Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/542—Mucosal route oral/gastrointestinal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/35—Mycoplasma

Definitions

• Mycoplasma gallisepticum is an infectious respiratory pathogen of chickens and turkeys. It is the most pathogenic and economically significant mycoplasma pathogen of poultry. Economic losses from condemnation or downgrading of carcasses, reduced feed and egg production efficiency, and increased medication costs are factors that make this one of the costliest disease problems confronting commercial poultry production worldwide. While different measures such as voluntary monitoring programs, treatment with different antibiotics, and vaccination have been implanted in the poultry industry to control Mycoplasma gallisepticum infection and to decrease economic losses in the poultry industry, MG is still considered an enduring challenge to the commercial poultry industry.
• the present invention includes an isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain is the K6067 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA-127168, or a progeny or derivative thereof.
• the present invention includes a composition including the isolated Mycoplasma gallisepticum strain.
• the composition may include water.
• the composition may include a pharmaceutically acceptable carrier.
• the composition may include an adjuvant.
• the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration.
• the composition may be formulated for spraying or aerosolizing.
• the present invention includes an essentially biologically pure culture of the K6067 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA-127168.
• the present invention includes a composition including the isolated Mycoplasma gallisepticum strain.
• the composition may include water.
• the composition may include a pharmaceutically acceptable carrier.
• the composition may include an adjuvant.
• the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration.
• the composition may be formulated for spraying or aerosolizing.
• the present invention includes a vaccine including an isolated Mycoplasma gallisepticum strain K6067 deposited at the ATCC under Patent Designation PTA-127168, or a progeny or derivative thereof, as described herein, an essentially biologically pure culture of the K6067 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA-127168 as described herein, or a composition as described herein.
• the vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry.
• the vaccine reduces the susceptibility of a birds of the order Galliformes to disease induced by Mycoplasma gallisepticum.
• the present invention includes a live vaccine for birds of the order Galliformes, the vaccine including an amount of the K6067 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Deposit Designation PTA-127168 or a progeny or derivative thereof, as described herein, sufficient to protect the birds from disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.
• the present invention includes an isolated Mycoplasma gallisepticum strain, wherein the isolated Mycoplasma gallisepticum strain is the K4110 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA-127282, or a progeny or derivative thereof.
• the present invention includes a composition including the isolated Mycoplasma gallisepticum strain.
• the composition may include water.
• the composition may include a pharmaceutically acceptable carrier.
• the composition may include an adjuvant.
• the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration.
• the composition may be formulated for spraying or aerosolizing.
• the present invention includes an essentially biologically pure culture of the K4110 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA-127282.
• the present invention includes a composition including the isolated Mycoplasma gallisepticum strain.
• the composition may include water.
• the composition may include a pharmaceutically acceptable carrier.
• the composition may include an adjuvant.
• the composition may be formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration.
• the composition may be formulated for spraying or aerosolizing.
• the present invention includes a vaccine including an isolated Mycoplasma gallisepticum strain K4110 deposited at the ATCC under Patent Designation PTA-127282, or a progeny or derivative thereof, as described herein, an essentially biologically pure culture of the K4110 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA-127282 as described herein, or a composition as described herein.
• the vaccine reduces one or more of the clinical signs induced by Mycoplasma gallisepticum infection in poultry.
• the vaccine reduces the susceptibility of a birds of the order Galliformes to disease induced by Mycoplasma gallisepticum.
• the present invention includes a live vaccine for birds of the order Galliformes, the vaccine including an amount of the K4110 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Deposit Designation PTA-127282 or a progeny or derivative thereof, as described herein, sufficient to protect the birds from disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.
• an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, o a vaccine as described herein may be lyophilized, freeze dried, frozen, or an effervescent tablet.
• the present invention includes a kit including 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.
• the present invention includes a method of producing an immune response to Mycoplasma gallisepticum in a bird, the method including administering an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein.
• administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo.
• administration is by eye drop, by aerosol, or by drinking water.
• the bird is of the order Galliformes.
• the bird is a chicken or a turkey.
• the present invention includes a method for reducing susceptibility of a bird against disease induced by Mycoplasma gallisepticum, the method including administering an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein to the bird.
• administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo.
• administration is by eye drop, by aerosol, or by drinking water.
• the bird is of the order Galliformes.
• the bird is a chicken or a turkey.
• the present invention includes a method for protecting a bird against Mycoplasma gallisepticum infection, the method including administering an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein to the bird.
• administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo.
• administration is by eye drop, by aerosol, or by drinking water.
• the bird is of the order Galliformes.
• the bird is a chicken or a turkey.
