Mycoplasma gallisepticum live vaccine

Abstract

The present invention provides a Mycoplasma gallisepticum strain K6067 preserved in ATCC in patent number PTA-127168, a Mycoplasma gallisepticum strain K4110 preserved in ATCC in patent number PTA-127282, and progeny and derivatives thereof, which are useful as vaccines for the prevention of virulent Mycoplasma gallisepticum infection in avian alliformes. Compositions and methods for administration to galliformes birds are also provided.

Description

[0001] Continuous application data

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 327,851, filed on April 6, 2022, and U.S. Provisional Application Serial No. 63 / 404,226, filed on September 7, 2022, each of which is incorporated herein by reference. Background Art

[0003] Mycoplasma gallisepticum (MG) is an infectious respiratory pathogen of chickens and turkeys. It is the most pathogenic and economically significant mycoplasma pathogen in poultry. Economic losses due to carcass waste or degradation, reduced feed and egg production efficiency, and increased drug costs are factors that make this disease one of the most expensive disease problems facing commercial poultry production worldwide. Although the poultry industry has adopted different measures (such as voluntary monitoring programs, treatment with different antibiotics, and vaccination) to control M. gallisepticum infections and reduce economic losses to the poultry industry, MG is still considered a persistent challenge facing the commercial poultry industry. Voluntary monitoring programs can be effective, but the value of long-lived flocks (breeders and laying hens) makes it difficult to justify culling and quarantine, especially in areas where MG is widespread. Increasing legal restrictions on the use of antibiotics in poultry, especially for preventive in-feed use, and increasing reports of resistance to antibiotics commonly used to control MG infections have reduced the applicability and effectiveness of treatment with different antibiotics. Therefore, vaccination has become an increasingly attractive option for MG control. There are currently three commercially available live attenuated MG vaccines (6 / 85, F strain, and ts-11). Each of these three vaccines has advantages and disadvantages in terms of safety and efficacy. Therefore, additional live attenuated vaccines are needed to control M. gallisepticum infections in poultry. Summary of the invention

[0004] The present invention includes an isolated strain of Mycoplasma gallisepticum, wherein the isolated strain of Mycoplasma gallisepticum is the K6067 strain of Mycoplasma gallisepticum deposited with the ATCC under patent number PTA-127168, or a progeny or derivative thereof. In some aspects, the present invention includes a composition comprising the isolated strain of Mycoplasma gallisepticum. In some aspects, the composition may include water. In some aspects, the composition may include a pharmaceutically acceptable carrier. In some aspects, the composition may include an adjuvant. In some aspects, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous or intraovo administration. In some aspects, the composition may be formulated for spraying or atomization.

[0005] The present invention includes a substantially biologically pure culture of the K6067 Mycoplasma gallisepticum strain deposited with the ATCC under patent number PTA-127168. In some aspects, the present invention includes a composition comprising an isolated Mycoplasma gallisepticum strain. In some aspects, the composition may include water. In some aspects, the composition may include a pharmaceutically acceptable carrier. In some aspects, the composition may include an adjuvant. In some aspects, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous or intraovo administration. In some aspects, the composition may be formulated for spraying or atomization.

[0006] The present invention includes a vaccine comprising an isolated Mycoplasma gallisepticum strain K6067 deposited with ATCC as Patent No. PTA-127168 as described herein, or a progeny or derivative thereof, a substantially biologically pure culture of the K6067 Mycoplasma gallisepticum strain deposited with ATCC as Patent No. PTA-127168 as described herein, or a composition as described herein. In some aspects, the vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry. In some aspects, the vaccine reduces the susceptibility of Galliformes birds to diseases induced by Mycoplasma gallisepticum.

[0007] The present invention includes a live vaccine for birds of the order Galliformes, the vaccine comprising: an amount of the K6067 Mycoplasma gallisepticum strain deposited with the ATCC under patent number PTA-127168 as described herein, or a progeny or derivative thereof, the amount being sufficient to protect the bird from disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.

[0008] The present invention includes an isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain is the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent number PTA-127282, or a progeny or derivative thereof. In some aspects, the present invention includes a composition comprising the isolated Mycoplasma gallisepticum strain. In some aspects, the composition may include water. In some aspects, the composition may include a pharmaceutically acceptable carrier. In some aspects, the composition may include an adjuvant. In some aspects, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or intraovo administration. In some aspects, the composition may be formulated for spraying or atomization.

[0009] The present invention includes a substantially biologically pure culture of the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent number PTA-127282. In some aspects, the present invention includes a composition comprising an isolated Mycoplasma gallisepticum strain. In some aspects, the composition may include water. In some aspects, the composition may include a pharmaceutically acceptable carrier. In some aspects, the composition may include an adjuvant. In some aspects, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous or intraovo administration. In some aspects, the composition may be formulated for spraying or atomization.

[0010] The present invention includes a vaccine comprising an isolated Mycoplasma gallisepticum strain K4110 deposited with ATCC as Patent No. PTA-127282 as described herein, or a progeny or derivative thereof, a substantially biologically pure culture of the K4110 Mycoplasma gallisepticum strain deposited with ATCC as Patent No. PTA-127282 as described herein, or a composition as described herein. In some aspects, the vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry. In some aspects, the vaccine reduces the susceptibility of Galliformes birds to diseases induced by Mycoplasma gallisepticum.

[0011] The present invention includes a live vaccine for birds of the order Galliformes, the vaccine comprising: an amount of the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under patent number PTA-127282 as described herein, or a progeny or derivative thereof, the amount being sufficient to protect the bird from disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.

[0012] In some aspects, an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein can be lyophilized, freeze-dried, frozen, or an effervescent tablet.

[0013] The present invention includes a kit comprising an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein and printed instructions, wherein the contents of the kit are contained within packaging material.

[0014] The present invention includes a method of generating an immune response against Mycoplasma gallisepticum in a bird, the method comprising administering an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein. In some aspects, the administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, the administration is by eye drops, by aerosol, or by drinking water. In some aspects, the bird is of the order Galliformes. In some aspects, the bird is a chicken or a turkey.

[0015] The present invention includes a method for reducing the susceptibility of a bird to a disease induced by Mycoplasma gallisepticum, the method comprising administering to the bird an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein. In some aspects, the administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, the administration is by eye drops, by aerosol, or by drinking water. In some aspects, the bird is of the order Galliformes. In some aspects, the bird is a chicken or a turkey.

[0016] The present invention includes a method for protecting a bird from infection with Mycoplasma gallisepticum, the method comprising administering to the bird an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein. In some aspects, the administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, the administration is by eye drops, by aerosol, or by drinking water. In some aspects, the bird is of the order Galliformes. In some aspects, the bird is a chicken or a turkey.

[0017] The present invention includes a method of reducing one or more of the clinical signs induced by Mycoplasma gallisepticum infection in a bird, the method comprising administering to the bird an effective amount of an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein. In some aspects, the administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, the administration is by eye drops, by aerosol, or by drinking water. In some aspects, the bird is of the order Galliformes. In some aspects, the bird is a chicken or a turkey.

[0018] As used herein, "isolated" refers to material that has been removed from its original environment (eg, the natural environment if it occurs naturally) and has thus been altered "by the hands of man" from its natural state.

[0019] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0020] The words "preferred" and "preferably" refer to embodiments of the present invention that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the description of one or more preferred embodiments does not mean that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present invention.

[0021] Where the terms "comprises" and variations thereof appear in the description and claims, these terms do not have a limiting meaning.

[0022] Unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably and mean one or more than one.

[0023] 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.).

[0024] For any method disclosed herein comprising discrete steps, the steps may be performed in any feasible order. Also, any combination of two or more steps may be performed simultaneously, if appropriate.

[0025] Unless otherwise indicated, all numbers used in the specification and claims indicating the number of components, molecular weight, etc., should be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters listed in the specification and claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and not attempting to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0026] 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. All numerical values, however, inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0027] In many places throughout the application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the enumerated lists serve only as representative groups and should not be construed as exclusive lists. It should be understood that specific examples, materials, amounts, and procedures should be broadly interpreted according to the scope and spirit of the invention as set forth herein.

[0028] All headings throughout the text are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless otherwise indicated. DETAILED DESCRIPTION

[0029] The bacterium Mycoplasma gallisepticum is a member of the genus Mycoplasma. 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 throughout the world, and infection may be referred to as infectious synovitis, avian mycoplasmosis, infectious sinusitis, or mycoplasmal arthritis. Mycoplasma gallisepticum is transmitted vertically from infected breeders to offspring in some eggs (transovarian), and horizontally on vectors (shoes, equipment, etc.) via infectious aerosols and through contamination of feed, water, and the environment, and through human activities. It is of economic importance because infection may lead to a decrease in egg production (El-Gazzar, "Mycoplasma gallisepticum Infection in Poultry," Merk Veterinary Manual, 2020).

[0030] The present invention provides Mycoplasma gallisepticum (MG) strain K6067 and its progeny and derivatives, which are immunogenic and stable when administered as live preparations. The present invention also provides Mycoplasma gallisepticum (MG) strain K4110 and its progeny and derivatives, which are immunogenic and stable when administered as live preparations. The Mycoplasma gallisepticum preparations of the present invention are safe and effective for inhibiting Mycoplasma gallisepticum infection and will help reduce the incidence and severity of Mycoplasma gallisepticum-infected diseases in birds.

[0031] Mycoplasma gallisepticum strain K6707 was deposited with the American Type Culture Collection as PTA-127168 on November 24, 2021 10801 University Boulevard, Manassas, VA 20110-2209, USA. This strain was deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. It was deposited as PTA-127168 The Mycoplasma gallisepticum strain K6067 is also referred to herein as Mycoplasma gallisepticum strain 6067, Mycoplasma 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 PTA-127168.

[0032] Mycoplasma gallisepticum strain K4110 was deposited at the American Type Culture Collection as PTA-127282 on July 20, 2022 10801 University Boulevard, Manassas, VA 20110-2209, USA. This strain was deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. It was deposited as PTA-127282 The Mycoplasma gallisepticum strain K4110 is also referred to herein as Mycoplasma gallisepticum strain 4110, Mycoplasma 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 PTA-127282.

[0033] The present invention includes an isolated Mycoplasma gallisepticum (MG) strain K6067 having ATCC Patent Deposit No. PTA-127168. As used herein, "isolated" refers to a material that has been removed from its original environment (e.g., the natural environment if it occurs naturally) and has thus been altered from its natural state "by the hand of man." Also included is a biologically pure culture of Mycoplasma gallisepticum (MG) strain K6067 having ATCC Patent Deposit No. PTA-127168.

[0034] The present invention includes an isolated Mycoplasma gallisepticum (MG) strain K4110 having ATCC patent deposit number PTA-127282. It also includes a biologically pure culture of the Mycoplasma gallisepticum (MG) strain K4110 having ATCC patent deposit number PTA-127282.

