Mycoplasma gallisepticum live vaccine

Abstract

The present invention provides Mycoplasma gallisepticum strain K6067, deposited with the ATCC under patent designation PTA-127168, Mycoplasma gallisepticum strain K4110, deposited with the ATCC under patent designation PTA-127282, and progeny and derivatives thereof, for use as a vaccine for the prevention of virulent Mycoplasma gallisepticum infection in Galliformes birds. Also provided are compositions and methods for administration to Galliformes birds.

Description

[Technical field]

[0001] Continuing Application Data This application claims the benefit of U.S. Provisional Patent Application No. 63 / 327,851, filed April 6, 2022, and U.S. Provisional Patent Application No. 63 / 404,226, filed September 7, 2022, each of which is incorporated by reference herein. [Background technology]

[0002] Mycoplasma gallisepticum (MG) is an infectious respiratory pathogen of chickens and turkeys. It is the most virulent and economically impactful mycoplasmal pathogen of poultry. Economic losses from condemnation or downgrading of carcasses, reduced feed and egg efficiency, and increased medication costs make MG one of the most costly disease problems facing commercial poultry production worldwide. Although various measures such as voluntary surveillance programs, treatment with different antibiotics, and vaccination have been incorporated in the poultry industry to control Mycoplasma gallisepticum infections and reduce economic losses in the poultry industry, MG is still considered a persistent challenge to the commercial poultry industry. Although voluntary surveillance programs can be effective, evaluation of long-lived flocks (breeders and layers) makes it difficult to justify culling and quarantine, especially in areas where MG is widely prevalent and endemic. Increasing legal restrictions on antibiotic use in poultry, especially preventive feed use, as well as increasing reports of resistance to antibiotics commonly used to control MG infectious disease, reduce the applicability and effectiveness of different antibiotic treatment options. Therefore, vaccination becomes an even more attractive option for MG control. Currently, there are three commercially available live attenuated vaccines against MG (6 / 85, F strain, and ts-11). All three vaccines have advantages and disadvantages in terms of safety and efficacy. Therefore, there is a need for additional live attenuated vaccines for the control of Mycoplasma gallisepticum infections in poultry. Summary of the Invention

[0003] The present invention includes an isolated Mycoplasma gallisepticum strain, the isolated Mycoplasma gallisepticum strain being the K6067 Mycoplasma gallisepticum strain deposited with the ATCC under patent designation PTA-127168, or a progeny or derivative thereof. In some embodiments, the present invention includes a composition comprising an isolated Mycoplasma gallisepticum strain. In some embodiments, the composition may include water. In some embodiments, the composition may include a pharma- ceutically acceptable carrier. In some embodiments, the composition may include an adjuvant. In some embodiments, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. In some embodiments, the composition may be formulated for spraying or aerosolization.

[0004] The present invention includes an essentially biologically pure culture of Mycoplasma gallisepticum strain K6067 deposited with the ATCC under patent designation PTA-127168. In some embodiments, the present invention includes a composition comprising an isolated Mycoplasma gallisepticum strain. In some embodiments, the composition may include water. In some embodiments, the composition may include a pharma- ceutically acceptable carrier. In some embodiments, the composition may include an adjuvant. In some embodiments, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. In some embodiments, the composition may be formulated for spraying or aerosolization.

[0005] The invention includes a vaccine comprising the isolated Mycoplasma gallisepticum strain K6067 deposited with the ATCC under patent designation PTA-127168 described herein, or a progeny or derivative thereof, an essentially biologically pure culture of Mycoplasma gallisepticum strain K6067 deposited with the ATCC under patent designation PTA-127168 described herein, or a composition described herein. In some embodiments, the vaccine reduces one or more clinical signs induced by Mycoplasma gallisepticum infection in poultry. In some embodiments, the vaccine reduces susceptibility of Galliformes birds to disease induced by Mycoplasma gallisepticum.

[0006] The present invention includes a live vaccine for birds of the order Galliformes, comprising a sufficient amount of Mycoplasma gallisepticum strain K6067 deposited with the ATCC under patent deposit designation PTA-127168, or a progeny or derivative thereof, as described herein, to protect the bird against disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.

[0007] The present invention includes an isolated Mycoplasma gallisepticum strain, the isolated Mycoplasma gallisepticum strain being the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent designation PTA-127282, or a progeny or derivative thereof. In some embodiments, the present invention includes a composition comprising an isolated Mycoplasma gallisepticum strain. In some embodiments, the composition may include water. In some embodiments, the composition may include a pharma- ceutically acceptable carrier. In some embodiments, the composition may include an adjuvant. In some embodiments, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. In some embodiments, the composition may be formulated for spraying or aerosolization.

[0008] The present invention includes an essentially biologically pure culture of Mycoplasma gallisepticum strain K4110 deposited with the ATCC under patent designation PTA-127282. In some embodiments, the present invention includes a composition comprising an isolated Mycoplasma gallisepticum strain. In some embodiments, the composition may include water. In some embodiments, the composition may include a pharma- ceutically acceptable carrier. In some embodiments, the composition may include an adjuvant. In some embodiments, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. In some embodiments, the composition may be formulated for spraying or aerosolization.

[0009] The invention includes a vaccine comprising the isolated Mycoplasma gallisepticum strain K4110 deposited with the ATCC under patent designation PTA-127282 described herein, or a progeny or derivative thereof, an essentially biologically pure culture of the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent designation PTA-127282 described herein, or a composition described herein. In some embodiments, the vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry. In some embodiments, the vaccine reduces susceptibility of Galliformes birds to disease induced by Mycoplasma gallisepticum.

[0010] The present invention includes a live vaccine for birds of the order Galliformes, comprising the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent deposit designation PTA-127282, or a progeny or derivative thereof, in an amount sufficient to protect the bird against disease induced by Mycoplasma gallisepticum, as described herein, and a pharmaceutically acceptable carrier.

[0011] In some aspects, the isolated Mycoplasma gallisepticum strains described herein, the compositions described herein, or the vaccines described herein are lyophilized, freeze-dried, frozen, or may be in an effervescent tablet form.

[0012] The invention includes a kit comprising an isolated Mycoplasma gallisepticum strain described herein, a composition described herein, or a vaccine described herein and printed instructions, wherein the contents of the kit are contained within packaging material.

[0013] The invention includes a method of generating an immune response to Mycoplasma gallisepticum in an avian, the method comprising administering an isolated Mycoplasma gallisepticum strain described herein, a composition described herein, or a vaccine described herein. In some embodiments, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some embodiments, administration is by eye drop, by aerosol, or by drinking water. In some embodiments, the avian is of the Galliformes order. In some embodiments, the avian is a chicken or a turkey.

[0014] The present invention includes a method for reducing the susceptibility of an avian to disease induced by Mycoplasma gallisepticum, comprising administering to the avian an isolated Mycoplasma gallisepticum strain described herein, a composition described herein, or a vaccine described herein. In some embodiments, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some embodiments, administration is by eye drop, by aerosol, or by drinking water. In some embodiments, the avian is of the Galliformes order. In some embodiments, the avian is a chicken or a turkey.

[0015] The present invention includes a method for protecting an avian against Mycoplasma gallisepticum infection comprising administering to the avian an isolated Mycoplasma gallisepticum strain described herein, a composition described herein, or a vaccine described herein. In some embodiments, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some embodiments, administration is by eye drop, by aerosol, or by drinking water. In some embodiments, the avian is of the Galliformes order. In some embodiments, the avian is a chicken or a turkey.

[0016] The present invention includes a method of reducing one or more clinical signs induced by Mycoplasma gallisepticum infection in an avian, comprising administering to the avian an effective amount of an isolated Mycoplasma gallisepticum strain described herein, a composition described herein, or a vaccine described herein. In some embodiments, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some embodiments, administration is by eye drop, by aerosol, or by drinking water. In some embodiments, the avian is of the Galliformes order. In some embodiments, the avian is a chicken or a turkey.

[0017] As used herein, "isolated" refers to material that has been removed from its original environment (e.g., the natural environment if it occurs in nature) and thus has been altered "by the hand of man" from its natural state.

[0018] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0019] The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0020] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0021] Unless otherwise noted, "a," "an," "the," and "at least one" are used interchangeably to mean one or more.

[0022] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0023] For any method disclosed herein that includes separate steps, the steps may be performed in any feasible order, and, if desired, any combination of two or more steps may be performed simultaneously.

[0024] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and without any attempt to limit the recitation of equivalents to the scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0025] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviations found in their respective testing measurements.

[0026] In several places throughout this application, guidance is provided through the enumeration of examples, which can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. The specific examples, materials, amounts, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0027] All headings herein are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The bacterium Mycoplasma gallisepticum is a member of the Mycoplasma genus. It is the causative agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys, chickens, game birds, pigeons, and passerine birds of all ages. It is found worldwide, and the infection may be referred to as infectious synovitis, avian mycoplasmosis, infectious sinusitis, or mycoplasmal arthritis. Mycoplasma gallisepticum is transmitted vertically (transovarially) within some eggs from infected breeders to their offspring, as well as horizontally via infectious aerosols, through contamination of feed, water, and the environment, and through human movement on fomites (shoes, equipment, etc.). Infection is economically important because it can cause a decline in egg production (El-Gazzar, “Mycoplasma gallisepticum Infection in Poultry,” Merk Veterinary Manual, 2020).

[0029] The present invention provides Mycoplasma gallisepticum (MG) strain K6067, and its progeny and derivatives, which are immunogenic and stable when administered as a live formulation. The present invention also provides Mycoplasma gallisepticum (MG) strain K4110, and its progeny and derivatives, which are immunogenic and stable when administered as a live formulation. The formulations of Mycoplasma gallisepticum of the present invention are safe and effective in inhibiting Mycoplasma gallisepticum infection and may be useful in reducing the incidence and severity of disease from Mycoplasma gallisepticum infection in birds.

