Mycoplasma synoviae live vaccine

Abstract

The present invention provides Mycoplasma synoviae strain K5885, deposited with the ATCC under patent designation PTA-127167, and progeny and derivatives thereof, for use as a vaccine for the prevention of virulent Mycoplasma synoviae infection in Galliformes birds. Also provided are compositions and methods for administration to Galliformes birds.

Description

[Technical field]

[0001] Continuing Application Data This application claims the benefit of U.S. Provisional Patent Application No. 63 / 319,532, filed March 14, 2022, which is incorporated herein by reference. [Background technology]

[0002] Mycoplasma synoviae (MS) infection most frequently occurs as a subclinical upper respiratory tract infection, but MS can become systemic causing air sac lesions, resulting in infectious synovitis in chickens and turkeys, an acute to chronic infectious disease primarily involving synovial and tenosynovitis of the joints producing exudative synovitis, tenosynovitis, or bursitis (Ferguson-Noel and Noormohammadi, “Mycoplasma synoviae infection.” In: Diseases of Poultry. DE S Wayne, J R Glisson, L R Mc Dougald, L K Nolan, DL Suarez and V. Nair, eds. Wiley-Blackwell, Ames, Iowa. pp 900-906. 2013).

[0003] Mycoplasma synoviae is egg-transmitted, and the most effective control method is to select chickens or turkeys from flocks that do not have MS and to use effective biosecurity to prevent the introduction of infection (Kleven, 2008, Avian Diseases;52:367-374). Alternatively, antibiotic treatment and vaccination may be used to improve the efficacy of MS infection. Inactivated oil emulsion bacterins are commercially available, but their role in the control of MS has not been well studied. Also, a temperature-sensitive MS live vaccine strain, MS-H, selected by mutagenesis of a field isolate from Australia, is used in many major poultry producing countries, but registration in the United States is pending. See Morrow et al., 1998, Avian Diseases; 42:667-670; Markham et al., 1998, Avian Diseases; 42:671-676; Markham et al., 1998, Avian Diseases; 42:677-681, and Markham et al., 1998, Avian Diseases; 42:682-689. Thus, there is a need for additional vaccines for the control of Mycoplasma synoviae infection in poultry. Summary of the Invention

[0004] The present invention includes an isolated Mycoplasma synoviae strain, the isolated Mycoplasma synoviae strain is the K5885 Mycoplasma synoviae strain deposited with the ATCC under patent designation PTA-127167, or a progeny or derivative thereof. In some aspects, the present invention includes a composition comprising an isolated Mycoplasma synoviae strain deposited with the ATCC under patent designation PTA-127167. In some aspects, the composition may include water. In some aspects, the composition may include a pharma- ceutically 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 in ovo administration. In some aspects, the composition may be formulated for spraying or aerosolization.

[0005] The present invention includes an essentially biologically pure culture of Mycoplasma synoviae strain K5885 deposited with ATCC under patent designation PTA-127167, or a progeny or derivative thereof. In some embodiments, the present invention includes a composition comprising an essentially biologically pure culture of Mycoplasma synoviae strain K5885 deposited with ATCC under patent designation PTA-127167, or a progeny or derivative thereof. In some embodiments, the composition may include water. In some embodiments, the composition may include a pharma- ceutically acceptable carrier. In some embodiments, the composition may include an adjuvant. In some embodiments, the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. In some embodiments, the composition may be formulated for spraying or aerosolization.

[0006] The invention includes a vaccine comprising an isolated Mycoplasma synoviae strain described herein, an essentially biologically pure culture of the K5885 Mycoplasma synoviae strain described herein, or a composition described herein. In some embodiments, the vaccine reduces one or more clinical signs induced by Mycoplasma synoviae infection in poultry. In some embodiments, the vaccine reduces susceptibility of Galliformes birds to disease induced by Mycoplasma synoviae.

[0007] The present invention includes a live vaccine for birds of the order Galliformes, comprising a sufficient amount of Mycoplasma synoviae strain K5885 deposited with the ATCC under patent deposit designation PTA-127167, or a progeny or derivative thereof, to protect the bird from disease induced by Mycoplasma synoviae, and a pharmaceutically acceptable carrier.

[0008] In some aspects, the isolated Mycoplasma synoviae strains described herein, the compositions described herein, or the vaccines described herein may be lyophilized, freeze-dried, frozen, or in an effervescent tablet form.

[0009] The invention includes a kit comprising an isolated Mycoplasma synoviae strain described herein, a composition described herein, or a vaccine described herein and printed instructions, wherein the contents of the kit are contained within packaging material.

[0010] The invention includes an effervescent tablet comprising an isolated Mycoplasma synoviae strain described herein, a composition described herein, or a vaccine described herein.

[0011] The present invention includes a method of generating an immune response against Mycoplasma synoviae in an avian, comprising administering to the avian an isolated Mycoplasma synoviae strain described herein, a composition described herein, or a vaccine described herein. In some aspects, in the methods described herein, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, in the methods described herein, administration is by eye drop, by aerosol, or by drinking water. In some aspects, in the methods described herein, the avian is of the Galliformes order. In some aspects, in the methods described herein, the avian is a chicken or a turkey.

[0012] The present invention includes a method for reducing susceptibility of an avian to disease induced by Mycoplasma synoviae, comprising administering to the avian an isolated Mycoplasma synoviae strain described herein, a composition described herein, or a vaccine described herein. In some aspects, in the methods described herein, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, in the methods described herein, administration is by eye drop, by aerosol, or by drinking water. In some aspects, in the methods described herein, the avian is of the Galliformes order. In some aspects, in the methods described herein, the avian is a chicken or a turkey.

[0013] The present invention includes a method for protecting an avian against Mycoplasma synoviae infection, comprising administering to the avian an isolated Mycoplasma synoviae strain described herein, a composition described herein, or a vaccine described herein. In some aspects, in the methods described herein, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, in the methods described herein, administration is by eye drop, by aerosol, or by drinking water. In some aspects, in the methods described herein, the avian is of the Galliformes order. In some aspects, in the methods described herein, the avian is a chicken or a turkey.

[0014] The present invention includes a method of reducing one or more clinical signs induced by Mycoplasma synoviae infection in an avian, comprising administering to the avian an effective amount of an isolated Mycoplasma synoviae strain described herein, a composition described herein, or a vaccine described herein. In some aspects, in the methods described herein, the clinical signs include weight suppression, reduced egg production, mortality, upper respiratory tract infection, lameness, swollen joints, ovarian involution, air sac lesions, tracheal lesions, foot pad lesions, and / or eggshell tip abnormalities. In some aspects, in the methods described herein, administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. In some aspects, in the methods described herein, administration is by eye drop, by aerosol, or by drinking water. In some aspects, in the methods described herein, the avian is of the Galliformes order. In some aspects, in the methods described herein, the avian is a chicken or a turkey.

[0015] As used herein, "isolated" refers to material that has been removed from its original environment (e.g., the natural environment if it occurs in nature) and has thus been altered "by the hand of man" from its natural state.

[0016] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0017] The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0018] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the present description and claims.

[0019] Unless otherwise noted, "a," "an," "the," and "at least one" are used interchangeably to mean one or more.

[0020] 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.).

[0021] For any method disclosed herein that includes separate steps, the steps may be performed in any feasible order, and, if desired, any combination of two or more steps may be performed simultaneously.

[0022] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and without any attempt to limit the recitation of equivalents to the scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0023] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviations found in their respective testing measurements.

[0024] In several places throughout this application, guidance is provided through the enumeration of examples, which can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. It should be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0025] All headings herein are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. [Brief description of the drawings]