[0035] The present invention also includes isolated progeny and isolated derivatives of Mycoplasma gallisepticum strain K6067 deposited with ATCC patent deposit number PTA-127168, which have 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 data in the examples included herein. More specifically, the progeny or derivatives of the K6067 strain deposited with ATCC as PTA-127168 may retain the particularly advantageous protective properties of the present invention. The progeny or derivatives of the Mycoplasma gallisepticum strain K6067 deposited with ATCC as PTA-127168 may be obtained by any of a variety of methods known in the art for breeding Mycoplasma gallisepticum, including but not limited to, for example, in vitro culture or back-transfer in birds. Derivatives of Mycoplasma gallisepticum strain K6067 ATCC PTA-127168 may include genetically modified versions of the deposited MG K6067 strain. Such manipulations include, but are not limited to, mutagenizing the MG strain, or introducing a gene or gene cassette encoding a replacement protein or a non-functional protein, or a non-coding nucleotide sequence into the MG organism.

[0036] The present invention also includes isolated progeny and isolated derivatives of Mycoplasma gallisepticum strain K4110 deposited with ATCC patent deposit number PTA-127282, which have 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 data in the examples included herein. More specifically, the progeny or derivatives of the K4110 strain deposited with ATCC as PTA-127282 may retain the particularly advantageous protective properties of the present invention. The progeny or derivatives of the Mycoplasma gallisepticum strain K4110 deposited with ATCC as PTA-127282 may be obtained by any of a variety of methods known in the art for breeding Mycoplasma gallisepticum, including but not limited to, for example, in vitro culture or back-transfer in birds. Derivatives of Mycoplasma gallisepticum strain K411 ATCC PTA-127282 may include genetically modified versions of the deposited MG K4110 strain. Such manipulations include, but are not limited to, mutagenizing the MG strain, or introducing a gene or gene cassette encoding a replacement protein or a non-functional protein, or a non-coding nucleotide sequence into the MG organism.

[0037] As described herein, Mycoplasma gallisepticum (MG) strain K6067 isolate, MG strain K4110, and progeny and derivatives thereof can be propagated by conventional methods, including but not limited to any of the methods described in the Examples section included herein. For example, the Mycoplasma gallisepticum strains of the present invention can be cultured with modified Freund's 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, ed. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. Pages 63-70; 2016).

[0038] MG strain K6067, MG strain K4110, and progeny and derivatives thereof can be identified and distinguished from other mycoplasmas and other strains of M. gallisepticum using any of a number of known techniques, including, for example, direct immunofluorescence with species-specific antibodies, serum plate agglutination (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 spacer region (IGSR) (Papazisi et al., 2003, Microbiology; 149: 2307-16) and mgc2 cell adhesin (Hnatow et al., 1998, Infect Immun; 66: 3436-42).

[0039] Mycoplasma gallisepticum (MG) strain K6067, Mycoplasma gallisepticum (MG) strain K4110 and their progeny and derivatives as described herein can be administered to poultry, including chickens and turkeys, as attenuated MG live vaccines. Although the poultry industry has adopted different measures (such as voluntary monitoring programs, vaccination and treatment with different antibiotics) to control Mycoplasma gallisepticum (MG) infection and reduce economic losses in the poultry industry, MG is still considered a persistent challenge facing the commercial poultry industry. Voluntary monitoring programs may be effective, but the value of long-lived flocks (breeders and laying hens) makes it difficult to justify culling and quarantine, especially in areas where MG is widely prevalent. Furthermore, increasing legal restrictions on the use of antibiotics in poultry, especially preventive feed use, in Europe and the United States (Veterinary Feed Directive Journal. Vol. 80, No. 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) have reduced the applicability and effectiveness of this option. In these circumstances, vaccination has become an increasingly attractive option for MG control.

[0040] Currently available immunizing agents include bacterins, live attenuated vaccines, and recombinant vaccines. Bacterins have some advantages because they are not contagious or transmissible and cannot restore virulence (Sasipreeyajan et al., 1987, Avian Dis; 31: 776-81); Bacterins have been shown to reduce egg production losses, ovarian degeneration, and egg transmission of MG (Glisson and Kleven, 1985, Avian Dis; 29: 408-15). However, bacterins provide very limited protection against infection, tracheal and air sac lesions (Talkington and Kleven, 1985, Avian Dis; 29: 998-1003). The human cost of intramuscular injections and the individual handling of birds may cause stress, and there is also a risk of local vaccine reactions. For MG recombinant vaccines, there is a commercially available MG vaccine vectored by fowlpox, but it is reported that the efficacy of this vaccine against respiratory lesions caused by pathogenic MG strains is significantly lower than that of bacterin and live attenuated vaccines (Ferguson-Noel et al., 2012, Avian Dis; 56: 272-275). Recently, a TM-1 protein-based adenovirus-based MG vaccine was developed, but further studies are needed to investigate its efficacy (Dongchao Zhang et al., 2018, Avian Pathology; 42(2): 213–222). There are multiple genes and virulence factors (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) that play different roles in MG pathogenicity and immunity, so selecting a specific gene to produce a sufficiently effective and protective MG vaccine may be challenging.

[0041] There are three types of attenuated live vaccines for MG currently on the market: 6 / 85, F strain, and ts-11. These vaccines have their own advantages and disadvantages in terms of safety and efficacy; the ideal MG vaccine should be immunogenic, non-toxic, affordable, and able to prevent colonization with 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, it is reported that the vaccine is not very effective and colonization is poor (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 evaluating the immune status of vaccinated flocks using serological assays (Throne Steinlage et al., 2003, Avian Dis; 47: 499-505).

[0042] 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 the least attenuated live MG vaccine currently available. The use of the F strain is not permitted in some regions (such as Minnesota, USA, Israel, and Japan) due to reports that the F strain is pathogenic to turkeys (Lin and Kleven, 1982, Avian Dis; 26: 360-4), can be transmitted to unvaccinated birds (Kleven, 1981, Avian Dis; 25: 1005-18), and has the risk of increased virulence when it circulates and returns to birds (Gharaibeh et al., 2011, Avian Dis; 55: 212-6). However, the F strain is very effective in replacing pathogenic MG strains (Kleven et al., 1990, Avian Dis; 34: 984-90) and reducing air sac and tracheal lesions (Kleven, 1986, Avian Diseases; 30 No. 1: 169-171) and reducing the transmission of virulent MG through eggs (Glisson and Kleven, 1984, Avian Dis; 28: 406-15). Another live MG vaccine that survives in the trachea of ​​vaccinated birds for life (Kleven, 1998, Poult Sci; 77: 1146-9) is ts-11 (Whithear et al., 1990, Aust Vet J; 67: 168-74). This vaccine is effective in preventing respiratory lesions (Abd-el-Motelib and Kleven, 1993, Avian Dis; 37:981-7), egg production loss (Barbour et al., 2000, Poult Sci; 79:1730-5), and ovarian degeneration (Whithear et al., 1990, IOM Lett; 1:361-362). Although ts-11 is safe for use in turkeys, it does not colonize well in the trachea to elicit protective immunity (Whithear et al., 1990, Aust Vet J; 67:159-65; and Wijesurendra et al., 2017, Avian Pathol; 1-10; 2017). Virulence recovery of ts-11 has also been reported (Armour et al., 2015, Avian Pathol; 44:296-304). Recently, a GapA+ts-11 vaccine, ts-304, was developed.Although the durability of the vaccine remains similar to ts-11 (Condello et al., 2020, Veterinary Microbiology; 251: 108883), it has been reported to be safe and effective for use in turkeys (Kanci et al., 2018, Vaccine; 36(18): 2487-2493).

[0043] The present invention includes compositions and vaccines of Mycoplasma gallisepticum isolates and progeny and derivatives thereof as described herein. In preferred embodiments, the Mycoplasma gallisepticum isolates are alive. In some embodiments, the Mycoplasma gallisepticum isolates may be inactivated or killed. The Mycoplasma gallisepticum strains and compositions and vaccines thereof of the present invention may be stored until use in any of a variety of forms. For example, such materials may be lyophilized or freeze-dried and may be rehydrated for use. In some embodiments, the Mycoplasma gallisepticum strains or compositions or vaccines thereof may be frozen.

[0044] In some embodiments, the Mycoplasma gallisepticum isolate or a composition or vaccine thereof can be formulated as an effervescent tablet. Such an effervescent tablet can be packaged, for example, in a lightweight aluminum blister. The tablet can be dissolved in water and administered, for example, orally, nasally, or by an aerosol spray, whereby the droplets enter through the mucous membranes of the bird.

[0045] The composition and vaccine of the present invention may include, for example, water or culture medium. Such composition and vaccine may include one or more suitable pharmaceutically 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). When dry vaccine formulations are prepared by lyophilization, stabilizers are particularly advantageous. 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).

[0046] The composition or vaccine of the present invention may also include one or more compounds having 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, for example, mineral oil (such as Bayol F TM or Marcol 52 TM ), complete Freund's adjuvant, incomplete Freund's adjuvant, or vegetable oils (such as vitamin E acetate and saponins).

[0047] The composition or vaccine of the present invention may further include one or more immunogens derived from other pathogens that infect poultry. Such immunogens may be derived from, for example, Mycoplasma synoviae (MS), Marek's disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), egg 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).

[0048] The compositions and vaccines of the invention may be substantially pure. As used herein, "substantially pure" shall mean a material that is substantially free of macromolecules or other biological entities normally found in nature.

[0049] The compositions and vaccines of the present invention can be applied to any bird of a variety of bird species susceptible to infection with Mycoplasma gallisepticum, including but not limited to poultry, Galliformes birds and exotic bird species. Galliformes birds include but are not limited to chickens, turkeys, grouse, quail and pheasants. As used herein, poultry includes domesticated birds raised for the purpose of collecting eggs or slaughtering for meat and / or feathers. These are most typically members of the Galliformes (birds), especially Galliformes (including, for example, chickens, quail, turkeys and grouse) and Anatidae (Anseriformes), commonly referred to as "waterfowl" (including, for example, ducks, geese and swans). Poultry can also include other birds that are slaughtered for meat, such as pigeons or doves or birds that are considered to be prey, such as pheasants. Chickens include but are not limited to hens, roosters, broilers, roast chickens, laying hens, breeders, offspring of mating hens and laying hens. As used herein, the term "susceptible" means the likelihood or reality of an adverse response to the microorganism in question, such as, for example, reduced viability or failure to grow, and / or one or more pathological conditions indicative of Mycoplasma gallisepticum infection, compared to a non-susceptible individual or population.

[0050] Compositions and vaccines of the present invention can be formulated for delivery by any of the multiple approaches known in the veterinary field, including but not limited to, for example, mucosal, intranasal, intraocular or oral administration. Compositions and vaccines of the present invention can be formulated for delivery to the respiratory mucosa, and can be used in a manner that makes it contact the respiratory mucosa of birds immediately or finally. Compositions or vaccines of the present invention can be used by any suitable known method for inoculating poultry, including but not limited to nasal, eye, by eye drops, by injection, in drinking water, in feed, in eggs, through mother, by respiratory tract inhalation, etc. When administered by injection, immunogenic compositions or vaccines can be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular or intraperitoneal injection.