[0030] Mycoplasma gallisepticum strain K6707 was deposited at the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110-2209, USA as PTA-127168 on November 24, 2021. The strain was deposited pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Mycoplasma gallisepticum strain K6067, deposited with the ATCC® as PTA-127168, is also referred to herein as Mycoplasma gallisepticum strain 6067, M. gallisepticum strain 6067, MG strain K6067, K6067, MG strain K6067 ATCC PTA-127168, K6067 ATCC PTA-127168, MG strain K6067 PTA-127168, K6067 PTA-127168, and ATCC® PTA-127168.

[0031] Mycoplasma gallisepticum strain K4110 was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110-2209, USA as PTA-127282 on July 20, 2022. The strain was deposited pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Mycoplasma gallisepticum strain K4110, deposited with the ATCC® as PTA-127282, is also referred to herein as Mycoplasma gallisepticum strain 4110, M. gallisepticum strain 4110, MG strain K4110, K4110, MG strain K4110 ATCC PTA-127282, K4110 ATCC PTA-127282, MG strain K4110 PTA-127282, K4110 PTA-127282, and ATCC® PTA-127282.

[0032] The present invention includes isolated Mycoplasma gallisepticum (MG) strain K6067, ATCC patent deposit designation PTA-127168. As used herein, "isolated" refers to material that has been removed from its original environment (e.g., the natural environment if it occurs in nature) and thus altered from its natural state "by the hand of man." Also included is a biologically pure culture of Mycoplasma gallisepticum (MG) strain K6067, ATCC patent deposit designation PTA-127168.

[0033] The present invention includes an isolated Mycoplasma gallisepticum (MG) strain K4110 having ATCC patent deposit designation PTA-127282. Also included is a biologically pure culture of Mycoplasma gallisepticum (MG) strain K4110 having ATCC patent deposit designation PTA-127282.

[0034] Also included in the present invention are isolated progeny and isolated derivatives of Mycoplasma gallisepticum strain K6067 deposited under ATCC patent deposit designation PTA-127168, having equivalent or similar biological, serological, and / or genetic characteristics. As used herein, serological, biological, and genetic characteristics may include one or more of the characteristics described in the Example data contained herein. More specifically, progeny or derivatives of K6067 strain deposited with ATCC as PTA-127168 may retain particularly preferred protective properties belonging to the present invention. Progeny or derivatives of Mycoplasma gallisepticum strain K6067 deposited with ATCC as PTA-127168 may be obtained by any of the various methods for propagating Mycoplasma gallisepticum known in the art, including, for example, but not limited to, in vitro culture or backpassage in avian. Derivatives of Mycoplasma gallisepticum strain K6067 ATCC PTA-127168 may include genetically modified versions of the deposited MG strain K6067. Such manipulations may include, but are not limited to, mutagenizing the MG strain or introducing genes or gene cassettes encoding alternative or non-functional proteins, or non-coding nucleotide sequences into the MG organism.

[0035] Also included in the present invention are isolated progeny and isolated derivatives of Mycoplasma gallisepticum strain K4110 deposited under ATCC patent deposit designation PTA-127282, having equivalent or similar biological, serological, and / or genetic characteristics. As used herein, serological, biological, and genetic characteristics may include one or more of the characteristics described in the Example data contained herein. More specifically, progeny or derivatives of K4110 strain deposited with ATCC under PTA-127282 may retain particularly preferred protective properties that belong to the present invention. Progeny or derivatives of Mycoplasma gallisepticum strain K4110 deposited with ATCC under PTA-127282 may be obtained by any of the various methods for propagating Mycoplasma gallisepticum known in the art, including, for example, but not limited to, avian in vitro culture or backpassage. Derivatives of Mycoplasma gallisepticum strain K411 ATCC PTA-127282 may include genetically modified versions of the deposited MG strain K4110. Such manipulations may include, but are not limited to, mutagenesis of the MG strain, or the introduction of genes or gene cassettes encoding alternative or non-functional proteins, or non-coding nucleotide sequences into the MG organism.

[0036] The Mycoplasma gallisepticum (MG) strain K6067 isolate, MG strain K4110, and their progeny and derivatives described herein can be grown by conventional methods, including, but not limited to, any of those described in the Examples section contained herein. For example, the Mycoplasma gallisepticum strains of the present invention can be cultured in Frey's modified broth and agar (Ferguson-Noel and Kleven, "Mycoplasma species," In: Williams SM, Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E. Woolcock PR, editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. p. 63-70; 2016).

[0037] MG strain K6067, MG strain K4110, and their progeny and derivatives, can be identified and differentiated from other Mycoplasma species and other M. gallisepticum strains using any of a number of known techniques, including, for example, direct immunofluorescence using species-specific antibodies, serum plate agglutination test (SPA) test, hemagglutination inhibition test (HI), enzyme-linked immunosorbent assay (ELISA), and real-time quantitative PCR (qPCR) (Raviv and Kleven, 2009, Avian Dis; 53: 103-107), whole genome sequencing, and targeted DNA sequencing of the 16S-23S rRNA intergenic space region (IGSR) (Papazisi et al., 2003, Microbiology; 149: 2307-16) and mgc2 cytadecine (Hnatow et al., 1998, Infect Immun; 66: 3436-42).

[0038] Mycoplasma gallisepticum (MG) strain K6067, Mycoplasma gallisepticum (MG) strain K4110, and their progeny and derivatives described herein can be administered as live attenuated MG vaccines to poultry, including chickens and turkeys. Although various measures such as voluntary surveillance programs, vaccination and treatment with different antibiotics have been incorporated in the poultry industry to control Mycoplasma gallisepticum (MG) infections and reduce economic losses in the poultry industry, MG is still considered a persistent challenge to the commercial poultry industry. Although voluntary surveillance programs can be effective, evaluation of long-lived flocks (breeders and layers) makes it difficult to justify culling and quarantine, especially in areas where MG is widely prevalent and endemic. Furthermore, in Europe and the United States, increasing legal restrictions on antibiotic use in poultry (especially prophylactic feed use) (Veterinary Feed Directive Journal.80 number 106, 2015) as well as increasing reports of resistance to antibiotics commonly used to control MG infections (de Jong et al., 2021, Avian Pathology:50(2):161-173) reduce the applicability and effectiveness of this option. In these situations, vaccination becomes an even more attractive option for MG control.

[0039] Currently available immunizing agents include bacterial vaccines, live attenuated vaccines, and recombinant vaccines. Bacteria have some advantages because they are not infectious or contagious and cannot revert to virulence (Sasipreeyajan et al., 1987, Avian Dis; 31: 776-81), and bacteria have been shown to reduce egg loss, ovarian regression, and egg transmission of MG (Glisson and Kleven, 1985, Avian Dis; 29: 408-15). However, bacteria offer very limited protection against infection, tracheal and air sac lesions (Talkington and Kleven, 1985, Avian Dis; 29: 998-1003). The labor costs for intramuscular injections and individual handling of birds can cause stress, and there is also a risk of local vaccine reactions. Regarding MG recombinant vaccines, a fowlpox vectored MG vaccine is commercially available, but its efficacy against respiratory lesions caused by pathogenic MG strains has been reported to be significantly lower than that of bacterial and live attenuated vaccines (Ferguson-Noel et al., 2012, Avian Dis; 56: 272-275). Recently, an adenovirus vectored MG vaccine based on the TM-1 protein has been developed, but further studies are needed to investigate its efficacy (Dongchao Zhang et al., 2018, Avian Pathology; 42(2): 213-222). There are several genes and virulence factors that play different roles in MG pathogenesis and immunity (Yu et al., 2019, Infection and Immunity; 87: e00248-19; Papazisi et al., 2002, Infect Immun; 70: 6839-45, and Indikova et al., 2014, Vet Res; 45: 99), and it can be difficult to select one specific gene to generate an appropriately effective and protective MG vaccine.

[0040] Currently, there are three commercially available live attenuated vaccines against MG, as 6 / 85, F strain and ts-11. All these vaccines have their advantages and disadvantages in terms of safety and efficacy, and the ideal MG vaccine above should be immunogenic and non-toxic, yet still be accessible and capable of preventing colonization of pathogenic MG strains after exposure (Cummings and Kleven, 1986, Avian Dis; 30: 169-71). The 6 / 85 vaccine is very safe (Evans and Hafez, 1992, Avian Dis; 36: 197-201; Ley et al., 1997, Avian Dis; 41: 187-94; and Zaki et al., 2004, Avian Dis; 48: 642-6), however, vaccine efficacy is not high and poor colonization has been reported (Evans and Hafez, 1992, Avian Dis; 36: 197-201; and Kleven et al., 1998, Avian Dis; 42: 300-6). In addition, poor serological responses may cause problems in assessing the immune status of vaccinated herds with serological assays (Throne Steinlage et al., 2003, Avian Dis; 47: 499-505).

