVAC-B52 vaccine. Birds wereweighed by pen, starter feed was weighed and issued, and birds were placed into pens with dirty litter blend. The birds were grouped into blocks of five (5) with 24 replicates per treatment block.[000324] SD 1-20 - IPV was delivered through DW for Treatment Groups 3-5.[000325] SD 14 - Booster IPV (LRCVP) was administered to Group 2 in DW.[000326] SD 15 - Birds were weighed by pens. Non-consumed starter feed was weighed and discarded and replaced with grower feed.[000327] SD 29 - Birds were weighed by pens. Non-consumed grower feed was discarded and replaced with finisher feed.[000328] SD 41 - Birds were weighed by pens, non-consumed feeder finisher feed was weighed, study was terminated, and chickens were euthanized.5. Conclusion Criteria5.1 Satisfactory Criteria and Efficacy Criteria:[000329] The study met the satisfactory criteria of the flock remaining free of c linical signs of Necrotic Enteritis.[000330] Any IVP would be considered efficacious if any of the Biologic groups effect a statistically significant reduction in feed conversion rate and / or body weights at a significance level (Type I Error rate) of 0.10. See Results section below for information about the results obtained in this case.5.2 Statistical Methods:[000331] Primary' variables include average daily weight gain, average daily feed intake, feed efficiency, and mortality (total & NE-specific). These variables were modelled using a generalized linear mixed model with fixed effect Treatment Group, random effect Block, and appropriate link function and error distribution. Least square means and confidence intervals were used to evaluate pairwise differences of the levels of fixed effect. All statistical tests were evaluated at the two-sided 0.10 significance level. 6.0 Results[000332] Body weight in Kg was assessed and evaluated for each of the Treatment Groups on each of three days, days 15, 28 and 41, in the unchallenged scenario of the study. The body weight results showed that the control group and the chassis group had a similar body weight result on each day tested, and notably all three groups administered LRCVP had a higher body weight than either of those two groups.[000333] Notably, feed intakes were higher in the LRCVP / Chassis groups through day 28 and overall. Mortality was 2% and morbidity 0.8%.[000334] This study demonstrates that performance criteria, particularly body weight, feed intake, and feed efficiency of broiler chickens housed in commercial-like conditions, were all improved with administration of LRCVP, in the absence of any purposeful NE challenge.EXAMPLE 5Efficacy Study for Conditional Licensure[000335] A further study was conducted for efficacy in reducing NE mortality in the face of moderate NE challenge. The study was designed to evaluate effects on NE mediated mortality. This relates to NE Mortality evaluation wherein the mortality in unvaccinated control animals is at least 10% and in the range of 10%-50% mortality. In accordance with the results of this study, a combination of a first L. reuteri bacteria expressing a Net B nanobody and a second L. reuteri bacteria strain expressing an alpha¬ toxin nanobody reduced NE mortality from 13.1% of the population to 6.65%, providing a mortality reduction overall of 48.7%.[000336] The study subjects were straight run Ross 308 chicks. No additional vaccines or medications were administered. Housing was Petersime Battery cages with 11 birds / cage for 28 days. In this study there were 44 cages per treatment. Ad lib access to food and water was provided, except for a pre-period before the day 13 administration of the NetB+aToxin Nanobody expressing L. Reuteri strains in drinking water. In this NE challenge model, a moderate NE mortality (10-20%) was aimed at for the challenge control group. Each mortality was necropsied to identify the cause. The Prevented Fraction was determined as the proportion of mortality that were prevented compared to unvaccinated challenge control.[000337] In this study, 2.4 xlO6CFU / chick was administered by coarse spray at day 0 or hatching. At day 13, 2.4 xlO6CFU / chick was administered in drinking water. On day 14, 2000 oocysts of Eimeria maxima were administered by oral lavage. C. perfringens challenge at a dose of 2x 108- 4x 108CFU / chick was administered by oral lavage at days 19, 20 and 21.[000338] The results are presented in the table below:Table 10Treatment No. of No. of Vaccination Challenge Total NE Percent Birds Cages Mortality' NE Mortality Unchallenged 11 1 Distilled Water none 00controlChallenged 484 44 Distilled Water Eimeria + C 63 13.1 control perfringes.LRCVP 484 44 NetB+aToxin Eimeria + C 32 6.55Nab L. Reuteri perfringesstrains[000339] This study demonstrates significant reduction in NE mortality in the face of moderate NE challenge. A highly significant Prevented Fraction of 48.7% was seen with administration of two doses ofNetB+aToxin Nab expressing L. Reuteri strains (oral lavage at dayO / hatch and in drinking water day 13) versus the challenge control.EXAMPLE 6Comparison Study versus Commercially Available C. Perfringes Vaccine [000340] To further evaluate the nanobody expressing L. reuteri strain combination, a study was conducted to compare it directly with a commercially available C. perfringens vaccine. The study was designed to evaluate effects on NE mediated mortality and relates to NE Mortality evaluation wherein the mortality in unvaccinated control animals is at least 10% and in the range of 10%-50% mortality. Avert® NE is a recombinant attenuated Salmonella vaccine expressing C. perfringens genes coding for an alpha¬ toxin fragment and NetB toxin.[000341] In this study, 1.28 x106CFU / chick of the L. reuteri strain combination was administered by coarse spray at day 0 or hatching (day of hatch, DOH), and again at day 13 (d13), 1.28 x106CFU / chick was administered in drinking water. This was compared with administration of a single dose of Avert® NE administered by coarse spray at day 0 or hatching (no further dosing of Avert® NE), On day 14, Eimeria maxima 5000 oocysts were administered by oral lavage. C. perfringens challenge at a dose of 2x 108- 4x 108CFU / chick was administered by oral lavage at days 19 and 20.