[0026] [Figure 1] Results from Study 1 (Safety I). Chicken mean tracheal mucosa measurements with K5885A, K4971B, K5805A, or K1968 at 10 and 14 DPI. Different lowercase superscripts are significantly different (P<0.05) at the particular time point (10 or 14 DPI). [Diagram 2] Results of Study 1 (Safety I). Air sac lesions from chickens with K5885A, K4971B, K5805A, or K1968 at 10 and 14 DPI. Different lowercase superscripts are significantly different (P<0.05) at the particular time point (10 or 14 DPI). [Diagram 3] Results of Clinical Trial 3 (Safety II). Air sac lesion scores from chickens with K5885A or K6677 at 14 DPI. [Figure 4] Results of Trial 3 (Safety II). Footpad lesion scores from chickens with K5885A or K6677 at 14 DPI. Different lowercase superscript letters are significantly different (P<0.05). [Diagram 5] Results from Trial 4 (Efficacy II). Air sac lesion scores for vaccinated and non-vaccinated chickens 14 days after challenge with K6677. Different lower case superscripts are significantly different (P<0.05). [Figure 6] Results from Trial 4 (Efficacy II). Footpad lesion scores of vaccinated and non-vaccinated chickens 14 days after challenge with K6677. [Figure 7] Results from Trial 5 (Efficacy III). Air sac lesion scores for vaccinated and non-vaccinated chickens 14 days after challenge with K6677A. Different lower case superscripts are significantly different (P<0.05). [Figure 8] Results of Trial 5 (Efficacy III). Footpad scores of vaccinated and non-vaccinated chickens 14 days after challenge with K6677. Different lower case superscripts are significantly different (P<0.05). [Figure 9] Air sac lesion scores in different groups 2-6 weeks after vaccination. * means the difference is significant at P<0.05. NV / NC: non-vaccinated non-challenged. NV / CK: non-vaccinated K6677-challenged. NV / CW: non-vaccinated WVU1853-challenged. V / CK: vaccinated K6677-challenged. V / CW: vaccinated WVU1853-challenged. V / NC: vaccinated non-challenged. [Figure 10] Footpad lesion scores between different groups 2-6 weeks after vaccination. * means the difference is significant at P<0.05. ** means the difference is significant at P<0.005. NV / NC: non-vaccinated non-challenged. NV / CK: non-vaccinated K6677-challenged. NV / CW: non-vaccinated WVU1853-challenged. V / CK: vaccinated K6677-challenged. V / CW: vaccinated WVU1853-challenged. V / NC: vaccinated non-challenged. [Figure 11] Percentage of ovarian involution in different groups 2-6 weeks after vaccination. * means the difference is significant at P<0.05. NV / NC: non-vaccinated non-challenged. NV / CK: non-vaccinated K6677-challenged. NV / CW: non-vaccinated WVU1853-challenged. V / CK: vaccinated K6677-challenged. V / CW: vaccinated WVU1853-challenged. V / NC: vaccinated non-challenged. [Figure 12] Mean genome copy number of MS in the trachea of ​​different groups 2-6 weeks after vaccination. NV / NC: non-vaccinated, unchallenged. NV / CK: non-vaccinated, K6677-challenged. NV / CW: non-vaccinated, WVU1853-challenged. V / CK: vaccinated, K6677-challenged. V / CW: vaccinated, WVU1853-challenged. V / NC: vaccinated, unchallenged. [Figure 13] Mean genome copy numbers of K5885 and K6677 in different groups 2-6 weeks after vaccination. * means the difference is significant at P<0.05. ** means the difference is significant at P<0.005. *** means the difference is significant at P<0.0005. NV / NC: non-vaccinated non-challenged. NV / CK: non-vaccinated K6677-challenged. NV / CW: non-vaccinated WVU1853-challenged. V / CK: vaccinated K6677-challenged. V / CW: vaccinated WVU1853-challenged. V / NC: vaccinated non-challenged. [Figure 14] Percentage of hens laying in all groups from start of lay to 6WPC. NV / NC: non-vaccinated non-challenged. NV / CK: non-vaccinated K6677-challenged. NV / CW: non-vaccinated WVU1853-challenged. V / CK: vaccinated K6677-challenged. V / CW: vaccinated WVU1853-challenged. V / NC: vaccinated non-challenged. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The bacterium Mycoplasma synoviae is a member of the Mycoplasma genus. It causes disease in the joints, bones, and respiratory system of birds. It is found worldwide, and infections may be referred to as infectious synovitis, avian mycoplasmosis, infectious sinusitis, or mycoplasmal arthritis. It is economically important because infection can lead to a decline in egg production. The disease is primarily seen in chickens and turkeys, but ducks, geese, guinea fowl, parrots, pheasants, and quails may also be susceptible. Transmission occurs both vertically and horizontally. Mycoplasma synoviae most commonly causes subclinical upper respiratory tract infections in chickens, turkeys, and other avian species, but can also cause exudative tendonitis and synovitis, known as infectious synovitis.

[0028] The present invention provides Mycoplasma synoviae (MS) strain K5885, and its progeny and derivatives, which are immunogenic and stable when administered as a live formulation. The formulations of Mycoplasma synoviae of the present invention are safe and effective in inhibiting Mycoplasma synoviae infection and may be useful in reducing the incidence and severity of disease from Mycoplasma synoviae infection in birds.

[0029] Mycoplasma synoviae strain K5885 was deposited at the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110-2209, USA as PTA-127167 on November 24, 2021. The strain was deposited pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Mycoplasma synoviae strain K5885, deposited with the ATCC® as PTA-127167, is referred to herein as Mycoplasma synoviae strain K5885, Mycoplasma synoviae strain K5885A, MS strain K5885, MS strain K5885A, K5885, K5885A, MS strain K5885 ATCC PTA-127167, MS strain K5885A ATCC PTA-127167, K5885 ATCC PTA-127167, K5885A ATCC PTA-127167, MS strain K5885 PTA-127167., MS strain K5885A PTA-127167., K5885 PTA-127167, K5885A Also referred to as PTA-127167, ATCC PTA-127167, and PTA-127167.

[0030] The present invention includes isolated Mycoplasma synoviae (MS) strain K5885 having ATCC patent deposit designation PTA-127167. As used herein, "isolated" refers to material that has been removed from its original environment (e.g., the natural environment if it occurs in nature) and thus altered from its natural state "by the hand of man." Also included is a biologically pure culture of Mycoplasma synoviae (MS) strain K5885 having ATCC patent deposit designation PTA-127167.

[0031] Also included in the present invention are isolated progeny and isolated derivatives of Mycoplasma synoviae strain K5885 deposited under ATCC patent deposit designation PTA-127167, having equivalent or similar biological, serological, and / or genetic characteristics. As used herein, serological, biological, and genetic characteristics may include one or more of the characteristics described in the Examples and Figures data contained herein. More specifically, progeny or derivatives of K5885 strain deposited with ATCC as PTA-127167 may retain particularly preferred protective properties belonging to the present invention. Progeny or derivatives of Mycoplasma synoviae K5885 deposited with ATCC as PTA-127167 may be obtained by any of the various methods for propagating Mycoplasma synoviae known in the art, including, for example, but not limited to, avian in vitro culture or backpassage. Derivatives of Mycoplasma synoviae strain K5885 ATCC PTA-127167 may include genetically modified versions of the deposited MS strain K5885. Such manipulations include, but are not limited to, mutagenesis of the MS strain, or the introduction of genes or gene cassettes encoding alternative or non-functional proteins, or non-coding nucleotide sequences into the MS organism.

[0032] The Mycoplasma synoviae (MS) strain K5885 isolate described herein may be grown by conventional methods, including, but not limited to, any of those described in the Examples section contained herein. For example, the Mycoplasma synoviae strains of the present invention may be cultured as described in detail by Avian Mycoplasmas, Harry W. Yoder Jr., in Diagnostic Procedure in Veterinary Bacteriology and Mycology (Fifth Edition), 1990. Briefly, Mycoplasma synoviae may be cultured at 37°C in Frey's broth or agar supplemented with 10-15% normal porcine serum. The porcine serum may be heat inactivated at 56°C for 30 minutes. In addition, Mycoplasma synoviae may require the addition of 0.1% reduced nicotinamide adenine dinucleotide (NAD) to broth and agar media.

[0033] MS strain K5885 and its progeny and derivatives can be identified and differentiated from other Mycoplasma synoviae strains using any of the many techniques developed for differentiation of Mycoplasma synoviae strains, including, for example, direct immunofluorescence and real-time quantitative PCR (qPCR) (Raviv and Kleven, 2009, Avian Dis; 53:103-107), multilocus sequence typing (MLST) (Dijkman et al., 2016, Avian Pathol; 45(4): 426-442, and El-Gazzar et al., 2017, Avian Dis: 61(1): 25-32), and PCR analysis of lipoprotein and hemagglutinin A (vlhA) genes (Wetzel et al., 2010, Avian Dis 54(4): 1292: 1297).

[0034] The present invention includes the Mycoplasma synoviae isolates described herein, as well as their progeny and derivatives. In preferred embodiments, the Mycoplasma synoviae isolates are live. In some embodiments, the Mycoplasma synoviae isolates may be inactivated or killed. The Mycoplasma synoviae strains of the present invention, as well as compositions and vaccines thereof, may be stored until use in any of a variety of forms. For example, such materials may be lyophilized or freeze-dried and rehydrated for use. In some embodiments, the Mycoplasma synoviae strains, or compositions or vaccines thereof, may be frozen.

[0035] In some embodiments, the Mycoplasma synoviae isolate, or composition or vaccine thereof, may be formulated as an effervescent tablet. Such effervescent tablets may be packaged, for example, in lightweight aluminum blisters. The tablet may be dissolved in water and administered, for example, orally, nasally, or by aerosol spray, whereby the droplets may enter via the mucous membranes of the bird.

[0036] The compositions and vaccines of the present invention may include, for example, water or a medium. Such compositions and vaccines may include one or more suitable pharma- ceutically acceptable carriers, stabilizers, preservatives, diluents, and / or buffers. Suitable stabilizers include, for example, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextrin, or glucose), or proteins (such as albumin or casein). Stabilizers are particularly advantageous when the dry vaccine preparation is prepared by lyophilization. Suitable preservatives include, for example, thimerosal, merthiolate, and gentamicin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol).

[0037] The composition or vaccine of the invention may also contain one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminum hydroxide, aluminum phosphate, aluminum oxide, vegetable oils, animal oils, oil-in-water or water-in-oil emulsions based on mineral oils such as Bayol F™ or Marcol 52™, complete Freund's adjuvant, incomplete Freund's adjuvant, or vegetable oils such as vitamin E acetate, and saponins.

[0038] The compositions or vaccines of the present invention may further comprise one or more immunogens derived from other pathogens that infect poultry, such as, for example, Mycoplasma gallisepticum (MG), Marek's disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), egg-laying drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV), poxvirus, reovirus, chicken parvovirus, and avian nephritis virus (including, but not limited to, ANV-1 and ANV-2).

[0039] The compositions and vaccines of the invention may be substantially pure. As used herein, "substantially pure" refers to material that is essentially free from naturally occurring macromolecules or other biological entities.