[0041] The F strain was the first live vaccine introduced into the poultry industry to control MG infection (Yamamoto and Adler, 1958, J Infect Dis; 102: 143-52), and it is currently the lowest attenuated live MG vaccine commercially available. The use of the F strain is not permitted in some areas (e.g., Minnesota, USA, Israel, and Japan) due to reports of its pathogenicity in turkeys (Lin and Kleven, 1982, Avian Dis; 26: 360-4), its contagiousness to unvaccinated birds (Kleven, 1981, Avian Dis; 25: 1005-18), and the risk of increased virulence as it circulates and backpassages within flocks (Gharaibeh et al., 2011, Avian Dis; 55: 212-6). However, the F strain is highly effective in displacing virulent MG strains (Kleven et al., 1990, Avian Dis; 34: 984-90), as well as reducing air sac and tracheal lesions (Kleven, 1986, Avian Diseases; 30 No. 1: 169-171), and reducing virulent MG egg transmission (Glisson and Kleven, 1984, Avian Dis; 28: 406-15). Another live MG vaccine that persists in the vaccinated trachea for the life of the flock, similar to the F strain (Kleven, 1998, Poult Sci; 77: 1146-9), is ts-11 (Whithear et al., 1990, Aust Vet J; 67: 168-74). The vaccine is effective in preventing respiratory disease (Abd-el-Motelib and Kleven, 1993, Avian Dis; 37:981-7), egg loss (Barbour et al., 2000, Poult Sci; 79:1730-5), and ovarian involution (Whithear et al., 1990, IOM Lett; 1:361-362).ts-11 can be used safely in turkeys, but it does not colonize the trachea well enough to elicit protective immunity (Whithear et al., 1990, Aust Vet J; 67: 159-65, and Wijesurendra et al., 2017, Avian Pathol; 1-10; 2017). Reversion of ts-11 to virulence has also been reported (Armour et al., 2015, Avian Pathol; 44: 296-304). Recently, a ts-304, GapA+ts-11 vaccine has been developed. Vaccine persistence remains similar to ts-11 (Condello et al., 2020, Veterinary Microbiology;251:108883) and has been reported to be safe and effective for use in turkeys (Kanci et al., 2018, Vaccine;36(18):2487-2493).

[0042] The present invention includes compositions and vaccines of the M. gallisepticum isolates described herein, as well as their progeny and derivatives. In preferred embodiments, the M. gallisepticum isolates are live. In some embodiments, the M. gallisepticum isolates may be inactivated or killed. The M. gallisepticum strains of the present invention, as well as compositions and vaccines thereof, may be stored until use in any of a variety of forms. For example, such materials may be lyophilized or freeze-dried and rehydrated for use. In some embodiments, the M. gallisepticum strains, or compositions or vaccines thereof, may be frozen.

[0043] In some embodiments, the M. gallisepticum isolate, or composition or vaccine thereof, may be formulated as an effervescent tablet. Such effervescent tablets may be packaged, for example, in lightweight aluminum blisters. The tablet is dissolved in water and administered, for example, orally, nasally, or by aerosol spray, whereby the droplets may enter through the mucous membranes of the bird.

[0044] The compositions and vaccines of the present invention may include, for example, water or a medium. Such compositions and vaccines may include one or more suitable pharma- ceutically acceptable carriers, stabilizers, preservatives, diluents, and / or buffers. Suitable stabilizers include, for example, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextrin, or glucose), or proteins (such as albumin or casein). Stabilizers are particularly advantageous when the dry vaccine preparation is prepared by lyophilization. Suitable preservatives include, for example, thimerosal, merthiolate, and gentamicin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol).

[0045] The composition or vaccine of the invention may also contain one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminum hydroxide, aluminum phosphate, aluminum oxide, vegetable oils, animal oils, oil-in-water or water-in-oil emulsions based on mineral oils such as Bayol F™ or Marcol 52™, complete Freund's adjuvant, incomplete Freund's adjuvant, or vegetable oils such as vitamin E acetate, and saponins.

[0046] The compositions or vaccines of the present invention may further comprise one or more immunogens derived from other pathogens that infect poultry, such as, for example, Mycoplasma synoviae (MS), Marek's disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), egg-laying drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV), poxvirus, reovirus, chicken parvovirus, and avian nephritis virus (including, but not limited to, ANV-1 and ANV-2).

[0047] The compositions and vaccines of the invention may be substantially pure. As used herein, "substantially pure" refers to material that is essentially free from naturally occurring macromolecules or other biological entities.

[0048] The compositions and vaccines of the present invention may be administered to any of a variety of avian species susceptible to Mycoplasma gallisepticum infection, including, but not limited to, poultry, galliformes, and exotic avian species. Galliformes include, but are not limited to, chickens, turkeys, grouse, quail, and pheasants. As used herein, poultry includes domesticated birds that are raised for the purpose of harvesting their eggs or killing for their meat and / or feathers. These are most typically members of the superorder Phasianidae (food birds), particularly the order Phasianidae (including, for example, chickens, quail, turkeys, and grouse) and the family Anatidae (Anatidae), commonly known as "waterfowl" (including, for example, ducks, geese, and swans). Poultry may also include pigeons or dabs, or other birds that are killed for their meat, such as pheasants, which are considered game birds. Chickens include, but are not limited to, hens, roosters, broilers, roasters, layers, breeders, progeny of breeding hens, and layer hens. As used herein, the term "susceptible" refers to the likelihood or reality of one or more pathological conditions indicative of an adverse response to a reference microorganism and / or Mycoplasma gallisepticum infection, e.g., reduced vigor or impaired growth, when compared to a susceptible individual or group.

[0049] The compositions and vaccines of the invention may be formulated for delivery by any of a variety of routes known in the veterinary art, including, for example, but not limited to, mucosal, intranasal, intraocular, or oral administration. The compositions and vaccines of the invention may be formulated for delivery to the respiratory mucosa and may be administered so that it comes into immediate or eventual contact with the respiratory mucosa of the bird. The compositions or vaccines of the invention may be administered by any suitable known method of inoculating poultry, including, but not limited to, intranasally, ophthalmically, by eye drop, by injection, in drinking water, in feed, in ovo, maternally, by respiratory inhalation, and the like. When administered by injection, the immunogenic composition or vaccine may be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.

[0050] The composition or vaccine may be administered by mass administration techniques, for example, by placing the vaccine in drinking water, or by spraying or aerosolization. The composition may be administered by spraying a solution onto an individual or flock, and such aerosol delivery may include administration of the composition incorporated into small liquid particles. Such spray-type particles may have a droplet size within the range of about 10 to about 100 microns, more preferably about <1 to about 50 microns. For generation of small particles, conventional spray equipment and aerosol generators may be used, such as commercially available spray generators for knapsack sprays, hatchery sprays, and atomist sprays. Administration via drinking water may be carried out using conventional equipment.

[0051] The compositions or vaccines of the present invention may be administered to poultry before or after hatching. In ovo vaccination may occur, for example, at about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or any range therein. For in ovo delivery, the laying or breeding stock may be vaccinated, for example, at about 6-12 weeks of age and boosted at about 16-20 weeks of age. Such laying or breeding stocks may be vaccinated at about 6 weeks of age, about 7 weeks of age, about 8 weeks of age, about 9 weeks of age, about 10 weeks of age, about 11 weeks of age, or about 12 weeks of age. Also, in some embodiments, such laying or breeding stocks may be vaccinated within the first 2 weeks of age. Such laying or breeding stocks may be boosted at about 16 weeks of age, about 17 weeks of age, about 18 weeks of age, about 19 weeks of age, or about 20 weeks of age. Progeny of such spawning or breeding stocks may exhibit antibody titers against Mycoplasma gallisepticum, which may prevent or ameliorate symptoms of Mycoplasma gallisepticum infection in the progeny.

[0052] Poultry may receive the compositions or vaccines described herein at various ages. For post-hatch delivery, the material may be delivered at any suitable age, including but not limited to, about 1-3 days of age, about 1 week after hatching, about 2 weeks after hatching, about 3 weeks after hatching, about 4 weeks after hatching, about 5 weeks after hatching, about 6 weeks after hatching, or any range therein. Chickens may be vaccinated only once. Or, if a two-dose vaccine is used, the first vaccine is, for example, when the chickens are 3 days to 1 week old, followed by another 1-10 weeks later.

[0053] Multiple doses of the composition can be administered throughout the life of the chicken. Typically breeder chickens are vaccinated since maternal immunity is the primary source of protection for the broiler offspring, however, broiler chickens can be vaccinated if desired.

[0054] The compositions and vaccines of the invention can be formulated to contain a specified concentration of Mycoplasma gallisepticum. The organism can be measured as color change units. Color change units of Mycoplasma gallisepticum, also referred to herein as "ccu", can be quantified using established standard techniques, including, for example, the protocols described in Rodwell and Whitcomb (In "Methods in Mycoplasmology," Eds. Razin and Tully, 1993). For example, a composition or vaccine can be formulated to contain about 50, about 100, about 1x10 2 ccu / ml, approx. 2.5x10 2 ccu / ml, approx. 5x10 2 ccu / ml, approx. 1x10 3 ccu / ml, approx. 2.5x10 3 ccu / ml, approx. 5x10 3 ccu / ml, approx. 1x10 4 ccu / ml, approx. 2.5x10 4 ccu / ml, approx. 5x10 4 ccu / ml, approx. 1x10 5 ccu / ml, approx. 2.5x10 5 ccu / ml, approx. 5x10 5 ccu / ml, approx. 1x10 6 ccu / ml, approx. 2.5x10 6 ccu / ml, approx. 5x10 6 ccu / ml, approx. 1x10 7 ccu / ml, approx. 2.5x10 7 ccu / ml, approx. 5x10 7 ccu / ml, approx. 1x10 8 ccu / ml, approx. 2.5x10 8 ccu / ml, approx. 5x10 8 ccu / ml, approx. 1x10 9 ccu / ml, approx. 2.5x10 9 ccu / ml, or approximately 5 x 10 9 ccu / ml, or any range therebetween (e.g., about 1x10 5 ccu / ml ~ approx. 1x10 6Concentrations of about 0.05 ml to about 0.1 ml (ccu / ml) may be used. In some applications, an effective amount may be administered as one drop per bird (per eye per bird). One drop may be about 0.05 ml to about 0.1 ml.

[0055] The Mycoplasma gallisepticum strains of the present invention may be administered to birds to reduce their susceptibility to Mycoplasma gallisepticum infection. Upon such administration, the material does not result in significant clinical signs or lesions indicative of Mycoplasma gallisepticum. Thus, it is an object of the present invention to provide an immunological material that upon administration does not result in significant clinical signs or lesions indicative of MS disease. It is another object to provide an immunological material with low toxicity.