[0051] Composition or vaccine can be formulated for use by a large number of application techniques, these techniques are such as by putting the vaccine into drinking water or by spraying or atomization. Composition can be used by the mode of spraying solution to individual or flock of birds, and this type of aerosol delivery may relate to the use of the composition mixed in the small liquid particles. This type of spray type particle can have a droplet size ranging from about 10 to about 100 microns, more preferably, from about <1 to about 50 microns. In order to generate small particles, conventional spraying devices and aerosol generators can be used, such as commercially available spray generators for backpack spraying, hatchery spraying and atomization spraying. Conventional devices can also be used to use by drinking water.

[0052] Compositions of the present invention or vaccine can be applied to poultry before or after hatching.In ovo vaccination can be carried out in, for example, 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 thereof.For delivery in ovo, egg-laying livestock or breeding livestock can be, for example, vaccinated at about 6-12 weeks of age, and booster immunization at about 16-20 weeks of age.Such egg-laying livestock or breeding livestock can 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.In addition, in some embodiments, such egg-laying livestock or breeding livestock can be vaccinated in about the first two weeks of age.Such egg-laying livestock or breeding livestock can be booster immunization 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. The offspring of such egg-laying or breeding livestock may show antibody titers against M. gallisepticum, which may prevent or reduce the symptoms of M. gallisepticum infection in the offspring.

[0053] Poultry can receive compositions or vaccines as described herein at various ages. For delivery after hatching, the material can be delivered at any suitable age, including but not limited to about one to three days old, about one week after hatching, about two weeks after hatching, about three weeks after hatching, about four weeks after hatching, about five weeks after hatching, about six weeks after hatching, or any range thereof. Chickens can only be vaccinated once. Alternatively, if two doses of vaccine are used, the first dose of vaccine is given, for example, when the chicken is 3 days to one week old, followed by another 1-10 weeks later.

[0054] Multiple doses of the composition may be administered throughout the life of the chicken. As maternal immunity is the primary source of protection for broiler offspring, breeder chickens are usually vaccinated, but broiler chickens may also be vaccinated if desired.

[0055] The compositions and vaccines of the invention can be adjusted to include 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 methods, including, for example, the protocol described by Rodwell and Whitcomb (in "Methods in Mycoplasmology," ed. Razin and Tully, 1993). For example, a composition or vaccine can have a color change unit of about 50, about 100, about 1 x 10 2 ccu / ml, about 2.5x10 2 ccu / ml, about 5x10 2 ccu / ml, about 1x10 3 ccu / ml, about 2.5x10 3 ccu / ml, about 5x10 3 ccu / ml, about 1x10 4 ccu / ml, about 2.5x10 4 ccu / ml, about 5x10 4 ccu / ml, about 1x10 5 ccu / ml, about 2.5x10 5 ccu / ml, about 5x10 5 ccu / ml, about 1x10 6 ccu / ml, about 2.5x10 6 ccu / ml, about 5x10 6 ccu / ml, about 1x10 7 ccu / ml, about 2.5x10 7 ccu / ml, about 5x10 7 ccu / ml, 1x10 8 ccu / ml, about 2.5x10 8 ccu / ml, about 5x10 8 ccu / ml, about 1x10 9 ccu / ml, about 2.5x10 9 ccu / ml or about 5x10 9 ccu / ml, and any range thereof (such as, for example, about 1×10 5 ccu / ml is about 1x10 6 ccu / ml). In some applications, an effective amount may be administered to a single bird as one drop per eye per bird. One drop may be about 0.05 ml to 0.1 ml.

[0056] The Mycoplasma gallisepticum strains of the present invention may be administered to birds to reduce susceptibility to Mycoplasma gallisepticum infection. Upon such administration, the material does not result in significant clinical signs or lesions indicative of Mycoplasma gallisepticum. Thus, one object of the present invention is to provide immune materials that, when administered, do not result in significant clinical signs or lesions indicative of MS disease. Another object is to provide immune materials of reduced toxicity.

[0057] 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 as described herein. In some aspects, the immunity comprises humoral immunity and / or cellular immunity. For humoral responses, anti-MS antibodies can be measured, for example, by serum plate agglutination (SPA) test (using, for example, commercial antigens (Charles River Laboratories International, Inc., Wilmington, MA); hemagglutination inhibition (HI) test; and enzyme-linked immunosorbent assay (ELISA) test (using, for example, commercial kits (IDEXX, Westbrook, Maine). Ferguson-Noel et al. describe the SPA and HI test procedures in more detail (Ferguson-Noel, N. and SHKleven Mycoplasma species. In: A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens, 6th edition SM Williams, L. Dufour-Zavala, MW Jackwood, MDLee, B. Lupiani, WM Reed, E. Spackman and PR Woolcock, ed. American Association of Avian Pathologists. Pages 63-70. 2016). In some aspects, immunization includes mucosal immunization.

[0058] The present invention includes a method of reducing, inhibiting or preventing MG infection in poultry, the method comprising administering an isolated Mycoplasma gallisepticum strain, composition or vaccine as described herein. Administration of an isolated Mycoplasma gallisepticum strain, composition or vaccine as described herein may result in reduction, inhibition or prevention of one or more of the disease manifestations of further infection with MG, including one or more of the disease manifestations of infectious MG. Such clinical signs and symptoms may include, for example, respiratory distress, tracheal rales, dyspnea, coughing, sneezing, runny nose, eyelid swelling, eye discharge, impaired vision, conjunctivitis, foaming around the eyes, swelling of the infraorbital sinus blebs, sinusitis, catarrhal sinusitis, tracheitis, air sacculitis, pneumonia, leg problems, stunted growth, depression, weight loss, loss of appetite, slow growth, reduced hatchability, reduced chick vitality, feather abnormalities, poor productivity, reduced feed efficiency, reduced weight gain, reduced egg production, discard during processing and / or increased mortality.

[0059] The present invention also provides a kit comprising the Mycoplasma gallisepticum strain K6067 and / or its progeny or derivatives as described herein. The kit may include one or more containers containing the Mycoplasma gallisepticum of the present invention. The Mycoplasma gallisepticum strain K6067 may be lyophilized.

[0060] The present invention also provides a kit comprising the Mycoplasma gallisepticum strain K4110 and / or its progeny or derivatives as described herein. The kit may include one or more containers containing the Mycoplasma gallisepticum of the present invention. The Mycoplasma gallisepticum strain K4110 may be lyophilized.

[0061] The kit may include additional, separate containers containing other strains of Mycoplasma gallisepticum or other poultry pathogens. In addition, the kit may include other reagents, such as buffers and solutions required for practicing the present invention. Optionally, associated with such containers may be notices or printed instructions. The kit of the present invention may include "packaging materials". 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, contamination-free environment. The packaging material can be a solid matrix or a material such as glass, plastic, paper, foil, etc. Thus, for example, the package can be a glass or plastic vial for containing ccu amounts of Mycoplasma gallisepticum strain K6067 or Mycoplasma gallisepticum strain K4110.

[0062] Exemplary embodiments of the present invention include, but are not limited to, the following.

[0063] 1. An isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain is the K6067 Mycoplasma gallisepticum strain deposited in ATCC under patent number PTA-127168 or a progeny or derivative thereof.

[0064] 2. A substantially biologically pure culture of the K6067 strain of Mycoplasma gallisepticum deposited with the ATCC under Patent No. PTA-127168.

[0065] 3. An isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain is the K4110 Mycoplasma gallisepticum strain deposited in ATCC under patent number PTA-127282 or a progeny or derivative thereof.

[0066] 4. A substantially biologically pure culture of the K4110 strain of Mycoplasma gallisepticum deposited with the ATCC under Patent No. PTA-127282.

[0067] 5. A composition comprising the isolated Mycoplasma gallisepticum of any one of embodiments 1 to 4.

[0068] 6. The composition of embodiment 5 comprising water.

[0069] 7. The composition of embodiment 5, comprising a pharmaceutically acceptable carrier.

[0070] 8. The composition of any one of embodiments 5 to 7, comprising an adjuvant.

[0071] 9. The composition of any one of embodiments 5 to 8, wherein the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration.

[0072] 10. The composition of any one of embodiments 5 to 9, wherein the composition is formulated for spraying or atomization.

[0073] 11. A vaccine comprising the isolated Mycoplasma gallisepticum of any one of embodiments 1 to 4 or the composition of any one of embodiments 5 to 10.

[0074] 12. The vaccine of embodiment 11, wherein said vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry.

[0075] 13. The vaccine of embodiment 11 or 12, wherein the vaccine reduces susceptibility of Galliformes birds to disease induced by Mycoplasma gallisepticum.

[0076] 14. A live vaccine for birds of the order Galliformes, the vaccine comprising: an amount of the K6067 strain of Mycoplasma gallisepticum deposited with ATCC under patent number PTA-127168 or a progeny or derivative thereof, the amount being sufficient to protect the birds from diseases induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.

[0077] 15. A live vaccine for birds of the order Galliformes, the vaccine comprising: an amount of the K4110 Mycoplasma gallisepticum strain deposited with ATCC under patent number PTA-127282 or a progeny or derivative thereof, the amount being sufficient to protect the birds from diseases induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.

[0078] 16. The isolated Mycoplasma gallisepticum, composition or vaccine of any one of embodiments 1 to 15, wherein the isolated Mycoplasma gallisepticum, composition or vaccine is lyophilized, freeze-dried, frozen or an effervescent tablet.

[0079] 17. A kit comprising the isolated Mycoplasma gallisepticum, composition or vaccine of any one of embodiments 1 to 16 and printed instructions, wherein the contents of the kit are contained within packaging material.

[0080] 18. An effervescent tablet comprising the isolated Mycoplasma gallisepticum, composition or vaccine of any one of embodiments 1 to 16.

[0081] 19. A method of generating an immune response against Mycoplasma gallisepticum in a bird, the method comprising administering to the bird the isolated Mycoplasma gallisepticum, composition or vaccine of any one of embodiments 1 to 16.

[0082] 20. A method for reducing the susceptibility of a bird to a disease induced by Mycoplasma gallisepticum, the method comprising administering to the bird the isolated Mycoplasma gallisepticum, composition or vaccine of any one of embodiments 1 to 16.

[0083] 21. A method for protecting a bird from infection with Mycoplasma gallisepticum, the method comprising administering to the bird the isolated Mycoplasma gallisepticum, composition or vaccine of any one of embodiments 1 to 16.

[0084] 22. A method of reducing one or more clinical signs induced by Mycoplasma gallisepticum infection in a bird, the method comprising administering to the bird an effective amount of the isolated Mycoplasma gallisepticum, composition or vaccine of any one of embodiments 1 to 16.

[0085] 23. The method of any one of embodiments 19 to 22, wherein administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo.