[0040] The compositions and vaccines of the present invention may be administered to any of a variety of avian species susceptible to Mycoplasma synoviae infection, including, but not limited to, poultry, galliformes, and exotic avian species. Galliformes include, but are not limited to, chickens, turkeys, grouse, quail, and pheasants. As used herein, poultry includes domesticated birds that are raised for the purpose of harvesting their eggs or killing for their meat and / or feathers. These are most typically members of the superorder Phasianidae (food birds), particularly the order Galliformes (including, for example, chickens, quail, turkeys, and grouse) and the family Anatidae (Anatidae), commonly known as "waterfowl" (including, for example, ducks, geese, and swans). Poultry may also include pigeons or dabs, or other birds that are killed for their meat, such as pheasants that are considered game birds. Chickens include, but are not limited to, hens, roosters, broilers, roasters, layers, breeders, progeny of breeding hens, and layer hens. As used herein, the term "susceptible" refers to the likelihood or reality of one or more pathological conditions indicative of an adverse response to a reference microorganism and / or Mycoplasma synoviae infection, e.g., reduced vigor or impaired growth, when compared to a susceptible individual or group.

[0041] The compositions and vaccines of the invention may be formulated for delivery by any of a variety of routes known in the veterinary art, including, for example, but not limited to, mucosal, intranasal, intraocular, or oral administration. The compositions and vaccines of the invention may be formulated for delivery to the respiratory mucosa and may be administered so that it comes into immediate or eventual contact with the respiratory mucosa of the bird. The compositions or vaccines of the invention may be administered by any suitable known method of inoculating poultry, including, but not limited to, intranasally, ophthalmically, by eye drop, by injection, in drinking water, in feed, by exposure, in ovo, maternally, by respiratory inhalation, and the like. When administered by injection, the immunogenic composition or vaccine may be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.

[0042] The composition or vaccine may be administered by mass administration techniques, for example, by placing the vaccine in drinking water, or by spraying or aerosolization. The composition may be administered by spraying a solution onto an individual or flock, and such aerosol delivery may include administration of the composition incorporated into small liquid particles. Such spray-type particles may have a droplet size within the range of about 10 to about 100 microns, more preferably about <1 to about 50 microns. For generation of small particles, conventional spray equipment and aerosol generators may be used, such as commercially available spray generators for knapsack sprays, hatchery sprays, and atomist sprays. Administration via drinking water may be carried out using conventional equipment.

[0043] The compositions or vaccines of the present invention may be administered to poultry before or after hatching. In ovo vaccination may occur, for example, at about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or any range therein. For in ovo delivery, the laying or breeding stock may be vaccinated, for example, at about 6-12 weeks of age and boosted at about 16-20 weeks of age. Such laying or breeding stocks may be vaccinated at about 6 weeks of age, about 7 weeks of age, about 8 weeks of age, about 9 weeks of age, about 10 weeks of age, about 11 weeks of age, or about 12 weeks of age. Also, in some embodiments, such laying or breeding stocks may be vaccinated within the first 2 weeks of age. Such laying or breeding stocks may be boosted at about 16 weeks of age, about 17 weeks of age, about 18 weeks of age, about 19 weeks of age, or about 20 weeks of age. Progeny of such spawning or breeding stocks may exhibit antibody titers against Mycoplasma synoviae, which may prevent or ameliorate symptoms of Mycoplasma synoviae infection in the progeny.

[0044] Poultry may receive the compositions or vaccines described herein at various ages. For post-hatch delivery, the material may be delivered at any suitable age, including but not limited to, about 1-3 days of age, about 1 week after hatching, about 2 weeks after hatching, about 3 weeks after hatching, about 4 weeks after hatching, about 5 weeks after hatching, about 6 weeks after hatching, or any range therein. Chickens may be vaccinated only once. Or, if a two-dose vaccine is used, the first vaccine is, for example, when the chickens are 3 days to 1 week old, followed by another 1-10 weeks later.

[0045] Multiple doses of the composition can be administered throughout the life of the chicken. Typically breeder chickens are vaccinated since maternal immunity is the primary source of protection for the broiler offspring, however, broiler chickens can be vaccinated if desired.

[0046] The compositions and vaccines of the invention can be formulated to contain a specified concentration of Mycoplasma synoviae. The organisms can be measured as color change units. Color change units of Mycoplasma synoviae, also referred to herein as "ccu", can be quantified using established standard techniques, including, for example, the protocols described in Rodwell and Whitcomb (In "Methods in Mycoplasmology," Eds. Razin and Tully, 1993). For example, a composition or vaccine can be formulated to contain about 50, about 100, about 1x10 2 ccu / ml, approx. 2.5x10 2 ccu / ml, approx. 5x10 2 ccu / ml, approx. 1x10 3 ccu / ml, approx. 2.5x10 3 ccu / ml, approx. 5x10 3 ccu / ml, approx. 1x10 4 ccu / ml, approx. 2.5x10 4 ccu / ml, approx. 5x10 4 ccu / ml, approx. 1x10 5 ccu / ml, approx. 2.5x10 5 ccu / ml, approx. 5x10 5 ccu / ml, approx. 1x10 6ccu / ml, approx. 2.5x10 6 ccu / ml, approx. 5x10 6 ccu / ml, approx. 1x10 7 ccu / ml, approx. 2.5x10 7 ccu / ml, approx. 5x10 7 ccu / ml, approx. 1x10 8 ccu / ml, approx. 2.5x10 8 ccu / ml, approx. 5x10 8 ccu / ml, approx. 1x10 9 ccu / ml, approx. 2.5x10 9 ccu / ml, or approximately 5 x 10 9 ccu / ml, or any range therebetween (e.g., about 1x10 5 ccu / ml ~ approx. 1x10 6 Concentrations of about 0.05 ml to about 0.1 ml (ccu / ml) may be used. In some applications, an effective amount may be administered as one drop per bird (per eye per bird). One drop may be about 0.05 ml to about 0.1 ml.

[0047] The Mycoplasma synoviae strains of the present invention may be administered to birds to reduce their susceptibility to Mycoplasma synoviae infection. Upon such administration, the material does not result in significant clinical signs or lesions indicative of Mycoplasma synoviae. It is therefore an object of the present invention to provide an immunological material that upon administration does not result in significant clinical signs or lesions indicative of MS disease. It is another object to provide an immunological material with low toxicity.

[0048] The present invention includes a method for generating an anti-MS immune response in poultry, comprising administering a Mycoplasma synoviae strain, composition, or vaccine described herein. In some embodiments, the immunity includes humoral immunity and / or cellular immunity. In the humoral response, anti-MS antibodies can be measured, for example, by serum plate agglutination test (SPA) test (e.g., using commercially available antigens (Charles River Laboratories International, Inc., Wilmington, MA)), hemagglutination inhibition test (HI) test (e.g., using antigens prepared from the WVU1853 strain), and enzyme-linked immunosorbent assay (ELISA) test (e.g., using commercially available kits (IDEXX, Westbrook, Maine)). SPA and HI testing procedures are described in detail by Ferguson-Noel et al. (Ferguson-Noel, N., and SH Kleven Mycoplasma species. In: A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens, Sixth ed. SM Williams, L. Dufour-Zavala, MW Jackwood, MD Lee, B. Lupiani, WM Reed, E. Spackman and PR Woolcock, eds. American Association of Avian Pathologists. pp63-70. 2016). In some embodiments, immunization comprises mucosal immunization.

[0049] Administration of an isolated Mycoplasma synoviae strain, composition, or vaccine described herein may result in reduction, inhibition, or prevention of one or more disease manifestations of challenge with further MS infection, including one or more disease manifestations of infectious MS. Such symptoms may include one or more of weight suppression, reduced egg production, mortality, clinical signs (e.g., upper respiratory tract infection, lameness, swollen joints, and / or ovarian involution), and / or histopathological indications (e.g., air sac lesions, tracheal lesions, and / or foot pad lesions). The present invention includes a method of reducing, inhibiting, or preventing MS infection in poultry, comprising administering an isolated Mycoplasma synoviae strain, composition, or vaccine described herein.

[0050] The present invention also provides kits comprising Mycoplasma synoviae K5885, and / or its progeny or derivatives, as described herein. The kits may include one or more containers filled with Mycoplasma synoviae of the present invention. Mycoplasma synoviae strain K5885 may be lyophilized. The kits may include additional separate containers of other strains of Mycoplasma synoviae or other pathogens of poultry. In addition, the kits may include other reagents, such as buffers, including solutions necessary to practice the present invention. Optionally associated with such container(s) may be notices or printed instructions. The kits of the present invention may include "packaging materials." As used herein, the term "packaging materials" refers to one or more physical structures used to contain the contents of the kit. The packaging materials are constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials may be solid matrices, or materials such as glass, plastic, paper, foil, and the like. Thus, for example, the package may be a glass or plastic vial used to contain a ccu quantity of Mycoplasma synoviae strain K5885.