[0056] The present invention includes a method of generating an anti-MG immune response in poultry, the method comprising administering a Mycoplasma gallisepticum strain, composition, or vaccine described herein. In some embodiments, the immunity comprises humoral immunity and / or cellular immunity. In a humoral response, anti-MS antibodies can be measured, for example, by serum plate agglutination test (SPA) test (e.g., using commercially available antigens (Charles River Laboratories International, Inc., Wilmington, MA)), hemagglutination inhibition test (HI) test, and enzyme-linked immunosorbent assay (ELISA) test (e.g., using commercially available kits (IDEXX, Westbrook, Maine)). SPA and HI testing procedures are described in detail by Ferguson-Noel et al. (Ferguson-Noel, N., and SH Kleven Mycoplasma species. In: A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens, Sixth ed. SM Williams, L. Dufour-Zavala, MW Jackwood, MD Lee, B. Lupiani, WM Reed, E. Spackman and PR Woolcock, eds. American Association of Avian Pathologists. pp63-70. 2016). In some embodiments, immunization comprises mucosal immunization.

[0057] The invention includes methods of reducing, inhibiting, or preventing MG infection in poultry, the methods comprising administering an isolated Mycoplasma gallisepticum strain, composition, or vaccine described herein. Administration of an isolated Mycoplasma gallisepticum strain, composition, or vaccine described herein may result in a reduction, inhibition, or prevention of one or more disease manifestations upon challenge with further MG infection, including one or more disease manifestations of infectious MG. Such clinical signs and symptoms may include, for example, one or more of: respiratory distress, tracheal rales, difficulty breathing, coughing, sneezing, runny nose, swollen eyelids, ocular discharge, impaired vision, conjunctivitis, foam around the eyes, swelling of the infraorbital sinus, sinusitis, catarrhal sinusitis, tracheitis, air sacitis, pneumonia, leg problems, stunting, depression, weight loss, poor appetite, reduced growth, reduced hatching, reduced chick survival, feather abnormalities, low productivity, reduced feed efficiency, reduced weight gain, reduced egg production, condemnation at processing, and / or increased mortality.

[0058] The present invention also provides a kit comprising Mycoplasma gallisepticum strain K6067 and / or its progeny or derivative as described herein. The kit may comprise one or more containers filled with Mycoplasma gallisepticum of the present invention. Mycoplasma gallisepticum strain K6067 may be lyophilized.

[0059] The present invention also provides a kit comprising the Mycoplasma gallisepticum strain K4110 and / or its progeny or derivative as described herein. The kit may comprise one or more containers filled with the Mycoplasma gallisepticum of the present invention. The Mycoplasma gallisepticum strain K4110 may be lyophilized.

[0060] The kit may include additional separate containers of other strains of Mycoplasma gallisepticum or other pathogens of poultry. In addition, the kit may include other reagents, such as buffers, including solutions necessary to practice the invention. Optionally associated with such container(s) may be notices or printed instructions. The kit of the invention may include "packaging material." As used herein, the term "packaging material" refers to one or more physical structures used to contain the contents of the kit. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging material may be a solid matrix, or a material such as glass, plastic, paper, foil, etc. Thus, for example, the package may be a glass or plastic vial used to contain ccu amounts of Mycoplasma gallisepticum strain K6067 or Mycoplasma gallisepticum strain K4110.

[0061] Exemplary embodiments of the present invention include, but are not limited to, the following. 1. An isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain is the K6067 Mycoplasma gallisepticum strain deposited with the ATCC under patent designation PTA-127168, or a progeny or derivative thereof. 2. An essentially biologically pure culture of Mycoplasma gallisepticum strain K6067 deposited with ATCC under patent designation PTA-127168. 3. An isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain is the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent designation PTA-127282, or a progeny or derivative thereof. 4. An essentially biologically pure culture of Mycoplasma gallisepticum strain K4110 deposited with ATCC under patent designation PTA-127282. 5. A composition comprising an isolated Mycoplasma gallisepticum according to any one of embodiments 1 to 4. 6. The composition of embodiment 5, comprising water. 7. The composition of embodiment 5, comprising a pharma- ceutically acceptable carrier. 8. A composition according to any one of embodiments 5 to 7, comprising an adjuvant. 9. The composition of any one of embodiments 5-8, wherein the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. 10. The composition of any one of embodiments 5-9, wherein the composition is formulated for spraying or aerosolization. 11. A vaccine comprising an isolated Mycoplasma gallisepticum according to any one of embodiments 1 to 4, or a composition according to any one of embodiments 5 to 10. 12. The vaccine of embodiment 11, wherein the vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry. 13. The vaccine of embodiment 11 or 12, wherein the vaccine reduces the susceptibility of Galliformes birds to disease induced by Mycoplasma gallisepticum. 14. A live vaccine for birds of the order Galliformes, the vaccine comprising a sufficient amount of Mycoplasma gallisepticum strain K6067 deposited with the ATCC under patent deposit designation PTA-127168, or a progeny or derivative thereof, to protect the bird against disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier. 15. A live vaccine for birds of the order Galliformes, comprising a sufficient amount of the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent deposit designation PTA-127282, or a progeny or derivative thereof, to protect the bird against disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier. 16. The isolated Mycoplasma gallisepticum, composition, or vaccine according to any one of embodiments 1 to 15, wherein the isolated Mycoplasma gallisepticum, composition, or vaccine is lyophilized, freeze-dried, frozen, or is an effervescent tablet. 17. A kit comprising an isolated Mycoplasma gallisepticum, composition, or vaccine described in any one of embodiments 1 to 16 and printed instructions, wherein the contents of the kit are contained within packaging material. 18. An effervescent tablet comprising an isolated Mycoplasma gallisepticum, composition or vaccine according to any one of embodiments 1 to 16. 19. A method for generating an immune response against Mycoplasma gallisepticum in a bird, comprising administering to the bird an isolated Mycoplasma gallisepticum, composition, or vaccine described in any one of embodiments 1 to 16. 20. A method for reducing the susceptibility of a bird to disease induced by Mycoplasma gallisepticum, comprising administering to the bird an isolated Mycoplasma gallisepticum, composition, or vaccine described in any one of embodiments 1 to 16. 21. A method for protecting a bird against Mycoplasma gallisepticum infection, comprising administering to the bird an isolated Mycoplasma gallisepticum, composition, or vaccine described in any one of embodiments 1 to 16. 22. A method for reducing one or more clinical signs induced by Mycoplasma gallisepticum infection in a bird, comprising administering to the bird an effective amount of an isolated Mycoplasma gallisepticum, composition, or vaccine described in any one of embodiments 1 to 16. 23. The method of any one of embodiments 19-22, wherein administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. 24. The method of any one of embodiments 19-22, wherein administration is by eye drops, by aerosol, or by drinking water. 25. The method of any one of embodiments 19-24, wherein the bird comprises a bird of the order Galliformes. 26. The method of any one of embodiments 19-25, wherein the bird comprises a chicken or a turkey.

[0062] The present invention is illustrated by the following examples. It is to be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. EXAMPLES

[0063] Example 1 Evaluation of Mycoplasma gallisepticum strains as live vaccines in chickens and turkeys Ten Mycoplasma gallisepticum (MG) isolates were selected based on the analysis of cases and targeted sequencing results from the isolate reservoir at the Poultry Diagnostic and Research Center (PDRC). The safety of the isolates was evaluated in turkeys, and two isolates (K4110A and K6067) were selected based on successful colonization of the trachea without clinical signs or gross lesions at necropsy (trials 1 and 2). In trial 3, the safety and efficacy of both isolates were evaluated in chickens, where SPF chickens were vaccinated via eye drop at 4 weeks of age and the R strain was used to challenge chickens via aerosol 4 weeks after vaccination. Both groups vaccinated with the vaccine candidate showed no significant difference in lesions compared to the negative control, indicating that the candidate is safe in chickens. Also, both vaccinated groups had significantly lower lesions after challenge, indicating an effective vaccine. Further investigations were performed to determine the most protective route of administration and dose required for each candidate. The minimum protective dose of 50% for both K6067 and K4110A was 10 mg / kg / day via the ophthalmic route. 4 -10 5ccu / mL (Trials 4 and 5). In conclusion, the results of the study showed that no vertical transmission was detected in Trial 6, and both K6067 and K4110A have potential live vaccine candidates as these initial studies indicate that they are safe and effective in turkeys and chickens.

[0064] Materials and Methods MG strains and isolates Ten vaccine candidates (Table 1) were selected from the MG culture repository at the Poultry Diagnostic and Research Center (University of Georgia, Athens, GA) based on criteria including indication of natural attenuation (no cases of clinical signs or mortality) and rapid and consistent in vitro growth rates. Targeted DNA sequencing results of the 16S-23S rRNA intergenic space region (IGSR) (Papazisi et al., 2003, Microbiology; 149:2307-16) and mgc2 cytadecine (Hnatow et al., 1998, Infect Immun; 66:3436-42) genes were compared to the PDRC MG sequence database to avoid isolates that were highly similar to commercial vaccines (Ferguson et al., 2005, Microbiology; 151:1883-1893). Analysis and comparison of sequences was performed using NCBI Blast (Johnson et al., 2008, Nucleic Acids Research; 36(suppl 2):W5-W9) and MegAlign Software (Lasergene package, DNAStar, Inc., Madison, WI). Whole genome sequencing is performed on both low (~10p) and high (~50p) passages of candidates to assess genetic stability after in vitro passaging and to compare whole genome sequences with MG genome sequences publicly available or available in the whole genome library of the Mycoplasma Laboratory at PDRC. For isolates selected for whole genome analysis, DNA was extracted after growth in Frey's modified broth, cells were centrifuged at 13,000xg for 3 min, the supernatant was discarded, and the cell pellet was reconstituted in 200 μl of phosphate-buffered saline (pH=7). Genomic DNA was extracted using the QIAGEN DNEASY® Blood and Tissue Kit (QIAGEN, Valencia, Calif.) or the Promega WIZARD® Genomic DNA Purification Kit (Promega, Madison, Wisconsin) according to the manufacturer's recommendations.Illumina-based sequencing (Illumina, San Diego, California) was performed at Novogene (Sacramento, CA). Comprehensive genomic analysis of the isolates, including de novo assembly and annotation, was performed in PATRIC (Wattam et al., 2017, Nucleic Acids Res;45:D535-D542). Whole genomes of isolates were compared to established vaccine and laboratory strains using the Similar Genome Finder and Phylogenetic Tree Builder tools in PATRIC.