[000342] The results are presented in the table below':Table 11Treatment No. of No. of Vaccination Total NE Percent |Birds Cages Mortality NE i... Mortality 1Challenge 385 35 Distilled Water 198 52.7 icontrolLRCVP 385 35 NetB+aToxin 159 41.8 1Nab L. Reuteristrains (DOH &di 3)Avert® NE 198 18 1 does (DOH) 94 48 |[000343] This study demonstrates that a combination of a first L. reuteri bacteria expressing a Net B nanobody and a second L. reuteri bacteria strain expressing an alpha-toxin nanobody reduced E mortality by over 20% (20.5%). This is in contrast to the directly compared commercially available vaccine Avert® NE, which reduced NE mortality by only 8.7%. The combination of NetB+aToxin Nab expressing L. Reuteri strains (LRCVP) demonstrated a reduction in NE mortality that was significantly better than the commercially available vaccine Avert® NE (over twice or 2 times reduction in NE mortality relatively).[000344] Notably, the NE mortality in this study was unanticipated and very significant overall, yet the LRC VP performed to deliver statistically significant reduction in mortality, and to a degree even surpassing the commercially available C. perfringens vaccine Avert® NE.EXAMPLE 7Administration via Gel Alternative versus Coarse Spray[000345] For prevention of parasitic and bacteria mediated disease in livestock animals, including particularly poultry, vaccines or other therapeutic agents can be a critical component of animal management to ensure control and management of the mediated diseases. Vaccines or therapeutic agents are often applied by coarse spray in a water based diluent. These are applied to ail the animals in a group, cage, pen etc, such as at birth or hatching. Yet, despite its widespread adoption, this method of has several limitations and drawbacks. Specifically, using chicks as an example, there can be a lot of waste as the vaccine or agent quickly dries on application, and a high proportion of the vaccine or agent often is administered in small droplets that are not big enough to be taken up by the chicks. Additionally, a large amount of vaccine or agent is wasted on the chicks’ feathers and the crate / pen floor. Should the vaccine or agent be unevenly distributed, then success of administration will be similarly uneven. Wet chicks also can experience a significant drop in body temperature after spraying.[000346] Alternative gel diluents have been developed and entered the commercial marketplace as an alternative to water based diluents (Albanese GA et al (2018) Poultry Science 97:1544-1553; Ritzi MM et al (2016) Vet Res 47:111). This results in less waste and more consistent administration and uptake / ingestion of the vaccine or agent. The gel diluents can have variable viscosity and can be ‘dropped’ onto animas at birth or hatch, such as onto chicks, for example using an application bar. Gel beads have also been shown to be effective or protective when delivered in the feed (Danforth HD et al (1997) Parasitol Res 83:445-451; Jenkins MC et al (2012) Avian Dis 56:306-309; Jenkins MC et al (2013) Avian Dis 57:622-626). Commercially available gel diluents include Hydrodrop gel (ClearH2O, Westbrook. ME) and CEVAGEL (Ceva Animal Health, Lanexa, KS).[000347] A study was conducted to evaluate the combination of NetB+aToxin Nab expressing L.Reuteri strains (LRCVP) administered in two different doses and administered as a coarse spray (in water based diluent) versus a gel (gel drop). In this study, doses of either 1 xlO5or 1 xlO6CFU / chick of the L. reuteri strain combination was administered by coarse spray or gel drop at day 0 or hatching (day of hatch, DOH), and again at day 13 (d 13), doses of either 1 xlO5or 1 xlO6CFU / chick was administered in drinking water. On day 14, Eimeria maxima 1500 oocysts were administered by oral lavage. C. perfringens challenge at a dose of 1 x 108CFU / chick was administered by oral lavage at days 19, 20 and 21. There were 107-110 birds in each group of: Challenge Control; I xlO6CFU / chick of the L. reuteri straincombination by coarse spray; 1 xlO3CFU / chick of the L. reuteri strain combination by coarse spray; 1 xlO6CFU / chick of the L. reuteri strain combination by gel drop; and 1 x105CFU / chick of the L. reuteri strain combination by gel drop.[000348] The results showed 14.8% NE mortality in the challenge control group. NE mortality was reduced by over 21% in all other groups. In this study, the 1 xlO5CFU / chick dose of the L. reuteri strain combination was more effective in reducing mortality than the 1 xlO6CFU / chick dose. Administered by coarse spray or gel, the 1 xlO5CFU / chick dose reduced mortality and demonstrated a Prevented Fraction of 48.7% and 50%, reducing NE mortality toon the order of 7% vs the challenge control mortality of 14%. This study shows that a lower dose of 1 xlO5CFU / chick of the L. reuteri strain combination is very effective and that gel application is effective versus coarse spray.EXAMPLE 8Productivity Studies with NE Challenge[000349] With econo ical ly-significant diseases of livestock animals, including particularly poultry, prevention, reduction, control and management of disease is critical. Disease can result in poor feed conversion and reduced weight gain. It can also lead to necrotic enteritis in severe disease situations. Thus, in large populations of livestock animals, such as poultry, even a subclinical or low % infection of a bacteria or parasite, leads to detrimental and costly effects. Subclinical or low % infection of a bacteria or parasite in a livestock population, such as poultry', on the order of 2%-5% NE mortality level, impacts the overall body weight of the population and also the feed consumed per body weight gain or FCR. A goal is to retain or maintain or increase overall body weight gain and reduce the FCR.[000350] The primary efficacy variable in this next set of studies was Productivity. Study subjects were Male Ross 708 chicks. No vaccines or medications were administered other than the herpes virus vaccine HVT / SB I (at hatchery) and Eimeria species Coccivac B52 at day 0 as part of the challenge model. Chicks were housed in concrete floor pens with fresh pine shavings at 25-26 chicks / pen. Ad lib access to food and water was provided, except for a pre-period before the administration of the NetB+aToxin Nanobody expressing L. Reuteri strains in drinking water. In this Productivity model, a mild NE mortality (2-5% in challenge control) was aimed at and was induced through Coccivac spray on DOH and C. perfringens administration on days 15, 16, 17 and 28 applied as top dressing onto feed. Animals were fed a three phased diet of pelleted com-SBM. The FCR was measured as feed consumed (kg) / body weight gain (kg).I. Efficacy versus C. Perfringes Vaccine[000351] In this study, a Coccivac B52 spray dose was administered