[0086] 24. The method of any one of embodiments 19 to 22, wherein the administration is by eye drops, by aerosol, or by drinking water.

[0087] 25. The method of any one of embodiments 19-24, wherein the bird comprises a Galliformes bird.

[0088] 26. The method of any one of embodiments 19-25, wherein the bird comprises a chicken or a turkey.

[0089] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0090] Examples

[0091] Example 1

[0092] Evaluation of Mycoplasma gallisepticum strains as live vaccines in chickens and turkeys

[0093] Ten Mycoplasma gallisepticum (MG) isolates were selected based on case histories and analysis of targeted sequencing results from the Poultry Diagnostic and Research Center (PDRC) isolate library. The isolates were evaluated for safety in turkeys and two isolates (K4110A and K6067) were selected based on successful colonization of the trachea while not showing any clinical signs or gross lesions at necropsy (Trials 1 and 2). In Trial 3, the safety and efficacy of these two isolates were evaluated in chickens; SPF chickens were vaccinated via eye drops at 4 weeks of age and challenged with the R strain via aerosol 4 weeks after vaccination. Neither group vaccinated with the vaccine candidate showed significant differences in lesions compared to the negative control, indicating that the candidates are safe for chickens. Both vaccinated groups also had significantly reduced lesions after challenge, indicating the presence of an effective vaccine. Further studies were conducted to determine the most protective route of administration and dose required for each candidate. The 50% minimum protective dose of 10 for both K6067 and K4110A was achieved by the eye drop route of administration. 4 –10 5 ccu / mL (Trials 4 and 5). No vertical transmission was detected in Trial 6. In conclusion, based on the results of the study, both K6067 and K4110A are potential live vaccine candidates, as these preliminary studies show that they are safe and effective in turkeys and chickens.

[0094] Materials and methods

[0095] MG strains and isolates

[0096] Ten vaccine candidates (Table 1) were selected from the MG culture repository at the Poultry Diagnostic and Research Center (University of Georgia, Athens, Georgia) based on criteria including signs of natural attenuation (no history of illness, clinical signs, or death), and rapid and consistent in vitro growth rates. Targeted DNA sequencing results of the 16S-23S rRNA intergenic spacer region (IGSR) (Papazisi et al., 2003, Microbiology; 149:2307-16) and mgc2 cell adhesin (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). Sequence analysis and comparison were performed using NCBI Blast (Johnson et al., 2008, Nucleic Acids Research; 36(Suppl 2):W5-W9) and MegAlign software (Lasergene software package, DNAStar, Inc., Madison, WI). Whole genome sequencing was performed on low (~10p) and high (~50p) passages of candidates to assess genetic stability after in vitro passage, and whole genome sequences were compared with MG genome sequences in the publically available or PDRC Mycoplasma Laboratory whole genome library. For isolates selected for whole genome analysis, DNA was extracted after growth in modified Freund's broth; cells were centrifuged at 13,000×g for 3 minutes, the supernatant discarded, and the cell pellet reconstituted in 200 μl phosphate-buffered saline (pH=7). QIAGEN ELISA was used according to the manufacturer's recommendations. Blood and tissue kits (QIAGEN, Valencia, CA) or Promega Genomic DNA was extracted using a genomic DNA purification kit (Promega, Madison, Wisconsin). Illumina-based sequencing was performed at Novogene (Sacramento, California) (Illumina, San Diego, California). A comprehensive genomic analysis (including de novo assembly and annotation) was performed on the isolates in PATRIC (Wattam et al., 2017, Nucleic Acids Res; 45: D535-D542). The entire genome of the isolates was compared with established vaccine and laboratory strains using the Similar Genome Finder and Phylogenetic Tree Generator tools in PATRIC.

[0097] After an initial screening of the ten candidates in turkeys, K4110A and K6067 were further evaluated for safety and efficacy in chickens (Trial 3), the most protective and effective doses and routes of administration for each (Trials 4 and 5), and the potential for vertical transmission of each (Trial 6). Vaccinated or unvaccinated birds were challenged using the R-strain, a well-described pathogenic MG strain (Rodriguez and Kleven, 1980, Avian Dis; 24:800-7) as a positive control. Aerosol and eye drop routes were the two routes of administration used to vaccinate birds; for the former, a commercial paint sprayer ( For the latter, 100 μl of actively growing culture was applied to the eye.

[0098] Serology

[0099] Sera were analyzed to confirm stimulation of the humoral immune system by serum plate agglutination (SPA) using commercial antigens (Charles River Laboratories, North Franklin, CT), hemagglutination (HI) test using antigens prepared from strain A5969 and chicken red blood cells (RBC), and enzyme-linked immunosorbent assay (ELISA) to assess the presence of anti-MG antibodies using commercial 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, ed. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens, Fifth Edition. American Association of Avian Pathologists. Pages 59-64; 2008), and scores ≥ 1 were considered positive for SPA, while for HI, a titer of 1:20 was considered equivocal, and a titer of ≥ 1:40 was considered positive. For ELISA, a sample / positive (S / P) ratio ≥ 0.5 was considered positive.

[0100] Isolation and identification of mycoplasma

[0101] Swabs were taken from the trachea, post-narial clefts, and air sacs and inoculated in modified Freund's 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, eds. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. pp. 63-70; 2016).

[0102] For trials 1 and 2, samples were also inoculated in 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, eds. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. Pages 63-70; 2016) for pre-inoculation when screening birds for the presence of mycoplasma. All cultures were incubated at 37C and the cultures were considered negative for mycoplasma if no growth was observed in the broth tubes or agar plates after four weeks. Mycoplasma isolates were identified using direct immunofluorescence (Talkington and Kleven, 1983, Avian Dis; 27:422-9).

[0103] qPCR and colonization rate calculation

[0104] Real-time quantitative PCR (qPCR) procedures for specific detection of MG and MS, 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 genomic copy number of MG present in tracheal samples collected at necropsy. In addition, strain-specific qPCR was performed for the R strain, K4110A, and K6067 to evaluate the efficacy of these two vaccine candidates in controlling colonization with the pathogenic challenge strain. Tracheal swabs were collected during the trial, and upper tracheal sections from individual birds were collected in 10 ml of sterile PBS at necropsy for subsequent DNA extraction. DNA was extracted using the R strain according to the manufacturer's recommendations. Blood and Tissue DNA HDQ 96 Kit (Omega Bio-tek, Inc., Norcross, GA) was used in MAGMAX TMGenomic DNA was extracted from 200 μl of throat wash and tracheal swab on an Express-96 magnetic particle processor (Thermo Fisher Scientific). Real-time PCR was performed using an Applied Biosystems 7500 fast real-time PCR system (Thermo Fisher Scientific), and cycle threshold (Ct) values ​​≤ 39 were considered positive. In order to quantify the assay, a plasmid containing a genomic target was constructed as a standard DNA control. The procedure for constructing a DNA control and a quantitative standard curve have been described in detail elsewhere (Raviv et al., 2008, Vet Microbiol; 129: 179-87).

[0105] Evaluation of Lesions

[0106] Total air sac lesions were scored from 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 under a microscope by measuring the width of the tracheal mucosa. The upper third of the trachea (about 1 inch from the larynx) was fixed in 10% neutral formalin. The thickness of the tracheal mucosa was measured at four equidistant points on a histological slide of the tracheal cross section (Whithear, 1996, Rev. Sci. Tech. Off. Int. Epiz. 15: 1527-1553). Any other autopsy findings were also recorded.

[0107] Animal Care and Use

[0108] 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 feed and water ad libitum and were euthanized with carbon dioxide.

[0109] Statistical analysis

[0110] One-way ANOVA was used to analyze air sac lesions and Mycoplasma gallisepticum isolates from chickens. Mean tracheal mucosal thickness, log10 MG DNA genome copy number, SPA score, S / P ratio, HI titer, fertility rate, and weekly egg production in hens were analyzed using Tukey-Kramer highly significant difference test (Minitab 17 Statistical Software. [Computer Software]. State College, Pennsylvania: Minitab Inc. (www.minitab.com). 2010). P values ​​≤ 0.05 were considered significant.

[0111] Experimental design

[0112] Trials 1 and 2 (Turkey Safety 1 and 2)

[0113] Trial 1 and Trial 2 had the same experimental design; each trial included 5 candidates, for a total of 10 vaccine candidates evaluated in both trials. For each trial, 42 turkey poults were obtained from a source known to be free of MG and MS at 1 day of age and housed in 2 isolators. At 2 weeks of age (WOA), turkeys were leg-tied with random numbers (www.randomlists.com) and randomly assigned to 7 treatment groups (7 isolators) with 6 poults per group. Ten turkeys distributed in each treatment group were screened for the presence of mycoplasma by culture, quantitative polymerase chain reaction (qPCR) of mycoplasma antibodies in tracheal samples and serum at this time. At 3WOA, turkeys were vaccinated by aerosol with the corresponding treatment or not vaccinated (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 washings.

[0114] Study 3 (Chicken Safety and Efficacy)

[0115] SPF chicks were obtained from a source known to be free of MG and MS at 125 days of age and housed in a pen (1.5 x 3 m 2). At 3WOA, the chicks were randomly wing-banded and assigned to four treatment groups. Fifteen chickens (distributed in each treatment group) were screened for the presence of mycoplasma by culture of tracheal samples, MG and Mycoplasma synoviae qPCR, and serology. At 4WOA, the chickens were vaccinated with the corresponding treatment (B, C, D, K6067, K4110A or R strain) by aerosol, or were not vaccinated as a negative control group, as shown in Table 4 below. Five chickens in groups B and C were not vaccinated and co-housed with vaccinated birds at 5WOA. Nineteen days after co-housed (8WOA), the 10 contact birds were autopsied and evaluated. Four weeks after vaccination, 10 chickens in each of groups B, C and D were directly challenged with the R strain by aerosol. The remaining vaccinated birds were co-housed with these birds, and 10 unvaccinated birds were directly contacted with the unvaccinated aerosol challenge group (contact attack). 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 washings.

[0116] Trial 4 (Dose Response 1)

[0117] SPF chicks aged 84 days were obtained from a source known to be free of MG and MS. At 2WOA, chicks were wing-banded and randomly assigned to 12 treatment groups in twenty-four isolators (Table 5). Ten birds distributed in each treatment group were screened for the presence of mycoplasma by culture, MG and Mycoplasma synoviae qPCR, and serology of tracheal samples. At 3WOA, chickens in groups C to G were vaccinated with K6067 vaccine by eye drops, and chickens in groups H to L were vaccinated with K6067 vaccine by aerosol. As shown in Table 5, each group received different doses of vaccine candidates. Groups A and B were not vaccinated and served as negative controls and challenge-only groups, respectively. One week after vaccination, nasal cleft and tracheal swabs were collected from all groups for culture and MG qPCR. Two weeks after vaccination, all eighty-four chickens were bled and swabped for MG serology, culture, and MG qPCR. Four weeks after vaccination and at 7 WOA, chickens from groups B to L were challenged with the R strain by aerosol. At 9 WOA, all birds were necropsied and evaluated by total air sac lesion scoring, serology, post-nasal cleft and air sac culture, tracheal histopathology, and MG and strain-specific qPCR of tracheal washings. The 50 minimum infectious dose (MID50) and 50 minimum protective dose (MPD50) were calculated based on the sampling and necropsy results (Cottey et al., 2001, Current Protocols in Immunology; 42: 19-11).