[0065] After preliminary screening of 10 candidates in turkeys, K4110A and K6067 were further evaluated for safety and efficacy in chickens (trial 3), the most protective and effective dose and route of administration of each (trials 4 and 5), and potential vertical transmission of each (trial 6). The R strain, a well-described pathogenic strain of MG (Rodriguez and Kleven, 1980, Avian Dis; 24:800-7), was used to challenge vaccinated or naive birds as a positive control group. The aerosol route and eye drop route were the two routes of administration used to inoculate birds; in the former, 1 mL of actively growing culture was aerosolized using a commercial paint sprayer (PREVAL® Sprayer Division, Precision Valve Corporation, Yonkers, NY), and in the latter, 100 μl of actively growing culture was administered to the eye.

[0066] Serology Serum was analyzed to confirm stimulation of the humoral immune system, and the presence of anti-MG antibodies was assessed by serum plate agglutination (SPA) using commercially available antigens (Charles River Laboratories, North Franklin, CT), hemagglutination inhibition (HI) tests using antigens prepared from the A5969 strain and chicken red blood cells (RBCs), and enzyme-linked immunosorbent assay (ELISA) using commercially available kits (IDEXX, Westbrook, ME; Biochek, Scarborough, ME). SPA and HI tests were performed using previously described procedures (Kleven SH, "Mycoplasmosis," In: Dufour-Zavala L, Swayne DE, Glisson JR, Pearson JE, Reed WM, Jackwood MW. Woolcock PR, editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens, Fifth Edition. American Association of Avian Pathologists. p. 59-64; 2008), with a score of ≥ 1 considered positive for SPA and for HI, a titer of 1:20 considered suspicious and a titer of ≥ 1:40 considered positive. For ELISA, a sample / positive (S / P) ratio of ≥ 0.5 was considered positive.

[0067] Isolation and Identification of Mycoplasma Cotton swabs were taken from the trachea, palate and air sacs and inoculated into Frey's modified broth and agar (Ferguson-Noel and Kleven, “Mycoplasma species,” In: Williams SM, Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E. Woolcock PR, editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. p. 63-70; 2016).

[0068] For trials 1 and 2, samples were also inoculated into modified PPLO broth and agar (Ferguson-Noel and Kleven, “Mycoplasma species,” In: Williams SM, Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E. Woolcock PR, editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. p. 63-70; 2016) (prior to inoculation when screening birds for the presence of Mycoplasma species). All cultures were incubated at 37C and cultures were considered negative for Mycoplasma if no growth was observed in the broth tubes or agar plates after 4 weeks. Direct immunofluorescence was used to identify mycoplasma isolates (Talkington and Kleven, 1983, Avian Dis; 27:422-9).

[0069] qPCR and calculation of colony formation efficiency Real-time quantitative PCR (qPCR) procedures for specific detection of MG and MS, as well as MM and MI, were performed using previously described protocols (Raviv and Kleven, 1983, Avian Dis; 53:103-7, and Callison et al., 2006, Avian Dis; 50:537-44). qPCR was performed to estimate the genome copy number of MG present in tracheal samples collected at necropsy. In addition, strain-specific qPCR was performed against R strains, K4110A and K6067 to evaluate the efficacy of these two vaccine candidates in controlling colonization of the virulent challenge strain. Tracheal swabs were taken during the trial and at necropsy, upper tracheal segments of individual birds were collected at necropsy in 10 ml of sterile PBS for later DNA extraction. Genomic DNA was extracted from 200 μl of laryngeal washings and tracheal swabs using MAG-BIND® Blood and Tissue DNA HDQ 96 Kits (Omega Bio-tek, Inc., Norcross, GA) on MAGMAX™ Express-96 Magnetic Particle Processors (Thermo Fisher Scientific) according to the manufacturer's recommendations. Real-time PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific), and a cycle threshold (Ct) of ≦39 was considered positive. To make the assay quantitative, a plasmid containing the genomic target was constructed as a standard DNA control. The procedures used for constructing DNA controls and standard curves for quantification have been described in detail elsewhere (Raviv et al., 2008, Vet Microbiol; 129: 179-87).

[0070] Lesion evaluation Gross air sac lesions were scored on a scale of 0 to 4 using the scoring system described by Kleven (Kleven et al., 1972, Avian Dis; 16:915-24, and Kleven et al., 1975, Avian Dis; 19:126-35). Tracheal lesions were assessed microscopically by measuring the width of the tracheal mucosa. A portion of the upper third of the trachea (approximately 1 inch distal to the larynx) was fixed in 10% neutral formalin. The thickness of the tracheal mucosa was measured at four equally spaced points on histological slides of cross sections of the trachea (Whithear, 1996, Rev. Sci. Tech. Off. Int. Epiz. 15:1527-1553). Other necropsy findings were also recorded.

[0071] Animal Care and Use All animal procedures in these experiments were approved by the Institutional Animal Care and Use Committee of the University of Georgia, Athens, Ga. All birds in these studies were provided with food and water ad libitum and were euthanized with carbon dioxide.

[0072] statistical analysis Air sac lesions and M. gallisepticum isolation from chickens were analyzed using one-way ANOVA. Mean tracheal mucosal thickness, MG DNA genome copy number log10, SPA score, S / P ratio, HI titer, fertility rate, and weekly egg production rate of hens were analyzed using the Tukey-Kramer highly significant difference test (Minitab 17 Statistical Software. [Computer software]. State College, PA: Minitab, Inc. (www.minitab.com). 2010). A p value ≤ 0.05 was considered significant.

[0073] Experimental design Clinical Trials 1 and 2 (Turkey Safety 1 and 2) Trials 1 and 2 had the same experimental design, with five candidates in each trial, for a total of 10 vaccine candidates evaluated across the two trials. For each of these trials, 42 turkeys were obtained at 1 day of age from a source known to be free of MG and MS and housed in two isolators. At 2 weeks of age (WOA), turkeys were leg-banded using random numbers (www.randomlists.com) and randomly assigned to 7 treatment groups (7 isolators) of 6 birds each. Ten turkeys distributed across treatment groups were screened for the presence of mycoplasma antibodies by culture, quantitative polymerase chain reaction (qPCR) of tracheal samples, and serum at that time. In the 3WOA, turkeys were inoculated via aerosol with their respective treatment or left uninoculated (negative control group) (Tables 2 and 3). At 5WOA, all birds were necropsied and evaluated by gross air sac lesion scoring, serology, tracheal histopathology, and MG-specific qPCR of tracheal washes.

[0074] Clinical trial 3 (safety and efficacy in chickens) 125-day-old SPF chicks were obtained from a source known to be free of MG and MS and housed in one pen (1.5 × 3 m) with pine shavings. 2) in a 2-bedroom house. In 3 WOA, chicks were randomly wing-banded and assigned to 4 treatment groups. Fifteen chickens (distributed among treatment groups) were screened for the presence of Mycoplasma species by culture, MG and Mycoplasma synovitis qPCR of tracheal samples, and serology. In 4 WOA, chickens were inoculated via aerosol with the respective treatment (B, C, D, K6067, K4110A or R strains) or left unvaccinated as negative control groups, as detailed in Table 4 below. Five chickens from groups B and C were not inoculated and mixed with vaccinated birds in 5 WOA. Nineteen days after mixing (8 WOA), these 10 contact birds were necropsied and evaluated. Four weeks after vaccination, 10 chickens each from groups B, C and D were directly challenged with the R strain via aerosol. The remaining vaccinated birds were mixed with these birds and 10 naive birds were placed in direct contact with the unvaccinated aerosol challenge group (contact challenge). At 10 WOA, all birds were necropsied and evaluated by gross air sac lesion scoring, serology, tracheal histopathology, and MG qPCR and strain-specific qPCR of tracheal washes.

[0075] Clinical Trial 4 (Dose Response 1) 84-day-old SPF chicks were obtained from a source known to be free of MG and MS. In 2WOA, chicks were feather banded and randomly assigned to 12 treatment groups in 24 isolators (Table 5). Ten chicks (distributed among treatment groups) were screened for the presence of Mycoplasma species by culture, MG and Mycoplasma synovitis qPCR of tracheal samples, and serology. In 3WOA, chickens in groups C-G were vaccinated with K6067 via eye drop and chickens in groups H-L were vaccinated with K6067 via aerosol. The groups received different doses of the vaccine candidate as shown in Table 5. Groups A and B were left unvaccinated as negative control and challenge-only groups, respectively. One week after vaccination, cleft palate and tracheal swabs were taken from all groups for culture and MG qPCR. Two weeks after vaccination, all 84 chickens were bled and swabbed for MG serology, culture, and MG qPCR. Four weeks after vaccination and at 7 WOA, chickens from groups B-L were challenged with the R strain via aerosol. At 9 WOA, all birds were necropsied and evaluated by gross air sac lesion scoring, serology, cleft palate and air sac cultures, tracheal histopathology, and MG and strain-specific qPCR of tracheal washes. Minimum infectious dose 50 (MID50) and minimum protective dose 50 (MPD50) were calculated based on the results of sampling and necropsy (Cottey et al., 2001, Current Protocols in Immunology; 42:19-11).