to all groups at day 0. Also, two (2) or three (3) doses of 1 x! 06CFU / chick of the L. reuteri strain combination was administered and compared. In the 2-dose treatment group, 1 xlO6CFU / chick of the L reuteri strain combination was administered by coarse spray on day 0 and again in drinking water on day 14. In the 3-dose treatment group, 1 x106CFU / chick of the L. reuteri strain combination was administered by coarse spray on day 0 and in drinking water on day 14 and again on day 21. The L. reuteri combination was also compared versus administration of Avert® NE administered by coarse spray at day 0 and again on day 11 in drinking water. C. perfringens challenge was administered by oral lavage at days 15, 16, 17 and 28. Body weight and feed intake were measured on day 15, day 28 and finally on day 42. Some details and results are depicted in the below Table 12:Table 12Group Treatment Birds / pen Coarse spray Drinking Cperf %NE Prevented Pens DOH water d21 on feed Mortality Fraction Drinking dayswater dl4 16, 16,17, 28T " Unchallenged 25 20 water watercontrol2 LRCVP 2 25 20 1 xlO6waterdose + no CFU / birdchallenge3 Challenge 25 20 water water + " T31” ~ controlLRCVP 2 25 20 1 xlO6water + 1.22 63.1 dose CFU / bird___5 LRCVP 3 25 20 1 xlO62.26 32.0 dose CFU / bird CFU / birdAvert® NE 25 20 1 dose / label water 3.47 | -5.562 dose[000352] From the above, it is evident that the Avert® NE 2 dose treatment group had a higher mortality than the challenge control. The 2 dose and 3 dose LRCVP both reduced NE mortality, with the 2 dose LRCVP particularly significant.[000353] Average FCR change relative to control (aFCR) and weight gain are depicted in the below table:...Treatment aFCR aFCR aFCR Mean Mean Mean change d 15 change d28 change d42 Weight Gain Weight Gain Weight Gain dl 5 d28 d42 LRCVP 2 dose: aFCR change d15 --, aFCR change d28 4-, aFCR change d42 --, Mean Weight Gain d15 +, Mean Weight Gain d28 3+, Mean Weight Gain d42 3+LRCVP 3 dose: aFCR change d15 -, aFCR change d28 -, aFCR change d42 --, Mean Weight Gain d15 ++, Mean Weight Gain d28 +, Mean Weight Gain d42 4+Avert® NE: aFCR change d15 -, aFCR change d28 5-, aFCR change d42 ---, Mean Weight Gain d15 ++, Mean Weight Gain d28 4+, Mean Weight Gain d42 5+For FCR, - indicates change -0.01; -- indicates change -0.02; --- indicates change -0.03; 4- indicates change -0.04; and 5- indicates change -0.05.For mean weight gain, + indicates minimal or no weight gain (0, 0.001, -0.0005); ++ indicates gain 0.001- 0.004; 3+ indicates gain 0.01; 4+ indicates gain 0.03; 5+ indicates gain >0.05.[000354] As described above in Example 3, improvement in FCR was observed with 2 doses of the combination of L. reuteri strains expressing NetB and atoxin nanobodies. In this study, all treatments demonstrated relevant effects on aFCR and weight gain. In this evaluation, the Avert® NE was numerically superior but not statistically different than the combination of L. reuteri strains expressing NetB and a toxin nanobodies. Notably, however, in this study 2 doses of Avert® NE were given, whereas only I dose is typically used in commercial settings and is indicated on the current label as dosing of the product.[000355] Looking at the unchallenged setting relative to untreated control and particularly in comparing Treatment Group 2 with Treatment Group 1 in terms of relative FCR (aFCR) and mean weight gain, this study’s results demonstrated improvement in aFCR at day 28 and up to 3 points FCR improvement day 42. Mean weight gain also improved at the day 28 assessment with further increased mean weight gain at day 42 (50g improved weight gain versus untreated control). As in the earlier Examples, this again demonstrates positive performance results and efficacy of the strain combination LRCVP alone and in the absence of challenge.II. Efficacy versus Anti-Bacterial / Antibiotic[000356] Another study was conducted comparing the combination of L. reuteri strains expressing NetB and atoxin nanobodies directly with the antibacterial Bacitracin Methylene Dicalicylate (BMD), which is an antibiotic labeled for control of NE. Earlier Example 2 showed some comparison results. This was designed to evaluate performance efficacy, although the mortality in the challenge control was greater than 10% (14.27%).[000357] LRCVP was administered by gel drop at day 0 and again in drinking water at day 14. Chicks were administered Coccivac B52 spray on day 0. C. perfringens was administered as a challenge on days 15 and 16. Measurements for body weight and feed intake were taken as day 15, day 28 and day 43. For comparison in this study, there was a challenge control treatment group and a treatment group administered Bacitracin Methylene Dicalicylate (BMD). The % NE mortality in the challenge control was 14.1%, with NE mortality in the BMD treatment group 13.67%, and NE mortality in the LRCVP treatment group 12.67%. Despite the unexpected high % NE mortality in the challenge control group, LRCVP reduced mortality in this study and showed greater mortality reduction than the BMD antibiotic in direct comparison.[000358] Looking at the performance characteristics and aFCR and mean weight gain, the LRCVP treatment group showed 6 points reduction in aFCR at day 28 amd day 43 as compared to challenge control. The aFCR observed with LRCVP was greater across all days of measurement versus the BMD treated group. Mean weight main relative to control was seen in both the LRCVP nd the BMD treatment groups at all days measured. At the end of the study, chickens in the LRCVP group were on average over 140g heavier than the challenge control.[000359] REFERENCESCOCCIVAC-B52 Product Bulletin, 2020, Merck Animal Health.Cochran, W. G., and G. M, Cox, 1992, Experimental Design, 2nd Ed. John Wiley & Sons, New York, NY. Pg. 582-583.Hofacre et al. 1998, “Use of Aviguard and other intestinal bioproducts in experimental Clostridium perfringns-associated necrotizing enteritis in broiler chickens,” Avian Disease, 42:579-584.Jenkins, M., et al., 2006, “Application of polymerase chain reaction based on ITS1 rDNAto speciate Eimeria. C Avian Disease, 50:110-114.Schneiders, G., et al., 2019, “Ontogeny of intestinal permeability in chickens infectedwith Eimeria maxima’. Implications for intestinal health,” J. Adv. Parasitol., 6:41-50.You, M. 2014, “Suppression of Eimeria tenella sporulation by disinfectants,” Korean J. Parasitol., 52:435-438.[000360] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.[000361] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