[0118] Trial 5 (Dose Response 2)

[0119] SPF chicks aged 79 days were obtained from a source known to be free of MG and MS. At 3WOA, the chicks were wing-banded and randomly assigned to seven treatment groups in nineteen isolators (Table 6). Ten birds distributed in each treatment group were screened for the presence of mycoplasmas by culture, MG and Mycoplasma synoviae qPCR, and serology of tracheal samples. At 3WOA, chickens in groups C to G were vaccinated with different doses of vaccine candidate K4110A by eye drops. Groups A and B were not vaccinated and served as negative controls and challenge-only groups, respectively. One week after vaccination, nasal slits and tracheal swabs were collected from all groups for culture and MG q-PCR. Two weeks after vaccination, all seventy-nine birds were bled and swabped for MG serology, culture, and MG qPCR. At 4 weeks and 7WOA after vaccination, chickens in groups B to G were challenged with the R strain by aerosol. At 9 WOA (2 weeks post-challenge), all birds were necropsied and evaluated by gross air sac lesion scoring, serology, post-narial cleft and air sac cultures, tracheal histopathology, and MG and strain-specific qPCR of tracheal washings. Specific dates for the experimental design and procedures are detailed in Table 6.

[0120] The 50 minimum infectious dose (MID50) and 50 minimum protective dose (MPD50) were calculated based on the sampling and necropsy results (Cottey et al., 2001, Current Protocols in Immunology; 42: 19-11).

[0121] Test 6 (Vertical Transmission)

[0122] One hundred and forty-four 4-week-old SPF chickens (131 females and 18 males) were obtained and housed in four pens (1.5 x 3 m 2). At 19WOA, the chickens were transferred to the chicken house and randomly assigned to 5 groups (Table 7). At 23WOA, the presence of mycoplasma in sixteen chickens distributed in each treatment group was screened by culture of tracheal samples, MG and synoviae qPCR and serology. At 25WOA, the chickens were inoculated with the corresponding treatment (K6067, K4110A, F strain or R strain) by eye drops, or not inoculated as a negative control group. Three weeks after inoculation, at 28WOA, MG qPCR was performed on nasal cleft swabs from five birds in the negative control group, group A, and 10 birds in groups B to E. Eggs were collected every day from one week before inoculation (24WOA) until 31WOA and kept at 4C until incubation began. Except for Monday, eggs laid on weekdays were hatched on Saturdays, and eggs laid on Saturdays, Sundays and Mondays were hatched on Mondays. Each group of eggs was examined with bright light at embryonic development (DOE) days 7, 9, 11, 13, and 15. Unfertilized eggs or early dead embryos (less than 7 DOE) were discarded, and the yolk sac of any dead embryos was sampled at 9, 11, 13, and 15 DOE and inoculated into modified Freund's broth for culture. At 18 DOE, all remaining embryonated eggs were sampled for culture.

[0123] In addition, yolk sac samples from the last set of eggs were subjected to HI and ELISA (IDEXX, Westbrook, Maine) assays (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 the NPIP recommended egg yolk standard procedure (National Poultry Improvement Plan, "Procedures for preparing egg yolk samples for diagnostic tests," National Poultry Improvement Plan Program Standards, 23-24; 2017). HI test procedures as described by Ferguson-Noel et al. (Ferguson-Noel and Kleven, "Mycoplasma species," see: Williams SM, Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E. Woolcock PR, ed. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists. Pages 63-70; 2016). Egg laying parameters for all groups during egg laying, including fertilization rate and weekly production rate of hens, were also recorded. The fertilization rate of 16 groups of eggs in all groups was calculated by dividing the number of fertilized eggs after 7 DOE by the total number of incubated eggs multiplied by 100 (Lin and Kleven, 1982, Avian Dis; 26: 487-95). The number of eggs laid during the period from 21 to 31 WOA was recorded and the weekly egg production rate of the hens was calculated by dividing the total number of eggs laid in a week by the total number of hens multiplied by 100.

[0124] At 31 WOA, all birds were necropsied and evaluated by gross air sac lesion scoring, serology, post-narial cleft and air sac cultures, tracheal histopathology, and MG qPCR of tracheal washings and oviduct swabs.

[0125] result

[0126] Test 1 (Turkey Safety 1)

[0127] Birds tested pre-vaccinated at 2WOA were negative for MG, MM, and MS antibodies and by qPCR for MG, MS, MM, and MI. All cultures were also negative for mycoplasma at that time. MG antibodies were detected in all vaccinated groups; the serological results for trial 1 are summarized in Table 8. The weakest antibody response was in group E, which was vaccinated with isolate K6837. The only group in which no air sac lesions were observed was the negative control group, however, the mean air sac lesion scores in the groups vaccinated with K6067 (group B) and K6837 (group E) were significantly lower than those in the positive control group (group G) (P≤0.05). The highest mean tracheal mucosal thickness measurements were in groups D and F. The only vaccinated groups with mean measurements lower than the positive control (R-strain; group G) were those vaccinated with K6067 (group B) and K6837 (group E). qPCR results showed that all vaccinated groups were infected with MG except Group E (vaccinated with K6837), and MG replicated at high levels in the trachea. Mycoplasmas were recovered from cultures (trachea and air sacs) of all vaccinated groups except 2 air sac cultures of Group E (vaccinated with K6837) and Group B (vaccinated with K6067) (Table 9). No mortality occurred during the trial, but the clinical signs observed are summarized in Table 10. No respiratory clinical signs were observed in birds of Groups B (vaccinated with K6067) and E (vaccinated with K6837).

[0128] Test 2 (Turkey Safety 2)

[0129] Ten birds tested pre-vaccinated at 3WOA were negative for MG, MM, and MS antibodies, and by qPCR, were also negative for MG, MS, MI, and MM. All cultures were also negative for mycoplasma. MG antibodies were detected in all vaccinated groups, and the weakest antibody response was in Group C, which was vaccinated with K4110A (Table 11). This was consistent with the mean air sac lesion scores. The only group in which no air sac lesions were observed was the negative control group. However, the mean air sac lesion scores of the groups vaccinated with K4110A (Group C) and the group vaccinated with K4179 (Group B) were lower than those of the positive control (Group G). The difference between the mean air sac lesion score of Group B (3.5) and the mean air sac lesion score of the positive control group (3.7) was not significant (P≤0.05). The highest mean tracheal mucosal thickness measurements were in Groups B and F. The mean measurements in any vaccinated group were higher than those in the positive control (Group G). The qPCR results showed that all vaccinated groups were infected with MG and that MG replicated at high levels in the trachea. These results also indicate that although K4110A vaccination did not result in significant lesions (P≤0.05), the birds were infected and replicated MG. Mycoplasmas were recovered from all cultures of the vaccinated groups, including the trachea and air sacs. These results are summarized in Table 12. No mortality occurred during the trial, but the clinical signs observed are summarized in Table 13. No respiratory clinical signs were observed in birds of Group C (vaccinated with K4110A) and Group F (vaccinated with K6836A).

[0130] Study 3 (Chicken Safety and Efficacy)

[0131] Fifteen birds tested pre-vaccination at 2WOA were negative for MG and MS antibodies and were also negative for both MG and MS by qPCR. All cultures were also negative for mycoplasma before vaccination. Three necropsies were performed in this trial to assess the safety of the vaccine candidate in chickens (necropsy 1), to assess the potential for horizontal transmission of the vaccine candidate (necropsy 2), and to assess the efficacy of the vaccine candidate after challenge (necropsy 3).

[0132] Necropsy 1 (Safety). Although the antibody response in the group vaccinated with the vaccine candidate was weaker compared to the positive control (R strain), MG antibodies were detected in all vaccinated groups. The mean SPA scores of Group B (K6067) and Group C (K4110A) were significantly lower than those of Group D (R strain) (P≤0.05). A summary of these serological results is shown in Table 14. 14 days after vaccination, no air sac lesions appeared in all groups except the positive control group, and these differences were statistically significant (P≤0.05). There was no significant difference in the mean tracheal mucosal thickness between the groups (P≤0.0.), although the positive control (R strain) was numerically higher. MG was isolated from the trachea of ​​all vaccinated birds 14 days after vaccination, but no MG was isolated from the air sacs of the vaccine candidate group, in contrast, MG was isolated from 100% of the air sacs of the R strain group. qPCR results showed that although all isolates colonized birds in their groups, the genome copy number (MCNlog10) was significantly lower in K4110A compared to the groups inoculated with K6067 and R strains (P≤0.05). A summary of these results is shown in Table 15.

[0133] Necropsy 2 (horizontal transmission to contacts). At 19 days after co-housing, birds in direct contact with vaccinated birds had lower levels of infection (2 of 5 K6067 and 1 of 5 K4110A detected from tracheal isolation). There were no significant differences in serological responses (Table 16), air sac lesions, or tracheal mucosal thickness between the groups (Table 17).

[0134] Autopsy 3 (Efficacy - Aerosol and Contact Challenge). The group vaccinated with the vaccine candidate showed a strong serological response 14 days after challenge with the R-strain (shown in Table 18). As shown 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 in tracheal measurements even between the negative and positive controls (Table 21) (P≤0.05); however, the positive control produced the highest mean measurements. The qPCR results are summarized in Table 20. The qPCR results showed that the average genome copy number log10 in the trachea of ​​the K6067 group was higher than that of the K4110A group. It is also clear that most of the MG detected in the trachea of ​​the vaccinated group were vaccine strains, rather than the challenge strains of the two vaccine candidates. Both vaccines appear to be able to prevent colonization of the challenge strain, with the MCNlog10 of the R-strain-specific qPCR in the vaccinated group significantly reduced (P≤0.05). For the contact-challenge group, there was some degree of R-strain transmission among the unvaccinated contact birds, and based on the isolation results we could infer that at least 70% (7 / 10) of these contact birds were infected with the R-strain but none of the vaccinated birds that were exposed to the R-strain through contact with the challenge birds were infected with the challenge strain (from the R-strain qPCR results).

[0135] Trial 4 (Dose Response 1)

[0136] All 10 birds tested pre-vaccination at 2 WOA were negative for MG and MS antibodies and were also negative for MG and MS by qPCR. All cultures at this time were also negative for mycoplasma. A summary of the sampling results at 4 WOA (1 week after vaccination) is shown in Table 22. All cultures of Groups E, F, and G (10 3 , 10 4 and 10 5 CCU / mL) were positive for mycoplasma. In the other groups, group D (by eye drops, 10 2 CCU / mL), group J and group K (via aerosol, 10 5 and 10 6 The qPCR results also showed that only K6067 in groups E, F, and G had some MG colonization.