[0076] Clinical Trial 5 (Dose Response 2) 79-day-old SPF chicks were obtained from a source known to be free of MG and MS. In 3WOA, chicks were feather banded and randomly assigned to 7 treatment groups in 19 isolators (Table 6). Ten chicks (distributed among treatment groups) were screened for the presence of Mycoplasma species by culture, MG and Mycoplasma synovitis qPCR of tracheal samples, and serology. In 3WOA, chickens in groups C-G were vaccinated with different doses of vaccine candidate K4110A via eye drop. Groups A and B were left unvaccinated as negative control and challenge-only groups, respectively. One week after vaccination, palate and tracheal swabs were taken from all groups for culture and MG q-PCR. Two weeks after vaccination, all 79 chickens were bled and swabbed for MG serology, culture, and MG qPCR. At 4 weeks post-vaccination and at 7 WOA, chickens in groups B-G were challenged with the R strain via aerosol. At 9 WOA (2 weeks post-challenge), all birds were necropsied and evaluated by gross air sac lesion scoring, serology, cleft palate and air sac cultures, tracheal histopathology, and MG and strain-specific qPCR of tracheal washes. The experimental design and specific dates of procedures are detailed in Table 6.

[0077] The minimum infectious dose 50 (MID50) and minimum protective dose 50 (MPD50) were calculated based on the results of sampling and necropsy (Cottey et al., 2001, Current Protocols in Immunology; 42:19-11).

[0078] Clinical Trial 6 (Vertical Transmission) One hundred and forty-four 4-week-old SPF chickens (131 females and 18 males) were obtained and housed in four pens (1.5 × 3 m) with pine shavings. 2) at 19 WOA. Chickens were transferred to a colony house and randomly assigned to five groups (Table 7). Sixteen chickens (distributed among treatment groups) were screened for the presence of Mycoplasma species by culture, MG and Mycoplasma synovitis qPCR of tracheal samples, and serology at 23 WOA. At 25 WOA, chickens were inoculated with the respective treatment (K6067, K4110A, F strain, or R strain) via eye drop or left uninoculated as a negative control group. Three weeks after inoculation at 28 WOA, MG qPCR was performed on palate swabs from the negative control group, five birds from group A, and ten birds from each of groups B-E. Eggs were collected daily from one week before inoculation (24 WOA) until 31 WOA and maintained at 4C until incubation began. Eggs laid on weekdays other than Monday were set on Saturdays, and eggs laid on Saturdays, Sundays, and Mondays were set on Mondays. Each set of eggs was candled on days 7, 9, 11, 13, and 15 of embryogenesis (DOE). Infertile eggs or early dead embryos (<7 DOE) were discarded, and the yolk sacs of any dead embryo eggs at 9, 11, 13, and 15 DOE were sampled and inoculated into modified Frey's broth for culture. All remaining embryonated eggs were sampled for culture at 18 DOE.

[0079] In addition, HI and ELISA (IDEXX, Westbrook, Marine) assays were performed on yolk sac samples from the last set of eggs (National Poultry Improvement Plan, "Procedures for preparing egg yolk samples for diagnostic tests," National Poultry Improvement Plan Program Standards, 23-24; 2017). Samples were prepared and tested using standard procedures recommended by NPIP for egg yolk (National Poultry Improvement Plan, "Procedures for preparing egg yolk samples for diagnostic tests," National Poultry Improvement Plan Program Standards, 23-24; 2017). The HI test procedure was as described by Ferguson-Noel et al. (Ferguson-Noel and Kleven, “Mycoplasma species,” In: Williams SM, Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E. Woolcock PR, editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. p. 63-70; 2016). Laying parameters, including fertility and weekly hen production rate, were also recorded for all groups during the laying period. Fertility rates for all groups for 16 sets of eggs were calculated by dividing the number of fertilized eggs after 7 DOE by the total number of eggs incubated and multiplying by 100 (Lin and Kleven, 1982, Avian Dis; 26: 487-95). Egg numbers were recorded from 21–31 WOA and hen weekly egg production was calculated by dividing the total number of eggs laid per week by the total number of hens and multiplying this by 100.

[0080] At 31 WOA, all birds were necropsied and evaluated by gross air sac lesion scoring, serology, cleft palate and air sac cultures, tracheal histopathology, and MG qPCR oviductal swabs of tracheal washes.

[0081] result Clinical Trial 1 (Turkey Safety 1) All birds tested pre-inoculated in the 2WOA were negative for MG, MM, and MS antibodies and were also negative for MG, MS, MM, and MI by qPCR. All cultures were also negative for Mycoplasma species at that time. MG antibodies were detected in all inoculated groups and serological results for trial 1 are summarized in Table 8. The weakest antibody response was in group E inoculated with isolate K6837. The only group in which air sac lesions were not observed was the negative control group, but groups inoculated with K6067 (group B) and K6837 (group E) had significantly lower mean air sac lesion scores than the positive control (group G) (P < 0.05). The highest mean tracheal mucosal thickness measurements were in groups D and F. The only inoculated groups with mean measurements lower than the positive control (R strain, group G) were inoculated with K6067 (group B) and K6837 (group E). qPCR results showed that all inoculated groups were infected with MG, with the exception of group E (inoculated with K6837), which replicated at high levels in the trachea. Mycoplasma was recovered from all cultures (trachea and air sac) from the inoculated groups, with the exception of two air sac cultures from group E (inoculated with K6837) and group B (inoculated with K6067) (Table 9). There were no mortalities during the trial, but the clinical signs observed are summarized in Table 10. No respiratory clinical signs were observed in birds from groups B (inoculated with K6067) and E (inoculated with K6837).

[0082] Clinical Trial 2 (Turkey Safety 2) All 10 birds tested pre-vaccinated in 3WOA were negative for MG, MM, and MS antibodies, and also negative for MG, MS, MI, and MM by qPCR. All cultures were also negative for Mycoplasma species. MG antibodies were detected in all inoculated groups, with the weakest antibody response in Group C inoculated with K4110A (Table 11). This was consistent with the mean air sac lesion score. The only group in which air sac lesions were not observed was the negative control group. However, groups inoculated with K4110A (Group C) and K4179 (Group B) had lower mean air sac lesion scores than the positive control (Group G). The difference in mean air sac lesion score between Group B (3.5) and the positive control (3.7) was not significant (P≦0.05). The highest mean tracheal mucosa thickness measurements were in Groups B and F. No inoculated group had a lower mean measurement than the positive control (Group G). qPCR results showed that all inoculated groups were infected with MG and that MG was replicating at high levels in the trachea. These results also showed that while K4110A inoculation did not result in significant lesions (P≦0.05), birds were infected and had replicated MG. Mycoplasma was recovered from all cultures in the inoculated groups, both from the trachea and air sacs. These results are summarized in Table 12. There was no mortality during the trial, but the clinical signs observed are summarized in Table 13. No respiratory clinical signs were observed in Group C (inoculated with K4110A) and Group F (inoculated with K6836A) birds.

[0083] Clinical trial 3 (safety and efficacy in chickens) Fifteen birds tested pre-vaccinated in 2 WOA were negative for MG and MS antibodies and also negative for MG and MS by qPCR. All cultures were also negative for Mycoplasma species pre-vaccinated. Three necropsies were performed in this study to assess the safety of the vaccine candidate in chickens (necropsy 1), the potential for horizontal transmission of the vaccine candidate (necropsy 2), and the efficacy of the vaccine candidate after challenge (necropsy 3).

[0084] Necropsy 1 (safety). MG antibodies were detected in all inoculated groups, but the antibody response in the vaccine candidate inoculated groups was weaker compared to the positive control (R strain). The mean SPA scores of groups B (K6067) and C (K4110A) were significantly lower (P ≤ 0.05) than that of group D (R strain). A summary of these serological results is shown in Table 14. All groups except the positive control group had no air sac lesions 14 days after inoculation, and these differences were statistically significant (P ≤ 0.05). There was no significant difference in the mean tracheal mucosa thickness between the groups (P ≤ 0.0). However, the positive control (R strain) had numerically higher MG isolated from the trachea of ​​all inoculated birds 14 days after inoculation, but no MG was isolated from the air sacs of the vaccine candidate group compared to MG isolation from 100% of the R strain group. qPCR results showed that all isolates colonized birds in their groups, but there were significantly lower genome copies (MCNlog10) for K4110A compared to groups inoculated with K6067 and R strains (P≦0.05). A summary of these results is shown in Table 15.

[0085] Necropsy 2 (horizontal transmission to contacts). 19 days after mixing, birds in direct contact with vaccinated birds had low levels of infection (detected by tracheal isolation in 2 of 5 K6067 and 1 of 5 K4110A birds). There were no significant differences in serological responses (Table 16), air sac lesions or tracheal mucosa thickness (Table 17) between the groups.

[0086] Necropsy 3 (Efficacy - Aerosol and Contact Challenge). The vaccine candidate vaccinated group showed a strong serological response 14 days after the R strain challenge (shown in Table 18). As can be seen in Table 19, the vaccinated group also had a significantly lower mean air sac lesion score after challenge (P < 0.05). There was no significant difference (P < 0.05) between the negative and positive controls for the tracheal measurements (Table 21), but the positive control produced the highest mean measurement. The qPCR results are summarized in Table 20. The qPCR results show that the K6067 group had a higher mean genome copy number log10 in its trachea compared to the K4110A group. It is also clear that the majority of MG detected in the trachea of ​​the vaccinated group was the vaccine strain rather than the challenge strain for both vaccine candidates. It appeared that both vaccines were able to prevent colonization with the challenge strain with a significantly lower MCNlog10 for the R strain-specific qPCR in the vaccinated group (P < 0.05). For the contact challenge group, it can be inferred that there was some transmission of the R strain to non-vaccinated contact birds and from the isolation results, at least 70% (7 / 10) of these contact birds were infected with the R strain, but none of the vaccinated birds exposed to the R strain through contact with the challenge birds were infected with the challenge strain (from the qPCR results for the R strain).