CLAIMS1. A method of increasing mortality, feed conversion ratio and body weight in an animal at risk of necrotic enteritis comprising administering to the animal at least two doses of a composition comprising at least two Lactobacillus strains of bacteria genetically engineered to express and secrete one or more nanobody directed against at least one toxin of Clostridium perfringens, wherein at least one toxin is selected from alpha toxin and NetB, wherein the composition comprises at least 1x105CFU of each of the one or more bacteria, and wherein the Lactobacillus strains are strains that have native probiotic capability.

2. The method of claim 1, wherein the Lactobacillus strains are a first and a second Lactobacillus reuteri strain, wherein the first Lactobacillus reuteri strain has a genomic nucleic acid sequence comprising least one of SEQ ID NOs: 49-55, or having at least one nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 49-55 and further having at least 98% sequence identity with one or more of SEQ ID NOs: 49-55; and wherein the second Lactobacillus reuteri strain has a genomic nucleic acid comprising at least one of SEQ ID NOs: 44-48, or having at least one nucleic acid sequence difference from the sequence of at least one of SEQ ID NOs: 44-48 and further having at least 98% sequence identity with one or more of SEQ ID NOs: 44-48.

3. The method of claim 1 or 2, wherein animal is housed in a commercial setting or breeding facility or pen and the mortality from necrotic enteritis is subclinical wherein there is less than 10% NE- associated mortality in the absence of the composition.