[0051] Exemplary embodiments of the present invention include, but are not limited to, the following. 1. An isolated Mycoplasma synoviae strain, wherein the isolated Mycoplasma synoviae strain is the K5885 Mycoplasma synoviae strain deposited with the ATCC under patent designation PTA-127167, or a progeny or derivative thereof. 2. An essentially biologically pure culture of Mycoplasma synoviae strain K5885 deposited with ATCC under patent designation PTA-127167. 3. A composition comprising an isolated Mycoplasma synoviae according to embodiment 1 or 2. 4. The composition of embodiment 3, comprising water. 5. The composition of embodiment 3 or 4, comprising a pharma- ceutically acceptable carrier. 6. A composition according to any one of embodiments 3 to 5, comprising an adjuvant. 7. The composition of any one of embodiments 3-6, wherein the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. 8. The composition of any one of embodiments 3-7, wherein the composition is formulated for spraying or aerosolization. 9. A vaccine comprising an isolated Mycoplasma synoviae according to any one of embodiments 1 or 2, or a composition according to any one of embodiments 3 to 8. 10. The vaccine of embodiment 9, wherein the vaccine reduces one or more of the clinical signs induced by Mycoplasma synoviae infection in poultry. 11. The vaccine of embodiment 9 or 10, wherein the vaccine reduces the susceptibility of birds of the order Galliformes to disease induced by Mycoplasma synoviae. 12. A live vaccine for birds of the order Galliformes, the live vaccine comprising the K5885 Mycoplasma synoviae strain deposited with the ATCC under patent deposit designation PTA-127167, or a progeny or derivative thereof, in an amount sufficient to protect the bird against disease induced by Mycoplasma synoviae, and a pharmaceutically acceptable carrier. 13. The isolated Mycoplasma synoviae according to embodiment 1 or 2, the composition according to any one of embodiments 3 to 8, or the vaccine according to any one of embodiments 9 to 12, wherein the isolated Mycoplasma synoviae, composition, or vaccine is lyophilized, freeze-dried, frozen, or is an effervescent tablet. 14. A kit comprising an isolated Mycoplasma synoviae according to embodiment 1 or 2, a composition according to any one of embodiments 3-8, or a vaccine according to any one of embodiments 9-12, and printed instructions, wherein the contents of the kit are contained within a packaging material. 15. An effervescent tablet comprising an isolated Mycoplasma synoviae according to embodiment 1 or 2, a composition according to any one of embodiments 3 to 8, or a vaccine according to any one of embodiments 9 to 12. 16. A method for generating an immune response against Mycoplasma synoviae in an avian, the method comprising administering to the avian an isolated Mycoplasma synoviae as described in embodiment 1 or 2, a composition as described in any one of embodiments 3 to 8, or a vaccine as described in any one of embodiments 9 to 12. 17. A method for reducing the susceptibility of a bird to a disease induced by Mycoplasma synoviae, the method comprising administering to the bird an isolated Mycoplasma synoviae as described in embodiment 1 or 2, a composition as described in any one of embodiments 3 to 8, or a vaccine as described in any one of embodiments 9 to 12. 18. A method for protecting a bird against Mycoplasma synoviae, the method comprising administering to the bird an isolated Mycoplasma synoviae as described in embodiment 1 or 2, a composition as described in any one of embodiments 3 to 8, or a vaccine as described in any one of embodiments 9 to 12. 19. A method for reducing one or more clinical signs induced by Mycoplasma synoviae infection in a bird, the method comprising administering to the bird an effective amount of an isolated Mycoplasma synoviae as described in embodiment 1 or 2, a composition as described in any one of embodiments 3 to 8, or a vaccine as described in any one of embodiments 9 to 12. 20. The method of embodiment 19, wherein the one or more clinical signs comprise weight suppression, reduced egg production, mortality, upper respiratory tract infection, lameness, swollen joints, ovarian involution, air sac lesions, tracheal lesions, foot pad lesions, and / or eggshell tip abnormalities. 21. The method of any one of embodiments 16-20, wherein administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo. 22. The method of any one of embodiments 16-20, wherein administration is by eye drops, by aerosol, or by drinking water. 23. The method of any one of embodiments 16-22, wherein the bird comprises a bird of the order Galliformes. 24. The method of any one of embodiments 16-23, wherein the bird comprises a chicken or a turkey.

[0052] The present invention is illustrated by the following examples. It is to be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. EXAMPLES

[0053] Example 1 Evaluation of a Potential Live Mycoplasma synoviae Vaccine in Chickens This example identifies a live Mycoplasma synoviae (MS) vaccine candidate and presents a safety and efficacy study of the vaccine candidate. This example summarizes data from five animal trials (see Table 1). An MS challenge model was also developed. [Table 1]

[0054] Mycoplasma synoviae (MS) vaccine candidates were evaluated in five experiments for safety and efficacy. In trials 1 and 2, the candidates were evaluated for safety and efficacy and it was clear that the challenge strategy resulted in very mild lesions and therefore required improvement, but two of the candidates (K5885A and K4971B) showed promise as safe and potentially effective vaccines as they resulted in mild lesions after inoculation of naive birds and a significant reduction in colonization with the challenge strain (P<0.05). In trials 3 and 4, vaccine candidate K5885A was further evaluated for safety and efficacy using an approach that resulted in more severe MS challenge and lesions. K5885A inoculation (at 1000× the dose of the positive control strain) resulted in reduced air sac lesions (and reduced severity) and significantly reduced footpad lesions (P<0.05). There were also significantly fewer air sac and footpad lesions in birds vaccinated with K5885A after virulent challenge compared to non-vaccinated controls (P<0.05). In the final trial (trial 5), the efficacy of K5885A was compared to the MS vaccine strain K3928 (MS-H) and, although protection from air sac lesions was comparable with both vaccines, K5885A vaccination also significantly reduced footpad lesions, unlike K3928 / MS-H (P<0.05). Although further studies are needed, these preliminary studies indicate that K5885 is likely to be a safe and highly effective vaccine.

[0055] Materials and Methods MS Strains and Isolates. Vaccine candidates were selected from MS field isolates in the Mycoplasma Culture Depository at PDRC based on cases in which clinical disease was absent. K5885A was isolated from a broiler breeder chicken in Arkansas in 2006, K4971B was isolated from a commercial layer chicken in Georgia in 2000, and K5805A was isolated from a broiler breeder chicken in Alabama in 2005. The laboratory strain K1968 is a virulent MS strain that has been previously characterized (Lockaby et al., 1998, Vet Pathol;35:178-190) and was used as a control to evaluate the safety of the vaccine candidate strain in trial 1 and as a challenge strain to evaluate the efficacy of the vaccine candidate in trial 2. In subsequent trials, isolate K6677 was used as a positive control / challenge strain. K6677 was isolated from a broiler chicken in Georgia in 2014 and is a remarkably virulent MS strain. In trial 5, the efficacy of the selected vaccine candidate (K5885A) was compared to the MS- H vaccine strain (K3928 in the PDRC repository). MS-H has been characterized previously (Morrow et al., 1998, Avian Diseases; 42:667-670; Markham et al., 1998, Avian Diseases; 42:677-681; Markham et al., 1998, Avian Diseases; 42:671-676; Markham et al., 1998, Avian Diseases; 42:682-689, and Noormohammadi et al., 2007, Avian Diseases; 51:550-554).

[0056] Inoculation and challenge procedures. For aerosol inoculation / challenge, MS isolates were administered using a commercial paint sprayer (Preval® Sprayer Division, Precision Valve Corporation, Yonkers, NY). Approximately 1 ml of actively growing culture was sprayed per bird. For eye instillation, intra-air sac, intra-footpad, and intratracheal inoculations of 100 μl of isolate were administered at the site. Isolates were titrated (CCU / ml) at the time of inoculation using previously described methods (Rodwell, AW, and RF Whitcomb. Methods of Direct and Indirect Measurement of Mycoplasma Growth. In: Methods in Mycoplasmology. Volume I, Mycoplasma Characterization. S. Razin and JG Tully, eds. Academic Press, New York. pp 85-196. 1983). Isolate titers and inoculated doses for all trials are summarized in Table 2. [Table 2]

[0057] Serology. Sera in all trials were tested for the presence of MS antibodies using the serum plate agglutination (SPA) test with commercially available antigen (Charles River Laboratories International, Inc., Wilmington, MA), the hemagglutination inhibition (HI) test with antigen prepared from the WVU1853 strain with a commercially available kit (IDEXX, Westbrook, Maine), and the enzyme-linked immunosorbent assay (ELISA) test.

[0058] SPA and HI test procedures were as described by Ferguson-Noel et al. (Ferguson-Noel, N., and SH Kleven Mycoplasma species. In: A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens, Sixth ed. SM Williams, L. Dufour-Zavala, MW Jackwood, MD Lee, B. Lupiani, WM Reed, E. Spackman and PR Woolcock, eds. American Association of Avian Pathologists. pp63-70. 2016). SPA scores >1 were considered positive. HI titers >1:20 were considered positive. For ELISA tests, a geometric mean sample / positive (S / P) ratio of >0.5 was considered positive.

[0059] Isolation and identification of mycoplasmas. Cotton swabs from the trachea, palate, and air sac were used for culture. They were inoculated into Frey's modified broth and agar and incubated at 37°C. Mycoplasma isolates were identified using direct immunofluorescence (10).

[0060] Real-time quantitative PCR. Real-time quantitative PCR (qPCR) was performed using the procedure described by Raviv (Raviv and Kleven, 2009, Avian Diseases; 53:103-107). At necropsy, the larynx of each bird was collected in 4 ml of sterile PBS. Genomic DNA was extracted from 100 μl of laryngeal washing or cleft palate swab using Mag-Bind® Blood and Tissue DNA HDQ 96 kits (Omega Bio-tek, Inc., Norcross, GA) on MagMAX™ Express-96 Magnetic Particle Processors (Thermo Fisher Scientific) according to the manufacturer's recommendations. Real-time PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) and a cycle threshold (Ct) of ≦39 was considered positive. To make the assay quantitative, a plasmid containing a genomic target (16S-23S rDNA ISR) was constructed as a standard DNA control. The procedures used for constructing DNA controls and standard curves for quantification have been described in detail elsewhere (Raviv et al., 2008, Vet Microbiol;129:179-187).

[0061] Assessment of Lesions. Gross air sac footpad lesions were scored on a scale of 0 to 4 using the scoring system described by Kleven (Kleven et al., 1972, Avian Diseases; 16:915-924, and Kleven et al., 1975, Avian Diseases; 19:126-135). Tracheal lesions were assessed microscopically by measuring the width of the tracheal mucosa. A portion of the upper third of the trachea (approximately 1 inch distal to the larynx) was fixed in 10% neutral formalin. The thickness of the tracheal mucosa was measured at four equally spaced points on histological slides of cross sections of the trachea (Whithear, 1996, Rev Sci Tech; 15:1527-15).