[0087] Clinical Trial 4 (Dose Response 1) All 10 birds tested pre-vaccinated at WOA 2 were negative for MG and MS antibodies and were also negative for MG and MS by qPCR. All cultures were also negative for Mycoplasma species at this time. A summary of findings from sampling at WOA 4 (1 week post vaccination) is shown in Table 22. All cultures from groups E, F and G (10 doses via eye drop, respectively) were negative for Mycoplasma spp. 3 , 10 4 , and 10 5 CCU / mL) were positive for Mycoplasma species. 2 CCU / mL), J and K (10 5 and 10 6Only one sample from each of the 10 groups (CCU / mL) was positive. qPCR results also showed some degree of MG colonization only in groups E, F, and G with K6067.

[0088] Sampling results at 6WOA (3 weeks after vaccination) are shown in Tables 23 and 24. All cultures from groups E, F, and G were again positive for mycoplasma, one sample each from groups J and K and two samples from group D were positive, the rest were all negative. MG colonization was also assessed by performing qPCR on tracheal swabs, with groups D, E, F, and G showing some degree of colonization and group F (10% positive via eye drops). 4 CCU / mL) showed a colony formation rate of 100%. For serology, serum plate agglutination (SPA) tests showed some antibody responses in groups D-G, J, and K, but few were positive in groups F and G by hemagglutination inhibition test (HI) and enzyme-linked immunosorbent assay (ELISA) tests. Six weeks after vaccination (two weeks after challenge with the R strain), MG antibodies were detected in all vaccinated groups (B-G) (Tables 25 and 26). The mean SPA scores of groups E-G (which all showed K6067 colonization in the trachea at early sampling) were lower compared to group B (challenge only group). All groups except the negative control group (group A) had some level of air sac lesions after virulent challenge, but the mean air sac lesion scores of groups D, E, F, G, J, K, and L were lower than the challenge only group (group B). Among them, the mean scores of groups E, F and G were significantly lower (P≦0.05) compared with other vaccinated and challenged groups. The mean tracheal mucosal thickness measurements of groups D, E, F, G, J and K were lower compared with the challenge-only group, and the differences were significant in groups E, F and G (P≦0.05) (Table 27).

[0089] The qPCR results summarized in Table 28 showed that all inoculated groups were infected and MG was replicating in the trachea. The mean colonization rates of these groups E, F, and G (vaccinated with the highest dose of K6067 via eye drop) were significantly lower (P≦0.05) than the other groups, including the challenge-only group (group B). Also, the mean colonization rates of groups D, J, and K were lower than group B. The strain-specific qPCR results correlated with the MG qPCR for the groups (Table 28). Mycoplasma was recovered from all cleft palate cultures from inoculated groups after R strain challenge, except for two birds in group J. For air sac cultures, mycoplasma was recovered from the air sacs of all groups vaccinated via aerosol (groups H-L). In groups vaccinated via eye drop, mycoplasma was isolated from all birds in groups C, D, and G, three birds in group E, and five birds in group F (Table 27). The MID50 of K6067 was calculated based on qPCR and culture results of sampling at 1 and 3 weeks after vaccination for all doses and both routes of administration. The qPCR results did not allow for the calculation of the MID50, but the culture results for the group vaccinated via the eye drop route showed a MID50 of 10 at 1 week after vaccination. 3.8 ccu / mL, 10 3 weeks after vaccination 3.53 CCU / mL. It was not possible to calculate the MID50 from culture results for the group vaccinated via the aerosol route (Table 29).

[0090] In addition, the MPD50 of K6067 was also calculated based on the air sac score and tracheal thickness measurements recorded at necropsy. It was not possible to calculate the MPD50 for either the air sac score or tracheal thickness measurements in the group vaccinated via aerosol, but the MPD50 for the group vaccinated via eye drop was 10 for the air sac score. 4 CCU / mL, tracheal thickness measurement 10 3.3 CCU / mL (Table 30).

[0091] Clinical Trial 5 (Dose Response 2) All 10 birds tested pre-vaccinated at 2 WOA were negative for MG and MS antibodies and also negative for MG and MS by qPCR. All cultures were also negative for Mycoplasma species at that time point. A summary of findings from sampling at 4 WOA (1 week post-vaccination) is shown in Table 31. Ten of the 13 cultures from group C and all cultures from groups D-G were positive for Mycoplasma. qPCR results also showed some level of MG colonization in all groups vaccinated with K4110A, with groups F and G (those receiving the highest vaccine doses) having significantly higher mean genome copy numbers (MCNlog10) than the negative controls (P<0.05).

[0092] Results from sampling at 6WOA (3 weeks after vaccination) are summarized in Tables 32 and 33. All cultures of the vaccinated groups (except for two birds in group C) were positive for Mycoplasma. Some level of colonization was detected in all vaccinated groups, with groups E, F and G having significantly higher mean genome copy numbers (MCNlog10) than the negative controls (P<0.05). Regarding serology, more positive samples were seen in the SPA test compared to the HI and ELISA tests. The SPA score of group C (received the lowest dose of vaccine) was significantly lower than the other vaccinated groups (P<0.05). Group C also had no positive results in the ELISA and HI tests.

[0093] At 9 WOA (6 weeks after vaccination and 2 weeks after challenge with R strain), seroconversion was observed in all vaccinated groups. The mean SPA scores of groups C-G were lower compared to group B (challenge only group). However, the s / p ratios and HI titers from these groups were all higher than group B (Table 34). All groups except groups A (negative control) and F had some air sac lesions. However, the mean air sac lesion scores of all vaccinated groups were significantly lower (P<0.05) than group B (challenge only control) (Table 35). The mean tracheal mucosal thickness measurements of all vaccinated groups were significantly lower (P<0.05) compared to the challenge only group (group B). Mycoplasma was isolated from all cleft cultures of all vaccinated and challenged groups and from the air sacs of all birds of all vaccinated and challenged groups except two birds of group D and one bird of group F.

[0094] MG qPCR and strain-specific qPCR results are summarized in Table 36. R strain mean genome copy number (MCNlog10) detected in the trachea was significantly higher in the challenge-only group compared to the vaccinated group (P<0.05). K4110A-specific qPCR showed that the vaccine candidate colonized the trachea of ​​all K4110A-vaccinated groups, although the MCNlog10 of the vaccine strain in group C (lowest vaccination dose) was significantly lower than the other vaccinated groups. No K4110A was detected in air sac cultures from vaccinated groups, except group G (Table 36). The MID50 of K4110A was calculated based on the qPCR and culture results 1-3 weeks after vaccination for all doses (Table 37). The MID50 of the qPCR results for the group vaccinated via eye drop route was 10% at 1 week after vaccination, whereas no MID50 was seen in the culture results. 5.3 CCU / mL, 10 3 weeks after vaccination 5.2 The mean CCU / mL.

[0095] The MPD50 of K4110A was also calculated based on the mean air sac score autopsy, 10 3.7CCU / mL. (Table 38) The MPD50 for tracheal thickness measurements in the vaccinated group could not be calculated.

[0096] Clinical Trial 6 (Vertical Transmission) All 16 birds tested pre-vaccinated at 23 WOA were negative for MG and MS antibodies and were also negative for MG and MS by qPCR, and all cultures were negative for Mycoplasma species at that time.

[0097] A total of 4436 eggs were tested, none of which were positive for mycoplasma. However, HI and ELISA results on egg yolk samples indicated transfer of antibodies to the embryos (Table 39). There were some positive MG ELISA results for groups C (K4110A), D (F strain) and E (R strain), but only groups C and E were positive in MG HI. No group B yolk sac samples were positive by either HI or ELISA. Fertility rates and weekly hens production averages for all groups of 16 sets of eggs are shown in Table 40. There were no significant differences between groups (P<0.05).

[0098] A summary of the serological results at necropsy is shown in Table 41. MG antibodies were detected in all vaccinated groups. The HI titers and S / P ratios of ELISA in group B (K6067) were significantly lower (P<0.05) than the other vaccinated groups, but there was no significant difference in the SPA scores of the vaccinated groups.

[0099] The mean air sac lesions in the groups vaccinated with the two vaccine candidates and the F strain were significantly lower than in group E (strain R) (Table 42) P<0.05). The highest mean tracheal mucosal thickness measurements were in group E vaccinated with the R strain. With regard to tracheal lesions, both vaccine candidates (groups B and C) and the group vaccinated with the F strain (group D) had mean measurements significantly lower than the positive control (groups E-strain R) P<0.05). MG qPCR results of tracheal wash samples showed that all vaccinated groups were infected with MG and that MG was replicating in the trachea. These results showed that the MG colonization rate in the trachea of ​​the groups vaccinated with both vaccine candidates was higher than the groups vaccinated with the F and R strains. None of the oviduct swabs were positive by MG q-PCR. Mycoplasma was recovered from all cleft cultures in all vaccinated groups except one bird from group C and another bird from group D (Table 42). Both negative samples were from birds that were positive by q-PCR and air sac culture. The number of positive air sac cultures in all vaccinated groups was significantly lower than in group E (vaccinated with the R strain).