• the present invention includes a method of reducing one or more clinical signs induced by a Mycoplasma gallisepticum infection in a bird, the method including administering an effective amount of an isolated Mycoplasma gallisepticum strain as described herein, a composition as described herein, or a vaccine as described herein to the bird.
• administration is intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo.
• administration is by eye drop, by aerosol, or by drinking water.
• the bird is of the order Galliformes.
• the bird is a chicken or a turkey.
• isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
• a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
• the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
• the bacteria Mycoplasma gallisepticum is a member of the mycoplasma genus. It is the causative agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys, chickens, game birds, pigeons, and passerine birds of all ages. It is found throughout the world and infection may be referred to as infectious synovitis, avian mycoplasmosis, infectious sinusitis, or mycoplasma arthritis. gallisepticum is transmitted vertically within some eggs (transovarian) from infected breeders to progeny, and horizontally via infectious aerosols and through contamination of feed, water, and the environment, and by human activity on fomites (shoes, equipment, etc). It is of economic importance because infection can cause a drop in egg production (El-Gazzar, “Mycoplasma gallisepticum Infection in Poultry,” Merk Veterinary Manual, 2020).
• the present invention provides Mycoplasma gallisepticum (MG) strain K6067 and progeny and derivatives thereof that are immunogenic and stable when administered as live formulations.
• the present invention also provides Mycoplasma gallisepticum (MG) strain K4110 and progeny and derivatives thereof that are immunogenic and stable when administered as live formulations.
• Formulations of Mycoplasma gallisepticum of the present invention are safe and efficacious to inhibit Mycoplasma gallisepticum infections and will be useful in reducing the incidence and severity of disease of Mycoplasma gallisepticum infections in birds.
• Mycoplasma gallisepticum strain K6707 was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110-2209, USA, as PTA- 127168 on November 24, 2021. This strain was deposited in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
• Mycoplasma gallisepticum strain K6067 as deposited with the ATCC® as PTA-127168 is also referred to herein as Mycoplasma gallisepticum strain 6067, M.
• Mycoplasma gallisepticum strain K4110 was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110-2209, USA, as PTA- 127282 on July 20, 2022. This strain was deposited in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
• Mycoplasma gallisepticum strain K4110 as deposited with the ATCC® as PTA- 127282 is also referred to herein as Mycoplasma gallisepticum strain 4110, M.
• gallisepticum strain 4110 MG strain K4110, K4110, MG strain K4110 ATCC PTA- 127282, K4110 ATCC PTA-127282, MG strain K4110 PTA-127282, K4110 PTA-127282, and ATCC® PTA-127282.
• the present invention includes isolated Mycoplasma gallisepticum (MG) strain K6067 with the ATCC Patent Deposit Designation PTA-127168.
• isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
• biologically pure cultures of Mycoplasma gallisepticum (MG) strain K6067 with the ATCC Patent Deposit Designation PTA-127168 are also included.
• the present invention includes isolated Mycoplasma gallisepticum (MG) strain K4110 with the ATCC Patent Deposit Designation PTA-127282. Also included are biologically pure cultures of Mycoplasma gallisepticum (MG) strain K4110 with the ATCC Patent Deposit Designation PTA-127282.
• isolated progeny and isolated derivatives of Mycoplasma gallisepticum strain K6067 deposited with the ATCC Patent Deposit Designation as PTA-127168 with equivalent or similar biological, serological, and/or genetic characteristics.
• serological, biological, and genetic characteristics may include one or more of the characteristics described in the data in the Examples included herewith. More particularly, progeny or derivative of the K6067 strain deposited with the ATCC as PTA-127168 may retain the particularly favorable protective properties belonging to the present invention.
• Progeny or derivatives of Mycoplasma gallisepticum strain K6067 deposited with the ATCC as PTA-127168 may be obtained by any of the various methods for propagating Mycoplasma gallisepticum known in the art, including, but not limited to, for example, in vitro culture or back passage in a bird.
• 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 genes or gene cassettes encoding alternative proteins or nonfunctional proteins, or noncoding nucleotide sequences into the MG organism.
• isolated progeny and isolated derivatives of Mycoplasma gallisepticum strain K4110 deposited with the ATCC Patent Deposit Designation as PTA-127282 with equivalent or similar biological, serological, and/or genetic characteristics.
• serological, biological, and genetic characteristics may include one or more of the characteristics described in the data in the Examples included herewith. More particularly, progeny or derivative of the K4110 strain deposited with the ATCC as PTA-127282 may retain the particularly favorable protective properties belonging to the present invention.