4. The composition of claims 1, 2 or 3, wherein the strains are genetically engineered to express and secrete a nanobody directed against C. perfringens alpha toxin and a nanobody directed against C. perfringens Net B, wherein the nanobody directed against C. perfringens alpha toxin is selected from (a) EAT-1F2: EVQLVESGGGLVQAGGSLRLSCAGSGRTGSLYSMGWFRQAPGKEREFV AAITWRPSSTYYADSVKGTPQAYDYWGQGTQVTVSS (SEQ ID NO:56);(b) EAT-1F2_R27H:EVQLVESGGGLVQAGGSLRLSCAGSGHTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:57);(c) EAT-1F2 T28P:EVQLVESGGGLVQAGGSLRLSCAGSGRPGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:58);(d) EAT-IG4:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHYGQGTQVTVSS (SEQ IDNO:59); and (e) EAT-1G4_Y103W:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHWGQGTQVTVSS (SEQ ID NO:60); and wherein the nanobody directed against C. perfringens NetB is selected from(f) ENB-1 A4:EVQLVESGGGLVQAGGSLRLSCAASGSIFSTNVMGWYRQAPGKQREFVAGITIGGTARYPDSVK GRFTISRDNTQNTVYLQMNNLKPEDTAVYYCNAVLPSDQRRWSWGQGTQVTVSS (SEQ ID NO:61);(g) ENB-1D11:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGRTNYADSVK GRFTISIDNAKNSIHLQMNSLKPDDTARYYCNSPYHALWGQGTQVTVSS (SEQ ID NO:62); and (h) ENB-ID11 R56H:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGHTNYADSVK GRFTIALWGQGTQVTVSS (SEQ ID NO:63).