[0137] The results of the sampling at 6 WOA (3 weeks after vaccination) are shown in Tables 23 and 24. All cultures of groups E, F, and G were again positive for mycoplasma, one sample each of groups J and K was positive, and two samples of group D were positive, the rest were negative. MG colonization was also evaluated by qPCR on tracheal swabs, and groups D, E, F, and G showed some degree of colonization, with group F (by eye drops, 104 CCU / mL) was 100%. In terms of serology, the serum plate agglutination (SPA) test showed that there were some antibody responses in groups D to G, J and K, but the hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) tests showed that there were almost no positive reactions in groups F and G. MG antibodies were detected in all vaccination groups (B to G) 6 weeks after vaccination (2 weeks after attack with R-strain) (Tables 25 and 26). Compared with group B (attack group only), the average SPA scores of groups E to G (all showed K6067 colonization in the trachea in early sampling) were lower. Except for the negative control group (group A), all groups showed a certain degree of air sac lesions after virulence attack, but the average air sac lesion scores of groups D, E, F, G, J, K and L were lower than those of the attack group only (group B). Among them, the average scores of groups E, F and G were significantly lower than those of other vaccination groups and the attack group (P≤0.05). Mean tracheal mucosal thickness measurements were lower in groups D, E, F, G, J, and K compared to the challenge-only group, and the differences were significant (P≤0.05) for groups E, F, and G (Table 27).

[0138] The qPCR results summarized in Table 28 show that all vaccinated groups were infected and MG replicated in the trachea. The mean colonization rates of groups E, F, and G (vaccinated with the highest dose of K6067 by eye drops) were significantly lower than those of the other groups, including the challenge-only group (Group B) (P≤0.05). The mean colonization rates of groups D, J, and K were also lower than those of Group B. The strain-specific qPCR results correlated with the MG qPCR and the groups (Table 28). Mycoplasmas were recovered from all post-nasal cleft cultures of the vaccinated groups after R-strain challenge, except for two birds in Group J. For air sac cultures, mycoplasmas were recovered from the air sacs of all groups (Groups H to L) vaccinated by aerosol. For the groups vaccinated by eye drops, mycoplasmas were 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 samples collected 1 and 3 weeks after vaccination for all doses and both routes of administration. Although the MID50 of the qPCR results could not be calculated, the MID50 of the culture results for the group vaccinated by the eye drop route was 10 3.8 ccu / mL, and 10 three weeks after vaccination 3.53 CCU / mL. It was not possible to calculate the MID50 from the culture results for the group vaccinated by the aerosol route (Table 29).

[0139] In addition, the MPD50 of K6067 was calculated based on the air cuff score and tracheal thickness measurements recorded at necropsy. Although the MPD50 could not be calculated based on the air cuff score and tracheal thickness measurements of the group vaccinated by aerosol, the MPD50 of the group vaccinated by eye drop was 10 based on the air cuff score. 4 CCU / mL, and based on tracheal thickness measurement, the value is 10 3.3 CCU / mL (Table 30).

[0140] Trial 5 (Dose Response 2)

[0141] All 10 birds tested pre-vaccination at 2WOA were negative for MG and MS antibodies and were also negative for MG and MS by qPCR. All cultures were also negative for mycoplasma at that time. A summary of the sampling results at 4WOA (1 week after vaccination) is shown in Table 31. Ten of the 13 cultures in Group C and all cultures in Groups D to G were positive for mycoplasma. The qPCR results also showed that all groups vaccinated with K4110A had some degree of MG colonization, with the mean genome copy number (MCNlog10) in Groups F and G (the groups receiving the highest vaccine dose) significantly higher than the negative control (P<0.05).

[0142] The results of sampling at 6WOA (3 weeks after vaccination) are summarized in Tables 32 and 33. Cultures from all vaccinated groups (except 2 birds in Group C) were positive for Mycoplasma. Some degree of colonization was detected in all vaccinated groups, with the mean genome copy number (MCNlog10) in Groups E, F, and G significantly higher than that in the negative control (P<0.05). In terms of serology, the SPA test found more positive samples compared with the HI and ELISA tests. The SPA score of Group C (vaccinated with the lowest dose of vaccine) was significantly lower than that of the other vaccinated groups (P<0.05). The ELISA and HI tests of Group C also did not show positive results.

[0143] At 9WOA (6 weeks after vaccination and 2 weeks after challenge with R-strain), seroconversion was observed in all vaccination groups. Compared with group B (attack group only), the average SPA scores of groups C to G were lower. However, the s / p ratio and HI titer of these groups were higher than those of group B (Table 34). Except for group A (negative control) and group F, all groups had some air sac lesions. However, the average air sac lesion scores of all vaccination groups were significantly lower than those of group B (attack control only) (P<0.05) (Table 35). The average tracheal mucosal thickness measurements of all vaccination groups were significantly lower than those of the attack group only (group B) (P<0.05). Mycoplasma was isolated from the air sacs of all post-narial cleft cultures of all vaccination groups or challenge groups and all birds of all vaccination groups or challenge groups (except two birds in group D and one bird in group F).

[0144] The MG qPCR and strain-specific qPCR results are summarized in Table 36. The average genome copy number (MCNlog10) of the R strain detected in the trachea of ​​the challenge-only group was significantly higher than that of the vaccinated group (P<0.05). K4110A-specific qPCR showed that the vaccine candidate colonized the trachea of ​​all groups vaccinated with the K4110A vaccine, although the MCNlog10 of the vaccine strain in group C (lowest vaccination dose) was significantly lower than that of the other vaccination groups. Except for group G, K4110A was not detected in air pouch cultures from the vaccinated groups (Table 36). Based on the qPCR and culture results at 1 week and 3 weeks after vaccination for all doses, the MID50 of K4110A was calculated (Table 37). Although no MID50 was found for the culture results, the MID 50 for the qPCR results of the group vaccinated by the eye drop route was 10 one week after vaccination. 5.3 CCU / mL, and 10 at three weeks after vaccination 5.2 CCU / mL.

[0145] The MPD50 of K4110A is also calculated based on the average air sac score at autopsy and is 10 3.7 CCU / mL. (Table 38). MPD50 could not be calculated from the tracheal thickness measurements in the vaccinated group.

[0146] Test 6 (Vertical Transmission)

[0147] All 16 birds tested pre-vaccination at 23 WOA were negative for MG and MS antibodies and by qPCR, were also negative for MG and MS. All cultures were also negative for mycoplasma at that time.

[0148] A total of 4436 eggs were tested, none of which were positive for mycoplasma. However, HI and ELISA results of the yolk samples indicated antibody transfer to the embryo (Table 39). Although there were some positive MG ELISA results in Groups C (K4110A), D (F-strain), and E (R-strain), only Groups C and E were MG HI positive. Group B yolk sac samples were not positive by either HI or ELISA. The mean values ​​of fertility and weekly egg production of the 16 eggs from all groups are shown in Table 40; there were no significant differences (P<0.05) between the groups.

[0149] A summary of the serological results at necropsy is shown in Table 41. MG antibodies were detected in all vaccination groups. Although the HI titer and ELISA S / P ratio of group B (K6067) were significantly lower than those of the other vaccination groups, there were no significant differences in the SPA scores among the vaccination groups (P<0.05).

[0150] The mean air sac lesions in the groups vaccinated with both vaccine candidates and the F strain were significantly lower than those in group E (R-strain) (Table 42) (P<0.05). The highest mean tracheal mucosal thickness measurements were found in group E vaccinated with the R strain. For tracheal lesions, the mean measurements for the vaccine candidates (groups B and C) and the F strain vaccinated group (group D) were significantly lower than those for the positive control (group E–R-strain) (P<0.05). The results of MG qPCR of tracheal wash samples showed that all vaccinated groups were infected with MG and that MG replicated in the trachea. These results indicate that the trachea was colonized more frequently by MG in the groups vaccinated with both vaccine candidates than in the groups vaccinated with the F-strain and the R-strain. None of the oviduct swabs tested positive by MG q-PCR. Mycoplasmas were recovered from all post-narial cleft cultures of all vaccinated groups, except for 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 vaccination groups was significantly lower than that in group E (vaccinated with R strain).

[0151] discuss

[0152] Ten MG isolates that met the initial criteria for vaccine candidates were first administered to turkeys in Trials 1 and 2. The rationale for selecting isolates recovered from turkeys and for conducting the first safety studies in turkeys was that, in general, turkeys are more susceptible to MG and are more severely affected by the disease than chickens. The overall goal of this study was to develop a live attenuated MG vaccine candidate that would be effective and safe for both turkeys and chickens. Vaccine candidates that have some degree of natural attenuation are considered more promising than virulent strains. Analysis of the results of the two turkey trials (clinical signs, air sac and tracheal lesions) led to the selection of K6067 and K4110A for further study, as these isolates showed evidence of (some degree of) natural attenuation. There was another isolate in Trial 1 (K6837) that had the weakest antibody response and lower levels of lesions compared to the other vaccinated groups. However, the inoculation titer of this isolate (10 6 CCU / ml) was much lower than the inoculation titer of other isolates (10 8 CCU / ml), and based on the qPCR results, this isolate does not appear to colonize turkeys as effectively as the other MG isolates. Although K6837 met the minimum requirements for robust growth in vitro, it was the slowest growing of the isolates selected for this trial, and further studies of this isolate are needed to determine whether the results of trial 1 were due to the low titer (low colonization rate) of this isolate or to low virulence.

[0153] Results from trial 1 demonstrated that turkeys were infected and replicated MG (a requirement for the generation of a protective immune response), although K6067 inoculation did not result in significant lesions. Studies have shown that 6 / 85 colonized the trachea poorly, which may explain the difference compared to the F-strain and ts-11 (16, 29). Air sac culture results also suggest that K6067 may not be as invasive as the other MG isolates in this trial. Similarly, in trial (Turkey Safety 2), K4110A was able to infect turkeys but did not cause severe clinical signs or lesions. All remaining trials were conducted in chickens, but further studies in turkeys are necessary to investigate efficacy and other parameters in this poultry species.

[0154] In Trial 3, the two selected candidates were evaluated for safety (including the potential for horizontal transmission) and efficacy in chickens. In the first part of this trial, it was determined that both vaccine candidates met the safety requirements for chickens, as there were no clinical signs and no air sac lesions following aerosol administration of either K6067 or K4110A, despite the isolates colonizing the respiratory system and generating a systemic antibody response. In this trial, K6067 was present at higher levels in tracheal samples compared to K4110A, but this may be due to the slightly lower dose of K4110A administered (10 7 CCU / ml and 108 CCU / ml). Another aspect of safety was assessed by co-housing vaccinated birds with unvaccinated contacts – the risk of horizontal transmission. There was some evidence that contacts became infected, but as the birds were only co-housed for 14 days, the transmissibility of the vaccine candidates should be investigated more thoroughly before conclusions are 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. Mean air sac scores were significantly lower in vaccinated birds compared to unvaccinated control birds challenged by aerosol.