[0062] Statistical analysis. Air sac lesion score, footpad lesion score, and SPA score were analyzed using the Kruskal-Wallis rank sum test. Tracheal mucosal thickness, mean copy number (MCN) Log 10 , s / p ratio, and HI titer Log 10 were analyzed using the Tukey-Kramer HSD test. JMP® Statistics Made Visual (SAS Institute Inc., SAS Campus Drive, Cary, NC 27513) software. A P value of <0.05 was considered significant.

[0063] Safety and Efficacy The aim of this part of the study was preliminary screening of vaccine candidates selected for evidence of safety in naive broiler chickens (Study 1) and efficacy in protecting against tracheal and air sac lesions following challenge with a virulent MS isolate (K1968) (Study 2).

[0064] Experimental design Clinical Trials (Safety I). For trials 1 and 2, 110-day-old commercial broiler-type chickens were obtained from a source known to be free of MS and Mycoplasma gallisepticum (MG). They were housed in naturally ventilated, side-curtained chicken houses in floor pens (1.5 x 3 m) with pine shavings. 2 At 14 days of age, they were randomly divided into five experimental groups and housed in five colony houses (3×3 m) with concrete floors and pine shavings. 2 At this point, 10 chickens were randomly selected (2 per group) and tested by SPA, HI, ELISA, and cleft palate swabs were tested by culture and PCR to confirm that they were Mycoplasma negative.

[0065] At 21 days of age, three groups of 25 birds each were administered K5885A (2.9 × 10 7 CCU / ml), K4971B (3.6 × 10 7 CCU / ml), or K5805A (1.9 × 10 8A fourth group of 15 birds was inoculated (via aerosol) with a broth culture of K1968 (1.9 × 10 8 CCU / ml) (control). A negative control group of 10 chickens remained uninfected.

[0066] At 10 days post infection (DPI), 10 birds from each of the MS-inoculated groups and 5 birds from the negative control groups were euthanized and evaluated by air sac and lesion scoring. Samples were also collected for serology (SPA, HI, and ELISA), mycoplasma culture (palate / tracheal and air sac swabs), and histopathology (tracheal sections). At 14 DPI, 5 birds from the MS-inoculated groups were euthanized and evaluated as described.

[0067] Trial 2 (Efficacy I). Six weeks after infection with the vaccine candidate, the remaining vaccinated birds from Trial 1 were challenged with K1968 (4.7 × 10 6 CCU / ml) of broth culture was inoculated (via aerosol). Five birds were non-vaccinated controls and five birds were negative controls (unchallenged). At 10 days post-combination (DPC), all birds were necropsied and evaluated as described above.

[0068] Results and Discussion Trial 1. Safety I. Groups challenged with K5885A and K5805A showed signs of seroconversion at 10 days post-challenge (DPC), with 90% (9 / 10 birds) and 100% (10 / 10 birds), respectively, responding to the SPA test. Lesions associated with MS infection in this trial were not severe, and even infection with the positive control K1968, a pathogenic MS strain, did not result in severe air sac or tracheal lesions. As a result (combined with the low number of birds in this pilot study), there were few significant differences in lesions associated with MS infection between groups (P<0.05).

[0069] However, one of the MS candidates, K5805A, produced air sac lesions that were more severe than K1968 and significantly higher than K5885A and K4971B at 10 DPI (P<0.05). With regard to tracheal lesions, there was no significant difference in mean tracheal mucosal thickness between the MS-vaccinated groups, but the mean measurement in the K1968 group was significantly higher than the negative control (P<0.05). Candidate K5885A produced minimal increases in tracheal mucosal thickness at 10 and 14 DPI in the MS-infected groups. K5805A was eliminated from future studies due to air sac lesions and lack of indications that the strain would produce a safe vaccine. These results are summarized in Table 3 and Figures 1 and 2. [Table 3]

[0070] Trial 2. Efficacy I. Safety As in the trial, there were no severe lesions associated with MS infection after challenge with K1968 by aerosol. There were no significant differences between groups (vaccinated, or negative or positive control) in terms of air sac or tracheal lesions, so few conclusions can be drawn from the data regarding efficacy (P<0.05). However, there were significantly fewer MS isolations from the air sacs in the vaccinated group, indicating that vaccination may have prevented systemic infection with the challenge strain (P<0.05). These results are summarized in Table 4. [Table 4]

[0071] Following this trial, a recently isolated positive control strain (K6677) was used to optimize the challenge method to produce more severe lesions. Changes in inoculation method (intratracheal, intraairbag, and intrafootpad) also increased the severity of challenge. Respiratory virus vaccines routinely used in poultry production (Newcastle Disease Virus (NDV) and Infectious Bronchitis Virus (IBV)) were also included. K4971B was excluded from future trials due to the difficulty of growing this strain in vitro.

[0072] Safety II and Efficacy II The objectives of this part of the study were to investigate the safety of selected vaccine candidates following high-dose inoculation by several invasive routes and co-administration of a respiratory virus vaccine (trial 3). Protection of vaccinated birds from tracheal, air sac and footpad lesions following challenge with a virulent MS isolate (K6677) was investigated (trial 4).

[0073] Experimental design Trial 3 (Safety II). For trials 3 and 4, 100-day-old commercial broiler-type chickens were obtained from a source known to be free of MS and MG. They were housed in naturally ventilated, side-curtained chicken houses in floor pens (1.5 x 3 m) with pine shavings. 2 At 21 days of age, they were randomly divided into 6 experimental groups and transferred to 6 floor pens. At this time, 20 chickens were randomly selected and tested by MG and MS SPA, HI, ELISA, and cleft palate swabs were tested by culture and PCR to confirm that they were negative for Mycoplasma, NDV and IBV.

[0074] Thirty birds were also administered the vaccine candidate K5885A (2.8 × 10 8 CCU / ml) (via intratracheal, intrafootpad, and intraair sac routes) and NDV vaccine (B1B1, via eye drop route). Fifteen birds were inoculated with K6677 (1.4x10 5 CCU / ml) and NDV vaccine (positive control), and 10 birds were inoculated with NDV alone, and an additional 10 birds were not inoculated to serve as negative controls.

[0075] At 14 DPI, 15 birds from each of the MS inoculated groups and 10 birds from each of the NDV only and negative control groups were euthanized and evaluated by air sac and lesion scoring. Samples were also collected for serology (SPA, HI, and ELISA), mycoplasma culture (air sac swabs), histopathology (air sac swabs), tracheal histopathology (tracheal segments), and quantitative real-time PCR (tracheal segments).

[0076] Trial 4 (Efficacy II). Six weeks after infection with the vaccine candidate, 10 birds remaining from Trial 1 were selected and serologically tested by PCR. At this time, K5885A vaccinated chickens (n=17) were also immunized with K6677 (3.6×10 6 CCU / ml) and a broth culture of the IBV vaccine (via aerosol). Eighteen birds were non-vaccinated controls, eleven birds were vaccinated with IBV only, and nine birds were negative controls (not challenged or vaccinated). At 14 DPC, all birds were necropsied and evaluated as described above.

[0077] Results and Discussion Trial 3. Safety II. There was evidence of seroconversion 14 days after infection with both K5885A and K6677 in SPA and ELISA tests (see Table 5). The challenge in this trial was much stronger as birds were infected via intratracheal, intraair sac, and intrafootpad inoculation. The challenge resulted in air sac lesions in both MS-infected groups, but fewer birds in the K5885A group developed disease (73% (11 of 15) vs. 40% (6 of 15). Also, significantly fewer birds developed footpad lesions (P<0.05). The titers of K5885A were much higher than those of the control strain (2.8×10 for K5885A). 8 CCU / ml, 1.4 × 10 for K6677 5It should be noted that the mean mean seroconversion rate (CCU / ml) was 1.27 and 1.28, respectively. Comparison of tracheal mucosa measurements is pending. Isolate K5885A was detected in all inoculated birds and replicated at comparable levels to the positive control K6677, as shown by qPCR and MS isolation. These results are summarized in Table 6 and Figures 3 and 4.

[0078] Trial 4. Efficacy II. At 6 weeks post-vaccination (at challenge), serological and PCR results showed that non-vaccinated birds remained negative for MS. The vaccinated group seroconverted all serological tests and they were also MS PCR positive at that time point. At 14 DPC, seroconversion was similar in both vaccinated and non-vaccinated groups, but the mean ELISA s / p ratio was significantly lower in the vaccinated group (see Table 7) (P<0.05). Despite the strong challenge with K6677, there was significantly less air sac inflammation and foot pad lesions in birds vaccinated with K5885A (P<0.05). There was also significantly less isolation of MS from the air sacs and lower MCNlog10 in the trachea, indicating less replication and systemic infection with the MS challenge strain (despite direct inoculation of these sites) (P<0.05). These results are summarized in Table 8 and Figures 5 and 6. [Table 5] [Table 6] [Table 7] [Table 8]

[0079] Efficacy III The objective of this part of the study was to compare the efficacy of the selected vaccine candidate with a commercial MS vaccine strain (MS-H) in terms of tracheal, air sac and footpad lesions after challenge with a virulent MS isolate (K6677). The dose and route of vaccination were compared with field MS vaccination practice when the vaccine was administered via eye drop at 3 weeks of age.