5. The method of claim 4, wherein the animal is not administered antibiotic.

6. The method of claim 1, 2, 3, 4 or 5, wherein the animal is poultry.

7. The method of claim 6, wherein the poultry is chicken or turkey.

8. The method of claim 7, wherein the chicken or turkey are raised in a commercial setting or hatchery.

9. The method of any one of claims 1-8, wherein the animal or poultry is administered at least two doses of the composition.

10. The method of any one of claims 1-9, wherein a first dose is administered on the day of birth or hatch.

11. The method of any one of claims 1-10, wherein at least one dose is administered in drinking water or is sprayed on feed or food.

12. The method of any one of claims 1-11, wherein mortality is reduced by at least 25%,13. The method of any one of claims 1-11, wherein mortality is reduced by at least 40%.

14. The method of any one of claims 1-11, wherein feed conversion ratio (FCR) is lowered by at least 4 points.

15. The method of any one of claims 1-11, wherein feed conversion ratio (FCR) is lowered by at least 5 points.

16. The method of any one of claims 1-11, wherein feed conversion ratio (FCR) is lowered by at least 7 points.

17. The method of any one of claims 1-16, wherein weight gain is significant.

18. The method of claim 17, wherein weight gain is significant in at least two phases of growth of the animal, poultry, or chicken.

19. A composition comprising at least two Lactobacillus strains of bacteria genetically engineered to express and secrete one or more nanobody directed against at least one toxin of Clostridium perfringens, wherein at least one toxin is selected from alpha toxin and NetB, wherein the composition comprises at least 1x106CFU of each of the two or more bacteria, wherein the Lactobacillus strains are strains that have native probiotic capability, and wherein the strains are genetically engineered to express and secrete a nanobody directed against C. perfringens alpha toxin and a nanobody directed against C. perfringens Net B, wherein the nanobody directed against C. perfringens alpha toxin is selected from(a) EAT-1F2: EVQLVESGGGLVQAGGSLRLSCAGSGRTGSLYSMGWFRQAPGKEREFV AAITWRPSSTYYADSVKGTPQAYDYWGQGTQVTVSS (SEQ ID NO:56);(b) EAT-1F2_R27H:EVQLVESGGGLVQAGGSLRLSCAGSGHTGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ ID NO:57);(c) EAT-1F2 T28P:EVQLVESGGGLVQAGGSLRLSCAGSGRPGSLYSMGWFRQAPGKEREFVAAITWRPSSTYYADS VKGRFTISRDDAKNTVYLQMNSLKPEDTAVYFCAARPRGGLSPTPQAYDYWGQGTQVTVSS(SEQ IDNO:58);(d) EAT-IG4:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFI'ISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHYGQGTQVTVSS (SEQ IDNO:59); and (e) EAT-IG4 Y103W:EVQLVESGGGLVQPGGSLRLSCAASGSIATINDMGWFRQAPGKQRDWVATIVSDGSTAYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARRHWGQGTQVTVSS (SEQ ID NO:60); and wherein the nanobody directed against C. perfringens NetB is selected from(f) ENB-1A4:EVQLVESGGGLVQAGGSLRLSCAASGSIFSTNVMGWYRQAPGKQREFVAGITIGGTARYPDSVK GRFTISRDNTQNTVYLQMNNLKPEDTAVYYCNAVLPSDQRRWSWGQGTQVTVSS (SEQ ID NO:61);(g) ENB-lDll:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGRTNYADSVK GRFTISIDNAKNS1HLQMNSLKPDDTARYYCNSPYHALWGQGTQVTVSS (SEQ ID NO:62); and (h) ENB-1D11 R56H:EVQLVESGGGLVQTGGSLRLSCTASGTIDMTYGLIWYRQAPGKERELVASIRRDGHTNYADSVK GRFTIALWGQGTQVTVSS (SEQ ID NO:63).

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