[0155] Although there were no significant differences in tracheal mucosal measurements between groups in this trial, this may be due to the combined data from aerosol-challenged and contact-challenged birds. Contact-challenged birds did not develop severe lesions even in the unvaccinated control group. This challenge method does not generally result in a robust challenge with high lesion scores, however, the qPCR results suggest that the vaccine candidates are able to prevent (contact infection) or reduce (direct aerosol challenge) infection and colonization with the challenge strain. Further studies of the efficacy of these vaccine candidates (including direct comparisons with commercial vaccines) should be conducted to confirm these preliminary results. However, the results from Trials 4 and 5 suggest that the vaccine candidates have comparable minimum infectious and protective doses to commercial vaccines when administered by the eye drop route. At 1 week post-vaccination, 10 3 Eye drop doses in the CCU / ml range were sufficient to infect 100% (for K6067) and 77% (for K4110A) of birds. 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 calculated to be 10 5.5CCU / mL (Lin and Kleven, 1984, Avian Dis; 28: 273-7). Aerosol administration of K6067 was less successful; the equipment and environmental complexity of administering the vaccine by aerosol to a small number of birds in each group in the trial may affect this part of the study. More investigations (for a larger number of birds) may indicate that aerosol may be an effective route of administration, but from the results of this trial, the eye drop route appears to be the preferred route for K6067 vaccination. Similar results found that eye drops were 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). The results of the eye drop vaccination group were more consistent, with an increased immune response (measured by serology). The better results obtained with the eye drop route may be due to a variety of factors, including the individual administration of the vaccine and more consistent volume and dose for each bird in the flock. Notably, in the dose response 2 (K4110A) strain-specific PCRs, not only tracheal samples but also air sac cultures were tested, and the genome copy number of the R-strain was very high; this may be due to the adaptation of the R-strain to in vitro culture.

[0156] In turkeys and chickens, both vaccine candidates elicited detectable and consistent serological responses; although serum antibody levels did not appear to correlate with protection with the live attenuated MG vaccine, it is useful to be able to monitor vaccination using a widely available and inexpensive serological test.

[0157] Vertical transmission may become a major safety issue for mycoplasma vaccines, and both F-strain and ts-11 vaccines have been shown to have some risk of transmission to offspring through eggs (Armour et al., 2015, Avian Pathol; 44: 296-304; and El Gazzar et al. 2011, Avian Dis; 55: 569-74). In this study, no vertical transmission was found for any candidate. However, no vertical transmission was detected in the groups vaccinated with F strain or R strain. These results may be due to the relatively small number of birds in the trial and the route of administration (eye drops). Although this route more closely simulates the possible field application of the vaccine, higher transmission rates may occur under more severe challenges (aerosol). The results suggest that vaccine candidates are unlikely to have high vertical transmission rates, but increasing the number of birds and the number of eggs should be considered to confirm these results. The detection of transfer of maternal antibodies in egg yolk samples indicates that there is some systemic immune response to MG. The role of passive maternal antibody in MG infection has been shown to be unhelpful, as it provides little protection against MG challenge (Lin and Kleven, 1984, Avian Dis; 28: 79-87) and embryonic death caused by virulent MG is blocked by the antibody (Levisohn et al., 1985, Avian Dis; 29: 188-97), so the hatchability of infected eggs may be increased, which is not ideal.

[0158] In conclusion, both candidates were safe for turkeys and chickens and were effective in reducing colonization with virulent challenge strains, as well as reducing lesions and effects of virulent MG strains in chickens. The eye drop route was confirmed to be an effective route of administration, and the infectious and protective doses corresponded to other MG vaccines. Vertical transmission studies showed that neither K6067 nor K4110A posed a high risk of transmission. These results are encouraging, and further studies on parameters such as duration of immunity, transmissibility, and efficacy in turkeys will provide more information for these vaccines.

[0159] Table 1. History of vaccine candidates selected for primary screening (Trials 1 and 2).

[0160]

[0161]

[0162] WOA = age in weeks

[0163] Table 2. Trial 1 (Turkey Safety 1). Experimental Design.

[0164]

[0165] Table 3. Trial 2 (Turkey Safety 2). Experimental Design.

[0166]

[0167] Table 4. Trial 3 (Chicken Safety and Efficacy). Experimental Design.

[0168]

[0169]

[0170] Note: The titer of R strain 13P used to attack groups B, C, and D after inoculation was 2.45 x 10 9 CCU / ml.

[0171] Table 5. Trial 4 (Dose Response 1). Experimental Design.

[0172]

[0173] Note: The titer of R strain 12P used to attack groups B to L after inoculation was 1.1 x 10 9 CCU / ml.

[0174] Table 6. Trial 5 (Dose Response 2). Experimental Design.

[0175]

[0176] Note: The titer of R strain 12P used to challenge groups B to L after inoculation was 3.5 x 10 8 CCU / ml.

[0177] Table 7. Experiment 6 (vertical transfer). Experimental design.

[0178]

[0179] Table 8. Trial 1 (Turkey Safety 1). MG serological responses in turkeys 2 weeks after vaccination with K6067, K6524, K6694, K6837, K6813 or R strains. A

[0180]

[0181]

[0182] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0183] BNumber of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0184] C Average agglutination grade (from 0 to 4)

[0185] D Average titer log10

[0186] E Average sample / positive ratio

[0187] Table 9. Trial 1 (Turkey Safety 1). Lesion scores in trachea and air sacs, tracheal mucosal thickness, log10 genome copy number, and MG isolation in turkeys 2 weeks after inoculation with K6067, K6524, K6694, K6837, K6813, or R strains. A

[0188]

[0189] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0190] B Number of positive samples / number of tested samples (airbag score ≥ 1)

[0191] C Average rating (macro rating from 0 to 4)

[0192] D Average thickness of each group ± SD (μm)

[0193] E Mean (genomic) copy number log10±SD

[0194] Table 10. Trial 1 (Turkey Safety 1). Clinical signs in turkeys 2 weeks after inoculation with K6067, K6524, K6694, K6837, K6813 or R strains.

[0195]

[0196] Table 11. Trial 2 (Turkey Safety 2). MG serological responses in turkeys two weeks after vaccination with K4179, K4110A, K5058E, K5792A, K6836A or R strains. A

[0197]

[0198] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0199] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0200] C Mean agglutination grade (from 0 to 4).

[0201] D Average titer log10

[0202] E Average sample / positive ratio

[0203] Table 12. Trial 2 (Turkey Safety 2). Lesion scores, tracheal mucosal thickness, MG isolation, and qPCR in turkey trachea and air sacs two weeks after inoculation with K4179, K4110A, K5058E, K5792A, K6836A, or R strains. A

[0204]

[0205] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0206] B Number of positive samples / number of tested samples (airbag score ≥ 1)

[0207] C Average rating (macro rating from 0 to 4)

[0208] D Average thickness of each group ± SD (μm)

[0209] E Mean (genomic) copy number log10±SD

[0210] Table 13. Trial 2 (Turkey Safety 2). Clinical signs in turkeys two weeks after inoculation with K4179, K4110A, K5058E, K5792A, K6836A or R strains.

[0211]

[0212] Table 14. Trial 3 (Safety and Efficacy in Chickens). MG serological responses in chickens 14 days after inoculation with K6067, K4110A or R strains (necropsy 1). A

[0213]

[0214] AThe values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0215] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0216] C Mean agglutination grade (from 0 to 4).

[0217] D Average titer log10

[0218] E Average sample / positive ratio

[0219] Table 15. Trial 3 (Safety and Efficacy in Chickens). Lesion scoring, MG isolation and qPCR in chickens 14 days after vaccination with vaccine candidates or R strain (necropsy 1). A

[0220]

[0221] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0222] B Number of positive samples / number of tested samples (airbag score ≥ 1)

[0223] C Average rating (macro rating from 0 to 4)

[0224] D Average thickness of each group ± SD (μm)

[0225] E Mean (genomic) copy number log10±SD

[0226] Table 16. Trial 3 (Safety and Efficacy in Chickens). MG serological responses in chickens 19 days after co-housing with the vaccinated groups (Groups B and C) (necropsy 2). A

[0227]

[0228] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0229] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0230] CMean agglutination grade (from 0 to 4).

[0231] D Average titer log10

[0232] E Average sample / positive ratio

[0233] Table 17. Trial 3 (Safety and Efficacy in Chickens). Lesion scoring, MG isolation and qPCR (necropsy 2) in chickens 19 days after co-housing with vaccinated groups (Groups B and C). A

[0234]

[0235] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0236] B Number of positive samples / number of tested samples (air sac score ≥ 1; tracheal MG separation; air sac MG separation)

[0237] C Average rating (macro rating from 0 to 4)

[0238] D Average thickness of each group ± SD (μm)

[0239] E Mean (genomic) copy number log10±SD

[0240] Table 18. Trial 3 (Safety and Efficacy in Chickens). MG serological responses in chickens 28 days after vaccination with vaccine candidates or R strain and 14 days after challenge with R strain (necropsy 3). A

[0241]

[0242] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0243] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0244] C Mean agglutination grade (from 0 to 4).

[0245] D Average titer log10

[0246] E Average sample / positive ratio

[0247] Table 19. Trial 3 (Safety and Efficacy in Chickens). Lesion scores and MG isolation results in chickens 28 days after vaccination with vaccine candidates or R strain and 14 days after challenge with R strain (necropsy 3). A

[0248]

[0249] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0250] B Number of positive samples / number of tested samples (air sac score ≥ 1; number of MG separation samples / number of tested samples)

[0251] C Average rating (macro rating from 0 to 4)

[0252] Table 20. Trial 3 (Chicken Safety and Efficacy). MG, R-strain-specific, K6067-specific and K4110A-specific qPCR in chickens 28 days after vaccination with vaccine candidates or R strain and 14 days after challenge with R strain (necropsy 3). A

[0253]

[0254] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0255] B Mean (genomic) copy number log10±SD

[0256] Table 21. Trial 3 (Safety and Efficacy in Chickens). Tracheal mucosal thickness in chickens 28 days after vaccination with vaccine candidates or R strain and 14 days after challenge with R strain or co-housing with R strain challenged chickens (necropsy 3). A

[0257]

[0258] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0259] B Average thickness of each group ± SD (μm)

[0260] Table 22. Trial 4 (Dose Response 1). MG isolation and qPCR in chickens 7 days after inoculation with K6067 (1st sampling). A

[0261]

[0262] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0263] B Number of positive samples / number of samples tested (color change due to mycoplasma growth)

[0264] C Mean (genomic) copy number log10±SD

[0265] Table 23. Trial 4 (Dose Response 1). MG serological response in chickens 21 days after vaccination with K6067 (2nd sampling). A

[0266]

[0267]

[0268] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0269] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0270] C Mean agglutination grade (from 0 to 4).