[0080] Experimental Design: 100-day-old commercial broiler-type chickens were obtained from a source known to be free of MS and MG. They were housed in a colony house (3 x 3 m) with a concrete floor and pine shavings. 2 At 21 days of age, they were randomly divided into five experimental groups and housed in five new colony houses (3 x 3 m) with concrete floors and pine shavings. 2 At this time, 15 chickens were randomly selected and tested by SPA and HI, and cleft palate swabs were tested by culture and PCR to confirm that they were negative for mycoplasma and NDV. Also, at 21 days of age, 20 birds were inoculated with vaccine candidate K5885A (1.6x10 7 CCU / ml) and 20 birds were inoculated with the MS-H vaccine strain (5.0x10 5 CCU / ml) (both via eye drops).

[0081] Five weeks after vaccination, birds were vaccinated with K6677 (8.0x10 7 CCU / ml) and NDV vaccine (B1B1) via eye drop. Twenty naive birds were vaccinated with NDV vaccine only and 20 birds remained uninfected as negative controls. One week after aerosol challenge, the challenged group was also vaccinated with K6677 (4.2 10 7 At 14 DPC, birds were euthanized and evaluated as previously described.

[0082] Results and Discussion In this trial, seroconversion after challenge was similar in both vaccinated groups, but there were slightly lower titers in the HI test and significantly lower titers in the ELSIA test in the group vaccinated with K5885A (see Table 9) (P<0.05). In this trial, there was significantly less air sac inflammation in birds vaccinated with K5885A and MS-H (P<0.05), however, only vaccination with K5885A resulted in a significant reduction in footpad lesions (P<0.05). There were significantly fewer MS isolated from air sacs from birds vaccinated with K5885A or MS-H attenuated, but only MS-H had less MS replication in the trachea with significantly lower MCNlog10 (P<0.05). These results are summarized in Table 10 and Figures 7 and 8. [Table 9] [Table 10]

[0083] The titer of the MS-H vaccine was lower than that of the K5885A vaccine (5.0x10 against the MS-H strain). 5 For CCU / ml, 1.6x10 for K5885A 7 CCU / ml), it should be noted that commercial vaccines were not used in this study, and higher titers as well as commercial products may affect efficacy. Strain-specific PCR protocols (currently under development) will allow for more useful analysis of these data, as they will be able to distinguish replicating and isolating vaccine strains compared to challenge strains.

[0084] This example shows that K5885A is a low virulence MS isolate and vaccination with this isolate confers protection against MS-related disease and subsequent MS infection with some challenge strains, and is therefore a safe and effective vaccine candidate.

[0085] Example 2 Mycoplasma synoviae Challenge Model to Assess the Impact of Vaccination on Fallopian Tube Colonization To evaluate the egg-laying efficacy of the Mycoplasma synoviae vaccine candidate in layer-type chickens, groups of chickens were vaccinated with 5.5 CCU log10 of K5885 by eye drop at 5 weeks of age and then challenged with two virulent Mycoplasma synoviae strains (K6677 and WVU1853) by aerosol and footpad inoculation on 2 consecutive days at 23 weeks of age. Groups were euthanized and examined for air sacitis, footpad lesions, and ovarian involution at 2, 3, 4, 5, and 6 WPC. Eggshell strength and egg abnormalities were recorded, and eggs were cultured for Mycoplasma synoviae before and after challenge. Vaccination with K5885 provided significant protection from air sacitis, footpad lesions (synovitis), and ovarian involution following challenge with either K6677 or WVU1853 compared to challenged non-vaccinated groups (P<0.05). There was also a significant reduction in K6677 colonization (as indicated by qPCR mean (genomic) copy number log10 from trachea) in vaccinated groups compared to non-vaccinated controls (P<0.05). A significant reduction in egg production was observed 2-5 weeks after challenge in non-vaccinated groups challenged with either K6677 or WVU1853, but not in vaccinated groups (P<0.05). There were no significant differences in eggshell strength, egg abnormalities, oviduct colonization, or isolation of Mycoplasma synoviae from eggs between groups during the study. These results indicate the potential of the K5885 vaccine candidate to prevent clinical signs and egg production losses associated with Mycoplasma synoviae.

[0086] Introduction Mycoplasma synoviae (MS) infection is an important poultry disease that can cause tracheitis, air sacitis, synovitis, and several negative reproductive effects including eggshell apex abnormalities and reduced egg production in poultry, although subclinical infection of the respiratory tract appears to predominate (Feberwee et al., 2009, Avian Pathology; 38(2): 187-187; Gole et al., 2012, Preventive Veterinary Medicine; 106(1): 75-78; Kleven et al., 1975, Avian Dis; 19(1): 126-135, and Landman et al., 2004, Avian Pathology; 33(2): 210-215). The clinical manifestations of MS infection are related to the MS strain, with MS isolates from air sac lesions inducing air sacitis, isolates from joint lesions inducing joint pathology, and more recently the emergence of a Dutch strain causing eggshell apex anomalies (EAA) and low egg production encountered worldwide (Feberwee et al., 2009, Avian Pathology; 38(2): 187-187; Ferguson-Noel et al., 2013, Mycoplasma synoviae infection. In DE Swayne, J R Glisson, L R McDougald, L K Nolan, DL Suarez, & V. Nair (Eds.), Diseases of Poultry (pp. 900-906). Wiley-Blackwell; and Landman, 2014, Avian Pathology; 43(1): 2-8). EAA is characterized by changes to the shell surface, thinning of the shell, and cracks and breaks limited to an area up to about 2 cm from the apex of the egg (Feberwee et al., 2009, Avian Pathology; 38(2):187-187).The pathogenic process of MS infection involves attachment and colonization of the upper respiratory tract and eventually of the air sacs (leading to air sacitis), followed by spread to the joints via the hematogenous route after colonization of the respiratory tract, and also colonization of the fallopian tubes, which has been hypothesized as a prerequisite for the induction of eggshell abnormalities (Feberwee et al., 2009, Avian Pathology; 38(2): 187-187; Ferguson-Noel et al., 2013, Mycoplasma synoviae infection. In DE Swayne, J R Glisson, L R McDougald, L K Nolan, DL Suarez, & V. Nair (Eds.), Diseases of Poultry (pp. 900-906). Wiley-Blackwell; and Kawakubo et al., 1980, J Comp Pathol; 90: 457-467).

[0087] Transmission of MS can occur vertically, in eggs, or horizontally by direct contact or airborne transmission, and vaccination has significantly reduced shedding and horizontal transmission (Jones et al., 2006, Avian Dis; 50(1):88-91; and Noormohammadi et al., 2007, Avian Dis; 51(2):550-554). A temperature-sensitive live MS vaccine strain, MS-H, selected by mutagenesis of a virulent wild-type strain from Australia (Morrow et al., 1998, Avian Dis; 42(4):667-670), is used in many countries to prevent Mycoplasma synoviae infections. Its safety and efficacy in reducing air sac inflammation has been established under laboratory and field conditions (Markham et al., 1998, Avian Dis; 42(4): 677-681; Markham et al., 1998, Avian Dis; 42(4): 671-676; and Markham et al., 1998, Avian Dis; 42(4): 682-689), but there is no documented efficacy in preventing MS footpad lesions (synovitis). Genomic analysis of MS-H reisolates recovered from vaccinated herds showed the presence of several mutations similar to the parental strain sequence, indicating that MS-H strains revert to the parental strain sequence (Kordafshari et al., 2020, Avian Pathology; 49(3): 275-285, and Kordafshari et al., 2019, Veterinary Microbiology; 231: 48-55). Concomitant reversion in ObgE, OppF, and GapdH proteins was associated with higher gross air sac lesion scores (and increased microscopic upper tracheal mucosal thickness in chickens directly inoculated with an MS-H reisolate from the field following intratracheal inoculation of a virulent strain of infectious bronchitis virus) (Klose et al., 2022, Front Microbiol;13:1042212).

[0088] Described herein is a new MS vaccine (K5885), isolated in 2006 from broiler breeder chickens in Arkansas, that significantly reduces challenge MS burden in the upper respiratory tract and prevents air sac inflammation and foot pad lesions associated with MS infection.

[0089] the purpose The primary objective of this study was to develop a Mycoplasma synoviae challenge model to evaluate the impact of the K5885 vaccine candidate on oviduct colonization by two virulent challenge strains (K6677 and WVU1853). Specific objectives included trials of birds to compare air sac lesions, footpad lesions, cleft palate, and challenge strain load in tracheal washings, egg laying rates, and egg abnormalities in chickens vaccinated with K5885 and unvaccinated chickens prior to challenge with two MS wild-type strains (K6677 and WVU1853).

[0090] Materials and Methods MS isolates. The vaccine candidate (K5885A) was isolated from a broiler breeder chicken in Arkansas in 2006 and selected from the mycoplasma culture depository at the Poultry Diagnostic and Research Center (PDRC) at the University of Georgia, Athens. Isolates K6677 and WVU1853 were used as positive control / challenge strains. K6677 was isolated from a broiler chicken in Georgia in 2014 and is a significantly virulent strain, while WVU1853 is a Mycoplasma synoviae reference strain isolated in the United States (Zhu et al., 2018).

[0091] Vaccination and challenge procedures. Each vaccinated chicken received a dose of 5.5 CCU log10 in 30 μl placed in the left eye. For aerosol inoculations, MS isolates (K6677 and WVU1853) were administered using a commercial paint sprayer (PREVAL® Sprayer Division, Precision Valve Corporation, Yonkers, NY). Approximately 1 ml of actively growing culture was sprayed per chicken. For intra-footpad inoculations, 100 μl of isolate was administered at the site. Isolates were titrated (CCU / ml) at the time of inoculation using the method previously described by Rodwell and Whitcomb, 1983. Titers of MS isolates and doses inoculated for the experiments are summarized in Table 11.