[0100] Consideration Ten MG isolates that met our initial criteria for vaccine candidates were initially administered to turkeys in trials 1 and 2. The rationale for selecting isolates recovered from turkeys as well as conducting initial safety trials in turkeys was based on the fact that turkeys are generally more susceptible to MG infection and more severely susceptible to disease compared to chickens. The overall goal of this study was to develop a live attenuated MG vaccine candidate that is effective and safe in both turkeys and chickens. Vaccine candidates with some degree of natural attenuation were considered more promising candidates than virulent strains. Analysis of the results (clinical signs, air sac and tracheal lesions) from the two turkey trials showed evidence that these isolates were (to some degree) naturally attenuated, leading to the selection of K6067 and K4110A for further studies. In trial 1 (K6837), there was another isolate that produced the weakest antibody response and low levels of lesions compared to the other inoculated groups. However, the inoculum titer of this isolate (10 6CCU / ml) was calculated based on the inoculum titer of other isolates (10 8 CCU / ml), and based on the qPCR results, it appears that this isolate did not colonize turkey as well as other MG isolates. K6837 met the minimum requirements for robust in vitro growth, but it was the slowest growing of the isolates selected for this trial, and this isolate requires further study to determine whether the results from Trial 1 were due to the low titer (and low colonization rate) or low virulence of the isolate.

[0101] Results from trial 1 indicate that K6067 inoculation did not result in significant lesions, but turkeys were infected and replicated MG (a requirement for generating a protective immune response). 6 / 85 were shown to poorly colonize the trachea, which may be explained in comparison with F strains and ts-11 (16, 29). Air sac culture results also indicated that in this trial, K6067 may not be as invasive as other MG isolates. Similarly, in trial (turkey safety 2), K4110A was able to infect turkeys but did not result in severe clinical signs or lesions. All remaining trials were performed in chickens, but further studies in turkeys are needed to investigate efficacy and other parameters in this poultry species.

[0102] In trial 3, the two selected candidates were evaluated for safety (including potential for horizontal transmission) and efficacy in chickens. In the first part of this trial, both vaccine candidates were determined to meet the requirements for safety in chickens, with isolates colonizing the respiratory tract and generating a systemic antibody response, but without clinical signs or air sac lesions following administration of K6067 or K4110A by aerosol. In this trial, K6067 was present at higher levels in tracheal samples compared to K4110A, although this was due to the slightly lower dose of K4110A administered (10 7 CCU / ml vs. 10 8CCU / ml). Another aspect of safety - risk of horizontal transmission was assessed by mixing contacts of vaccinated and naive birds. There was some evidence of infection in the contacts, but as birds were only mixed for 14 days, the transmissibility of the vaccine candidate needs to be investigated more thoroughly before any conclusions can be drawn. Regarding the efficacy of the vaccine candidates, the results of this trial following a virulent challenge with the MG R strain showed that both candidates induced protective immunity in chickens. Vaccinated birds had significantly lower mean air sac scores compared to unvaccinated controls for aerosol challenged birds.

[0103] In this trial, there were no significant differences in tracheal mucosa measurements between groups, likely due to the combined data from aerosol-challenged and contact-challenged birds. Contact-challenged birds also did not have severe lesions in the unvaccinated control group. Although this challenge method is not generally expected to result in a strong challenge with high lesion scores, the qPCR results indicate that the vaccine candidates were able to prevent (with contact infection) or reduce (with direct aerosol challenge) infection and colonization against the challenge strain. Further investigation of the efficacy of these candidates (including direct comparison with the commercial vaccine) needs to be done to confirm these preliminary results. However, the results of trials 4 and 5 indicate that the vaccine candidates have a minimum infectious and protective dose equivalent to the commercial vaccine when administered by the eye drop route. A 10% dose reduction was achieved by administering the vaccine via eye drop. 3 Doses in the range of CCU / ml were sufficient to infect 100% (for K6067) and 77% (for K4110A) of the birds one week after vaccination. This dose was also sufficient to provide some protection against air sac and tracheal lesions after virulent challenge. The minimum effective dose for the F strain was 10 5.5Calculated in CCU / mL (Lin and Kleven, 1984, Avian Dis;28:273-7). Aerosol administration of K6067 was less successful, and the equipment and environmental complexities of aerosol administration of the vaccine to the small number of birds in each group in the trial likely influenced this portion of the study. Although a larger number of studies (larger number of birds) may indicate that aerosol may be an effective route of administration, the results of this trial suggest that the eye drop route is preferred for K6067 vaccination. These same results found eye drop to be the most effective route of administration for the F strain (Leigh et al., 2018, Poultry Science;97(9):3072-3075, and Evans et al., 2015, Poult Sci;94:1849-52). More consistent results and increased immune responses measured by serology for the eye drop vaccinated groups. The better results seen with the eye drop route may be due to several factors, including individual administration of vaccine and more consistent volume and dose for each bird in the flock. It should be noted that for the dose response 2 (K4110A) strain, strain-specific PCRS was performed not only on tracheal samples but also on air sac cultures, and genome copy numbers were very high for the R strain, likely due to the R strain being adapted to in vitro culture.

[0104] In both turkeys and chickens, the two vaccine candidates elicited detectable and consistent serological responses, and although serum antibody levels do not appear to correlate with protection with the live attenuated MG vaccine, it is useful to be able to monitor vaccination using widely available and inexpensive serological tests.

[0105] Vertical transmission can be a major concern for the safety of mycoplasma vaccines, and both the F and ts-11 vaccines have been shown to have some risk of transmission to offspring via eggs (Armour et al., 2015, Avian Pathol; 44: 296-304, and El Gazzar et al., 2011, Avian Dis; 55: 569-74). No vertical transmission was found in any of the candidates in this study. However, vertical transmission was also not detected in flocks inoculated with the F or R strains. These results may be due to the relatively small number of birds in the trial and the route of administration (eye drops). This route better mimics the potential field application of the vaccine, although with a more severe challenge (aerosol), a higher transmission rate may be seen. The results indicate that the vaccine candidate is unlikely to have a high vertical transmission rate, but that an increase in the number of birds and eggs should be considered to confirm these results. The detection of maternal antibody transfer in egg yolk samples indicated some systemic immune response against MG. The role of passive maternal antibodies in MG infection has been shown to be unbeneficial as they offer little protection against MG challenge (Lin and Kleven, 1984, Avian Dis;28:79-87) and embryonic lethality caused by virulent MG is blocked by the antibodies (Levisohn et al., 1985, Avian Dis;29:188-97), which may result in an increased hatch rate of infected eggs that is not ideal.

[0106] In conclusion, both candidates were safe in turkeys and chickens and effective in reducing colonization with virulent challenge strains and in reducing the lesions and effects of virulent MG strains in chickens. The eye drop route was confirmed as an effective route of administration, and the infectious and protective doses correspond to other MG vaccines. Vertical transmission studies showed that neither K6067 nor K4110A pose a high risk of transmission. These results are promising, and further studies evaluating parameters such as duration of immunity, transmissibility, and efficacy in turkeys will provide further information on these vaccines.

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34

Table 35

Table 36

Table 37

Table 38

Table 39

Table 40

Table 41

Table 42

Claims

1. An isolated Mycoplasma gallicepticum strain, wherein the isolated Mycoplasma gallicepticum strain includes Mycoplasma gallicepticum strain K6067 deposited with ATCC under patent designation PTA-127168, or its offspring or derivatives, or Mycoplasma gallicepticum strain K4110 deposited with ATCC under patent designation PTA-127282, or its offspring or derivatives.

2. Essentially biologically pure cultures of the K6067 Mycoplasma galliseptum strain deposited with ATCC under patent designation PTA-127168.

3. Essentially biologically pure cultures of the K4110 Mycoplasma galliseptum strain deposited with ATCC under patent designation PTA-127282.

4. A composition comprising the isolated Mycoplasma galliseptum strain described in claim 1.

5. The composition according to claim 4, comprising water.

6. The composition according to claim 4, comprising a pharmaceutically acceptable carrier.

7. The composition according to claim 4, comprising an adjuvant.

8. The composition according to claim 4, wherein the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or intraocular administration.

9. The composition according to claim 4, wherein the composition is formulated for spraying or aerosolizing.

10. A vaccine comprising the isolated Mycoplasma galliculum strain described in claim 1, or the composition described in claim 4.

11. The vaccine according to claim 10, wherein the vaccine reduces one or more clinical signs induced by Mycoplasma gallisepticum infection in birds, or reduces the susceptibility of Galliform birds to diseases induced by Mycoplasma gallisepticum.

12. A live vaccine for birds of the order Galliformes, comprising: a sufficient amount to protect the birds from diseases induced by Mycoplasma gallisepticum, comprising: the K6067 Mycoplasma gallisepticum strain deposited with the ATCC under patent deposit designation PTA-127168 or its offspring or derivatives; or the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent deposit designation PTA-127282 or its offspring or derivatives; and a pharmaceutically acceptable carrier.

13. The isolated Mycoplasma galliculis strain according to claim 1, the composition according to claim 4, or the vaccine according to claim 10, wherein the isolated Mycoplasma galliculis strain, composition, or vaccine is lyophilized, freeze-dried, frozen, or in the form of an effervescent tablet.

14. A kit comprising an isolated Mycoplasma galliseptum strain according to claim 1, the composition according to claim 4, or the vaccine according to claim 10, and printed instructions, wherein the contents of the kit are contained within a packaging material.

15. A method for generating an immune response against Mycoplasma galliseptum in birds, for reducing the susceptibility of birds to diseases induced by Mycoplasma galliseptum, for protecting birds from Mycoplasma galliseptum infection, or for reducing one or more clinical signs induced by Mycoplasma galliseptum infection in birds, the method comprising administering to the birds an isolated Mycoplasma galliseptum strain according to claim 1, the composition according to claim 4, or the vaccine according to claim 10.

16. The method according to claim 15, wherein the administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or intraocular.

17. The method according to claim 15, wherein administration is by eye drops, by aerosol, or by drinking water.

18. The method according to claim 15, wherein the bird includes birds of the order Galliformes.

19. The method according to claim 15, wherein the bird includes a chicken or a turkey.