[0271] D Average titer log10

[0272] E Average sample / positive ratio

[0273] Table 24. Trial 4 (Dose Response 1). MG isolation and qPCR in chickens 21 days after inoculation with K6067 (2nd sampling). A

[0274]

[0275] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0276] B Number of positive samples / number of samples tested (color change due to mycoplasma growth)

[0277] C Mean (genomic) copy number log10±SD

[0278] Table 25. Trial 4 (Dose Response 1). MG serological responses in chickens (necropsy) 6 weeks after vaccination with K6067 and 2 weeks after challenge with the R strain. A

[0279]

[0280] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0281] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0282] C Mean agglutination grade (from 0 to 4).

[0283] D Average titer log10

[0284] E Average sample / positive ratio

[0285] Table 26. Trial 4 (Dose Response 1). Lesion scores and MG isolation results (necropsy) 6 weeks after inoculation with K6067 and 2 weeks after challenge with R strain. A

[0286]

[0287]

[0288] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0289] B Number of positive samples / number of tested samples (airbag score ≥ 1)

[0290] C Average rating (macro rating from 0 to 4)

[0291] D Average thickness of each group ± SD (μm)

[0292] E Number of positive samples / number of tested samples (culture)

[0293] Table 27. Trial 4 (Dose Response 1). Lesion scores and MG isolation results (necropsy) 6 weeks after inoculation with K6067 and 2 weeks after challenge with R strain. A

[0294]

[0295] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0296] B Number of positive samples / number of tested samples (airbag score ≥ 1)

[0297] C Average rating (macro rating from 0 to 4)

[0298] D Average thickness of each group ± SD (μm)

[0299] E Number of positive samples / number of tested samples (culture)

[0300] Table 28. Trial 4 (Dose Response 1). MG, R-strain specific and K6067 specific genome copy numbers 6 weeks after vaccination with vaccine candidates and 2 weeks after challenge with R strain (necropsy). A

[0301]

[0302] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0303] B Mean (genomic) copy number log10±SD

[0304] Table 29. Trial 4 (Dose Response 1). 50% Minimum Infectious Dose (MID50) of K6067 based on qPCR and mycoplasma culture results from the 1st and 2nd samplings.

[0305]

[0306] A Eye drop vaccination group

[0307] B One week after inoculation, the first sampling was performed at 4 WOA.

[0308] C Aerosol vaccination group

[0309] D The second sampling was performed at 6 WOA, 3 weeks after inoculation.

[0310] Table 30. Trial 4 (Dose Response 1). 50% Minimum Protective Dose (MPD50) of K6067 based on air sac lesion scores and tracheal thickness measurements at necropsy.

[0311]

[0312] A Eye drop vaccination group

[0313] B Aerosol vaccination group

[0314] C To compare tracheal thickness measurements and to consider affected and unaffected measurements, the highest measurement in Group A (negative control) (118.2 μm) was considered the threshold.

[0315] Table 31. Trial 5 (Dose Response 2). MG isolation and qPCR in chickens 7 days after K4110A vaccination (1st sampling). A

[0316]

[0317] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0318] B Number of positive samples / number of samples tested (color change due to mycoplasma growth)

[0319] C Mean (genomic) copy number log10±SD

[0320] Table 32. Trial 5 (Dose Response 2). MG serological response in chickens 21 days after vaccination with K4110A (2nd sampling). A

[0321]

[0322] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0323] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0324] C Mean agglutination grade (from 0 to 4).

[0325] D Average titer log10

[0326] E Average sample / positive ratio

[0327] Table 33. Trial 5 (Dose Response 2). MG isolation and qPCR in chickens 21 days after inoculation with K4110A (2nd sampling).A

[0328]

[0329] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0330] B Number of positive samples / number of samples tested (color change due to mycoplasma growth)

[0331] C Mean (genomic) copy number log10±SD

[0332] Table 34. Trial 5 (Dose Response 2). MG serological responses in chickens (necropsy) 6 weeks after vaccination with K4110A and 2 weeks after challenge with the R strain. A

[0333]

[0334] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0335] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0336] C Mean agglutination grade (from 0 to 4).

[0337] D Average titer log10

[0338] E Average sample / positive ratio

[0339] Table 35. Trial 5 (Dose Response 2). Lesion scores, MG isolation and qPCR results (necropsy) 6 weeks after inoculation with K4110A and 2 weeks after challenge with R strain. A

[0340]

[0341]

[0342] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0343] B Number of positive samples / number of tested samples (airbag score ≥ 1)

[0344] C Average rating (macro rating from 0 to 4)

[0345] D Average thickness of each group ± SD (μm)

[0346] E Number of positive samples / number of tested samples (culture)

[0347] Table 36. Trial 5 (Dose Response 2). MG, R-strain specific and K4110A specific qPCR results of tracheal washes and air pouch cultures 6 weeks after inoculation with K4110A and 2 weeks after challenge with R strain (necropsy). A

[0348]

[0349]

[0350] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0351] B Mean (genomic) copy number log10±SD

[0352] Table 37. Experiment 5 (Dose Response 2). 50% Minimum Infectious Dose (MID50) of K4110A based on qPCR and mycoplasma culture results from the 1st and 2nd samplings.

[0353]

[0354] A Eye drop vaccination group

[0355] B One week after inoculation, the first sampling was performed at 4 WOA.

[0356] C The second sampling was performed at 6 WOA, 3 weeks after inoculation.

[0357] Table 38. Trial 5 (Dose Response 2). 50% Minimum Protective Dose (MPD50) of K4110A based on air cuff scores and tracheal thickness measurements at necropsy.

[0358]

[0359] A Eye drop vaccination group

[0360] BTo compare tracheal thickness measurements and to consider affected and unaffected measurements, the highest measurement in Group A (negative control) (91.3 μm) was considered the threshold.

[0361] Table 39. Trial 6 (vertical transfer). MG yolk antibodies from the last set of eggs laid at 31 WOA; 6 weeks after vaccination with vaccine candidate, F-strain or R-strain. A

[0362]

[0363] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0364] B Number of positive samples / number of tested samples (HI: ≥20, ELISA: ≥0.5)

[0365] C Average titer log10

[0366] D Average sample / positive ratio

[0367] Table 40. Experiment 6 (vertical transfer). The mean values ​​of fertility and weekly egg production of hens were 24-31 WOA and 21-31 WOA, respectively. A .

[0368]

[0369]

[0370] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0371] B The number of fertilized eggs after 7DOE divided by the total number of hatching eggs multiplied by 100

[0372] C Total number of eggs laid this week divided by total number of hens multiplied by 100

[0373] D Percent ± SD

[0374] Table 41. Trial 6 (vertical transmission). MG serological responses in 31WOA chickens at 6 weeks post-vaccination (necropsy). A

[0375]

[0376] AThe values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0377] B Number of positive samples / number of tested samples (SPA: ≥1, HI: ≥20, ELISA: ≥0.5, air sac score ≥1)

[0378] C Mean agglutination grade (from 0 to 4).

[0379] D Average titer log10

[0380] E Average sample / positive ratio

[0381] Table 42. Trial 6 (vertical transmission). Lesion scoring, MG isolation and qPCR (necropsy) in chickens 6 weeks after inoculation with K6067, K4110A, F-strain or R-strain. A

[0382]

[0383]

[0384] A The values ​​in the columns with different lowercase letters at specific time points are significantly different (P≤0.05)

[0385] B Number of positive samples / number of tested samples (airbag score ≥ 1)

[0386] C Average rating (macro rating from 0 to 4)

[0387] D Average thickness of each group ± SD (μm)

[0388] E Average (genome) copy number log10±

Claims

1. An isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain comprises the K6067 Mycoplasma gallisepticum strain deposited in ATCC under patent number PTA-127168 or its progeny or derivatives, or comprises the K4110 Mycoplasma gallisepticum strain deposited in ATCC under patent number PTA-127282 and its progeny and derivatives.

2. A substantially biologically pure culture of the K6067 strain of Mycoplasma gallisepticum deposited with the ATCC under Patent No. PTA-127168.

3. A substantially biologically pure culture of the K4110 Mycoplasma gallisepticum strain deposited with the ATCC under Patent No. PTA-127282.

4. A composition comprising the isolated Mycoplasma gallisepticum according to any one of claims 1 to 3.

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

6. The composition according to claim 4 or 5, comprising a pharmaceutically acceptable carrier.

7. A composition according to any one of claims 4 to 6 comprising an adjuvant.

8. The composition of any one of claims 4 to 7, wherein the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous or in ovo administration.

9. A composition according to any one of claims 4 to 8, wherein the composition is formulated for spraying or atomization.

10. A vaccine comprising the isolated Mycoplasma gallisepticum according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 9.

11. The vaccine of claim 10, wherein the vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry.

12. The vaccine according to claim 10 or 11, wherein the vaccine reduces the susceptibility of Galliformes birds to disease induced by Mycoplasma gallisepticum.

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

14. The isolated Mycoplasma gallisepticum according to any one of claims 1 to 3, the composition according to any one of claims 4 to 9, or the vaccine according to any one of claims 10 to 13, wherein the isolated Mycoplasma gallisepticum, the composition or the vaccine is lyophilized, freeze-dried, frozen or an effervescent tablet.

15. A kit comprising the isolated Mycoplasma gallisepticum according to any one of claims 1 to 3, the composition according to any one of claims 4 to 9 or the vaccine according to any one of claims 10 to 13 and printed instructions, wherein the contents of the kit are contained within packaging material.

16. A method of generating an immune response against Mycoplasma gallisepticum in a bird, the method comprising administering to the bird an isolated Mycoplasma gallisepticum according to any one of claims 1 to 3, a composition according to any one of claims 4 to 9, or a vaccine according to any one of claims 10 to 13.

17. A method for reducing the susceptibility of a bird to a disease induced by Mycoplasma gallisepticum, the method comprising administering to the bird an isolated Mycoplasma gallisepticum according to any one of claims 1 to 3, a composition according to any one of claims 4 to 9, or a vaccine according to any one of claims 10 to 13.

18. A method for protecting a bird from infection with Mycoplasma gallisepticum, the method comprising administering to the bird the isolated Mycoplasma gallisepticum according to any one of claims 1 to 3, the composition according to any one of claims 4 to 9, or the vaccine according to any one of claims 10 to 13.

19. A method of reducing one or more of the clinical signs induced by Mycoplasma gallisepticum infection in a bird, the method comprising administering to the bird an effective amount of the isolated Mycoplasma gallisepticum according to any one of claims 1 to 3, the composition according to any one of claims 4 to 9, or the vaccine according to any one of claims 10 to 13.

20. The method of any one of claims 16 to 19, wherein administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous or in ovo.

21. The method according to any one of claims 16 to 19, wherein the administration is by eye drops, by aerosol or by drinking water.

22. The method of any one of claims 16 to 21, wherein the birds comprise birds of the order Galliformes.

23. The method of any one of claims 16 to 22, wherein the bird comprises a chicken or a turkey.

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