[0092] Lesion assessment. Gross air sac and footpad lesions were scored on a scale of 0 to 4 using a previously described scoring system (Kleven et al., 1975, Avian Dis; 19(1):126-135). Ovarian involution was assessed by macroscopic examination and ovaries were scored as immature (juvenile), normal (multiple follicles at various stages of development) or involuted (multiple atretic follicles that were atonic and / or discoloured) (Ferguson-Noel et al., 2012, Avian Dis: 56(2):272-275).

[0093] Egg strength measurements. Eggs were collected 2 weeks before and 6 weeks after challenge for egg strength testing. Egg strength was determined by the shell-break method using an Egg force reader (Orka Technology Ltd, Manchester, England).

[0094] Serology. Sera were tested for the presence of MS antibodies by serum plate agglutination (SPA) tests using commercially available antigens (Charles River Laboratories International, Inc., Wilmington, MA), hemagglutination inhibition (HI) tests using antigens prepared from the WVU18531853 strain, and enzyme-linked immunosorbent assay (ELISA) tests using commercially available kits (IDEXX, Westbrook, Maine). SPA and HI test procedures were as described by Ferguson-Noel et al. 2016. An SPA score >1 was considered positive. An HI titer of >1:20 was considered positive. For ELISA tests, a geometric mean sample / positive (S / P) ratio of >0.5 was considered positive.

[0095] Isolation and identification of mycoplasma. Egg yolk, cleft palate swabs and oviduct swabs were used for culture. Eggs were collected and incubated every other day for 6 days, starting 2 days after challenge until 6WPC. Yolks were pooled (3 in 1) from incubated eggs for MS culture. Swabs and egg yolks were inoculated into Frey's modified broth and agar and incubated at 37°C. Mycoplasma isolates were identified using direct immunofluorescence (Ferguson-Noel et al. 2016).

[0096] MS real-time quantitative PCR. Real-time quantitative PCR (qPCR) was performed using the procedure described by Raviv and Kleven, 2009. At necropsy, a portion of the upper trachea of ​​each chicken was collected in 9 ml of sterile PBS as well as an oviduct swab. Genomic DNA was extracted from 200 μl of upper tracheal wash or oviduct swab using MAG-BIND® Blood and Tissue DNA HDQ 96 kit (Omega Bio-tek, Inc., Norcross, GA) on MagMAX™ Express-96 Magnetic Particle Processors (Thermo Fisher Scientific) according to the manufacturer's recommendations. Real-time PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) and a cycle threshold (Ct) ≦39 was considered positive. To make the assay quantitative, a plasmid containing a genomic target (16S-23S rDNA ISR) was constructed as a standard DNA control. The procedures used for the construction of DNA controls and standard curves for quantification have been described in detail elsewhere (Raviv et al., 2008, Vet Microbiol; 129(1-2):179-187).

[0097] Strain-specific real-time PCR. Comparative genomics of the three MS strains in this study (K5885 (vaccine candidate) and K6677 (challenge strain)) was used to identify targets and develop quantitative PCR protocols for the specific detection of these strains. Strain-specific real-time PCR protocols were performed on samples prepared as described above using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific), and a cycle threshold (Ct) of ≤ 39 was considered positive. To make the assay quantitative, plasmids containing specific genomic targets were constructed as standard DNA controls, as previously described (Raviv et al., 2008, Vet Microbiol; 129(1-2): 179-187).

[0098] Chickens and Experimental Design. Three hundred and eighteen specific pathogen free (SPF) chicks were obtained from Charles River SPAFAS (Wilmington, DE) at 4 weeks of age (WOA). Chicks were randomly selected and placed into six groups: unvaccinated unchallenged (NV / NC), unvaccinated K6677 challenged (NV / CK), unvaccinated WVU1853 challenged (NV / CW), vaccinated K6677 challenged (V / CK), vaccinated WVU1853 challenged (V / CW), and vaccinated unchallenged (V / NC). Sixty chicks were randomly selected and placed into each of the NV / NC, NV / CK, NV / CW, V / CK, and V / CW groups, and 18 chicks were placed into the V / NC group. Chickens were housed in floor pens with pine shavings in a naturally ventilated, side-curtained chicken house. At 5 weeks of age (WOA), 30 chickens were screened to confirm that they were negative for avian Mycoplasma species by serology (SPA, HI, and ELISA), and culture of palate swabs, and qPCR of tracheal washes. At 6WOA, all chicks in V / CK, V / CW, and V / NC groups were vaccinated with K5885 (MS vaccine candidate) via the intraocular route. At 4 weeks post-vaccination (WPV) and 15WPV, 30 chickens (15 non-vaccinated and 15 vaccinated) were randomly selected to confirm vaccination and negative status. At 2 weeks pre-challenge and 3-6 weeks post-challenge (WPC), all eggs were collected every other day for eggshell strength testing. At 23 WOA, when egg production was approximately 80% in all groups, hens were challenged on two consecutive days (aerosol inoculation on the first day only, aerosol and intra-footpad inoculation on the second day). Chickens in NV / CK and V / CK groups were challenged with K6677, and hens in NV / CW and V / CW groups were challenged with WVU1853 (challenge doses per group are summarized in Table 11). At 2, 3, 4, 5, and 6 WPC, 12 hens from all groups except V / NC were randomly selected and euthanized. In the V / NC group, 6 hens were selected and euthanized at 2, 4, and 6 WPC.All eggs were collected every other day from 2 days after challenge until the end of the study and incubated for 6 days before being cultured for MS.

[0099] The experimental design and procedures are summarized in Table 12. Chickens were provided with food and water ad libitum throughout the study and were euthanized by cervical dislocation and carbon dioxide in accordance with the animal care and use policy of the University of Georgia, Athens, GA.

[0100] Statistical analysis. Air sac lesion score, footpad lesion score, HI titer Log10, ELISA s / p ratio, SPA mean aggregation grade, and eggshell strength were analyzed using two-way ANOVA with multiple comparison test in turkeys. For the number of ovarian regressions observed per group, a paired T-test was used to compare values ​​between two different groups. Percentage of weekly egg production and MS mean copy number (MCN) tracheal washes and oviduct swabs were analyzed using two-way ANOVA (turkey multiple comparison test), and tracheal K6677 mean copy number (MCN) was analyzed using two-way ANOVA (Sidak's multiple comparison test). All statistical analyses were performed using GraphPad Prism version 9.5.1 for macOS®, GraphPad Software, www.graphpad.com. P values ​​<0.05 were considered significant.

[0101] result Pre-vaccination and pre-challenge testing. Chickens tested prior to MS and MG vaccination (5 WOA) were negative by SPA, HI, and culture using ELISA, qPCR, and serology. At 10 WOA (4 weeks after vaccination and prior to challenge), all samples from the non-vaccinated group were negative, while samples from the vaccinated group were all MS positive by SPA, ELISA, culture, and PCR (4.0±0.4 MCNlog10). Only one sample (6.7%) from the vaccinated group was positive by HI at that time point with a titer of 1.30 log10. At 21 WOA (15 weeks after vaccination and prior to challenge), chickens from the vaccinated group were MS positive by ELISA, 15 positive by SPA, 17 positive by HI, and 18 positive by both culture and PCR (3.19±1.74 MCNlog10) (see Table 13 for summary of serological results).

[0102] Serology. Chickens tested prior to vaccination (5 WOA) for MS and MG were negative for antibodies using SPA, HI, and ELISA. At 10 WOA (4 WPV), samples from the non-vaccinated (NV) group were all negative, samples from the vaccinated group were all MS positive by SPA and ELISA, but only one (6.7%) of the samples from the vaccinated group was positive by HI at that time point, with a titer of 1.30. At 21 WOA (15 WPV), chickens from the vaccinated group were MS positive by ELISA (20 / 20), SPA (15 / 20), and HI (17 / 20). Serum samples from the non-vaccinated group were all negative.

[0103] At 4WPC, all chickens in the negative control group (NV / NC) were negative for MS antibodies. Although the vaccinated only group (V / NC) had the lowest ELISA titers, there were no significant differences between the other groups in the number of chickens and hens that tested positive for MS by SPA, HI, and ELISA.

[0104] At 6WPC (29WOA), serological results were similar to those observed at 4WPC, as the negative control (NV / NC) remained negative in all serological tests and there were no significant differences in the number and titers of MS positives among the other groups. Fewer chickens (2 / 6 and 3 / 6, respectively) were MS positive by SPA in the vaccinated WVU1853 challenged (V / CW) and K6677 challenged (NV / CK) groups, but the WVU1853 challenged (NV / CW and V / CW) groups had the lowest SPA and ELISA titers. Serological results are summarized in Table 13, see Table 13.

[0105] Air sac lesion scores. The severity of air sac lesions was highest in the unvaccinated group challenged with K6677 (NV / CK) group at 2 WPC, but the severity of the lesion score in this group progressively decreased to zero at 6 WPC. Air sacitis was significantly higher at 2 WPC, but not at 3, 4, 5, or 6 WPC, compared with the unvaccinated group challenged with WVU1853 (NV / CW). Also, the severity of air sacitis was significantly higher in the unvaccinated groups challenged with K6677 and WVU1853 (NV / CK and NV / CW) compared with the vaccinated and challenged groups (V / CK and V / CW) at 2 and 3 WPC. None of the three groups vaccinated with K5885 (V / CK, V / CW, and V / NC) developed air sacitis (Figure 9).

[0106] Footpad lesions. The number of chickens with footpad lesions as well as the severity of footpad lesions were significantly higher in the unvaccinated groups challenged with K6677 and WVU1853 (NV / CK and NV / CW) compared to the three K5885 vaccinated groups (V / NC, V / CK and V / CW) at 2, 3 and 4 WPC (P<0.05). There was no significant difference in the number of chickens with footpad lesions or the severity of lesions caused by K6677 compared to WVU1853 in the unvaccinated groups (NV / CK and NV / CW) (P<0.05) (Figure 10).

[0107] Ovarian regression. Ovarian regression was not observed in any of the vaccinated groups, including the negative control group or groups challenged with K6677 or WVU1853 (V / NC, V / CK, V / CW). The rate of ovarian regression was higher in K6677-challenged chickens than in WVU1853-challenged chickens, but the difference was not statistically significant (P<0.05) (Figure 11).

[0108] MS isolation. As shown in Tables 16-20, MS was isolated from the cleft palate of at least 83% (10 / 12) of chickens in both WVU1853-challenged groups (NV / CW, V / CW) at all time points after challenge, but in the K6677-challenged only group (NV / CK), the isolation rate of MS from the cleft palate decreased from 100% (12 / 12) at 3 WPC to 33.3% (4 / 12) at 4 WPC, and 0% at 5 and 6 WPC. In the oviduct, MS was isolated in only one sample each from the two groups challenged with WVU1853 (V / CW, NV / CW) at 2 WPC, and also in one sample from the vaccinated only group (V / NC) at 4 WPC.

[0109] Real-time PCR (qPCR). At least 92% (11 / 12) of tracheal washes from chickens in all groups except the negative control (NV / NC) were MS positive, and there was no significant difference in MS mean genome copy number (MCNlog10) between the positive groups (Figure 12) (P<0.05). No oviduct samples from any group were MS positive (Tables 16-20). As shown in Table 19, the number of K6677 positive chickens was higher at 2, 3, 4, 5, and 6 WPC in the non-vaccinated K6677-challenged group (NV / CK) compared to the vaccinated K6677-challenged group (V / CK) using K6677-specific PCR primers, but the difference was not statistically significant (P<0.05). However, there was a significantly higher K6677 mean genome copy number log10 in the non-vaccinated group challenged with K6677(NV / CK) compared to the vaccinated group challenged with K6677(V / CK) at 2, 3, 4, 5, and 6 WPC (P<0.05) (Figure 13). Meanwhile, in Table 20, there was a higher K5885 mean genome copy number log10 in the vaccinated group (V / NC) compared to the vaccinated group challenged with K6677(V / CK) using K5885-specific primers, but the difference was not significant (P<0.05). WVU1853-specific PCR results are pending. Egg laying. At 25 WOA (2 WPC), the weekly egg laying rate was significantly reduced (P<0.05) in the K6677-challenged group (NV / CK) compared to the negative control group, vaccinated group, and vaccinated groups (NV / NC, V / CK, V / CW). There was also a significant reduction in weekly egg-laying percentage in the WVU1853-challenged group (NV / W / CW) when compared to the negative control, the WVU1853-challenged group after vaccination, and the vaccinated groups (NV / NC, V / CW, V / NC) (Table 21). This significant reduction in weekly egg-laying percentage continued for 3 weeks (3, 4, and 5 WPC) in the K6677 (NV / CK) group until 28 WOA, unlike the WVU1853-challenged group (NV / CW) where the significant reduction was only at 3 WPC (P<0.05).Egg production in the K6677-challenged group (NV / CK) was significantly lower than that in the WVU1853 group (NV / CW) at 26WOA, 27WOA, and 28WOA, whereas at 29WOA, egg production in all groups was similar (P<0.05) (Figure 14).

[0110] Eggshell strength test and egg abnormalities. There was no significant difference (p<0.05) in eggshell strength before and after challenge with K6677 and WVU1853. Also, there was no significant difference (p<0.05) in eggshell strength in the non-vaccinated (NV / CK and NV / CW) groups compared to the vaccinated (V / CK and V / CW) groups. Egg abnormalities were not significantly different between the different groups after K6677 and WVU1853 challenge when compared to the NV / NC and V / NC groups.

[0111] Consideration Administration of a single dose of K5885 intraocularly stimulated immune responses in chickens 4 weeks post vaccination (WPV) as detected by circulating anti-MS antibodies by serology. The antibody response to K5885 vaccination increased to higher levels at 15 WPV from 4 WPV and titers were maintained until the end of the study at 24 WPV.

[0112] The colonization and replication rates of challenge strains K6677 and WVU1853 were similar in tracheal washes as detected by qPCR from 2 WPC to 6 WPC, but isolation rates from cleft palate were not similar between strains. Unlike WVU1853, which was isolated up to 6 WPC, K6677 could not be isolated from birds challenged after 4 WPC. The K6677 strain was also not isolated or detected via qPCR from oviducts, and despite isolation of WVU1853 and K5885 from some oviducts, they were also not detected via qPCR from oviduct swabs.

[0113] Challenge with K6677 strain resulted in more severe MS-related lesions in chickens than WVU1853 strain. Air sac inflammation, foot pad lesions, and ovarian involution were all observed at higher levels in K6677-challenged chickens compared to the WVU1853-challenged group. Egg laying was also significantly reduced in K6677-challenged chickens compared to WVU1853-challenged chickens (P<0.05). This suggests that K6677 strain is more virulent than WVU1853.

[0114] Vaccination with K5885 prevented MS-associated lesions in all vaccinated groups challenged with either K6677 or WVU1853. K5885 vaccination also reduced the replication of K6677 in the trachea, which may explain the absence of MS-associated lesions in the vaccinated groups.

[0115] Eggshell strength and egg abnormalities were not significantly affected in the groups challenged with K6677 and WVU1853. Because eggshell defects seem to be associated with certain strains of MS, the results of this study suggest that none of the MS strains used have a tropism for the reproductive system and therefore do not cause eggshell apical abnormalities (Feberwee et al., 2009, Avian Pathology; 38(2): 187-187; Ferguson-Noel et al., 2013, Mycoplasma synoviae infection. In DE Swayne, J R Glisson, L R Mc Dougald, L K Nolan, DL Suarez, & V. Nair (Eds.), Diseases of Poultry (pp. 900-906). Wiley-Blackwell; and Landman, 2014, Avian Pathology; 43(1): 2-8). In addition to the lack of effects on the eggshell, MS was not isolated from eggs from K6677- and WVU1853-challenged chickens.

[0116] This study shows that the vaccine candidate K5885 prevented MS-associated lesions, including air sacitis, ovarian involution, and footpad lesions (synovitis), reduced MS shedding, and reduced egg production losses associated with virulent MS strain infection. Further studies need to be performed to evaluate the ability of K5885 to prevent eggshell apical abnormalities (EAA) caused by EAA-associated MS strains and to understand the mechanisms by which K5885 may confer protection from disease due to MS infection in chickens. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23]

[0117] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated herein by reference. In the event of a discrepancy between the disclosure of this application and the disclosure(s) of any document incorporated herein by reference, the disclosure of this application shall govern. The foregoing detailed description and examples are given only for clarity of understanding. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, and variations obvious to one skilled in the art will be included within the invention as defined by the claims.

Claims

1. An isolated Mycoplasma sinobie strain, wherein the isolated Mycoplasma sinobie strain is Mycoplasma sinobie strain K5885 deposited with ATCC under patent designation PTA-127167, or its offspring or derivative.

2. Essentially biologically pure cultures of Mycoplasma sinobie strain K5885, deposited with ATCC under patent designation PTA-127167.

3. A composition or vaccine comprising the isolated Mycoplasma sinobie strain described in claim 1.

4. The composition or vaccine according to claim 3, comprising water.

5. The composition or vaccine according to claim 3, comprising a pharmaceutically acceptable carrier.

6. The composition or vaccine according to claim 3, comprising an adjuvant.

7. The composition or vaccine according to claim 3, formulated for spraying or aerosolizing.

8. The composition or vaccine according to claim 3, which is freeze-dried, frozen, or is an effervescent tablet.

9. The composition or vaccine according to claim 3, formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or intraocular administration.

10. The composition or vaccine according to claim 3, which reduces one or more clinical signs induced by Mycoplasma sinensis infection in poultry, or reduces the susceptibility of Galliformes birds to diseases induced by Mycoplasma sinensis.

11. A live vaccine for birds of the order Galliformes, comprising a sufficient amount to protect the birds from diseases induced by Mycoplasma sinobie, the K5885 Mycoplasma sinobie strain deposited with the ATCC under patent deposit designation PTA-127167, or its offspring or derivatives, and a pharmaceutically acceptable carrier.

12. A kit comprising an isolated Mycoplasma sinobie strain according to claim 1 and printed instructions, wherein the contents of the kit are contained within a packaging material.

13. A kit comprising the composition or vaccine described in claim 3 and printed instructions, wherein the contents of the kit are contained within a packaging material.

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

15. A method for generating an immune response against Mycoplasma sinensis in birds, for reducing the susceptibility of birds to diseases induced by Mycoplasma sinensis, for protecting birds from Mycoplasma sinensis infection, or for reducing one or more clinical signs induced by Mycoplasma sinensis infection in birds, wherein the method comprises administering to the birds the composition or vaccine according to claim 3.

16. The method according to claim 14 or 15, wherein the administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or intraocular.

17. The method according to claim 14 or 15, wherein administration is by eye drops, by aerosol, or by drinking water.

18. The method according to claim 14 or 15, wherein the bird includes birds of the order Galliformes.

19. The method according to claim 14 or 15, wherein the bird includes a chicken or a turkey.

Images