• Progeny or derivatives of Mycoplasma gallisepticum strain K4110 deposited with the ATCC as PTA-127282 may be obtained by any of the various methods for propagating Mycoplasma gallisepticum known in the art, including, but not limited to, for example, in vitro culture or back passage in a bird.
• 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 genes or gene cassettes encoding alternative proteins or nonfunctional proteins, or noncoding nucleotide sequences into the MG organism.
• a Mycoplasma gallisepticum (MG) strain K6067 isolate, a MG strain K4110, and progeny and derivatives thereof, as described herein may be propagated by conventional methods, including, but not limited to, any of those described in the examples section included herewith.
• a Mycoplasma gallisepticum strain of the present invention may be cultured with Frey’s modified broth and agar (Ferguson-Noel and KI even, “Mycoplasma species ” In: Williams SM, Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E. Woolcock PR, editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists, p. 63-70; 2016).
• MG strain K6067, MG strain K4110, and progeny and derivatives thereof may be identified and differentiated from other Mycoplasma spp. and other AL gallisepticum strains using any of many 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 KI even, 2009, Avian Dis; 53 : 103-107), whole genome sequencing, and targeted DNA sequencing of the 16S-23S rRNA intergenic space region (IGSR) (Papazisi et al., 2003, Microbiology’; 149:2307-16) and mgc2 cytadhesin (Hnatow et al., 1998, Infect Immun; 66:3436- 42).
• SPA serum plate agglutination
• HI hemagglutination
• Mycoplasma gallisepticum (MG) strain K6067, Mycoplasma gallisepticum (MG) strain K4110, and progeny and derivatives thereof as described herein may be administered as a live attenuated MG vaccine to poultry, including chickens and turkeys. While different measures such as voluntary monitoring programs, vaccination and treatment with different antibiotics have been implanted in the poultry industry to control Mycoplasma gallisepticum (MG) infection and to decrease economic losses in the poultry industry, MG is still considered an enduring challenge to the commercial poultry industry. Voluntary monitoring programs can be effective, but the value of the long-lived flocks, (breeders and layers), has made the justification to cull and quarantine difficult, especially in areas where MG is widespread and endemic.
• immunizing agents include bacterins, live attenuated and recombinant vaccines.
• bacterins There are some advantages with bacterins as they are not infectious or transmissible and cannot revert to virulence (Sasipreeyajan et al., 1987, Avian Dis; 31 :776-81); bacterins have been shown to reduce egg production losses, ovarian regression, and egg transmission of MG (Glisson and KI even, 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 labor cost and individual handling of the birds for intramuscular injection may cause stress and there is also a rick of local vaccine reactions.
• a fowl pox-vectored MG vaccine is commercially available but the efficacy against respiratory lesions caused by pathogenic MG strain was reported to be significantly less than bacterins and live attenuated vaccines (Ferguson-Noel et al., 2012, Avian Dis; 56:272-275).
• an adenovirus- vectored MG vaccine based on the TM-1 protein was developed but further study is needed to investigate the efficacy (Dongchao Zhang et al., 2018, Avian Pathology; 42(2):213-222).
• 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 was reported that the vaccine is not highly efficacious and colonizes weakly (Evans and Hafez, 1992, AvianDis; 36: 197-201; and Kleven et al., 1998, Avia Dis; 42:300-6). Additionally, poor serological response might pose problems in evaluating the immunization status of vaccinated flocks with serological assays (Throne Steinlage et al., 2003, Avian Dis; 47:499-505).
• F-strain was the first live vaccine introduced to the poultry industry to control MG infections (Yamamoto and Adler, 1958, J Infect Dis; 102: 143-52) and it is currently the least attenuated live MG vaccine commercially available. Due to reports of being pathogenic in turkeys (Lin and KI even, 1982, Avian Dis 26:360-4), being transmissible to the non-vaccinated birds (KI even, 1981, Avian Dis; 25: 1005-18), and the risk of increased virulence when it circulates and backpassages in a flock (Gharaibeh et al., 2011, Avian Dis; 55:212-6) the use of F- strain is not allowed in some areas (such as Minnesota, USA, Israel, and Japan).
• F- strain is very effective for displacement of the pathogenic MG strains (Kleven et al., 1990, Avian Dis; 34:984-90) as well as reducing air sac and tracheal lesions (Kleven, 1986, Avian Diseases; 30 No. 1 :169-171) and decreasing virulent MG egg transmission (Glisson and Kleven, 1984, Avian Dis; 28:406-15).
• the other live MG vaccine that persists in the trachea of the vaccinated for the life of the flock similar to F strain is ts-11 (Whithear et al., 1990, Aust Vet J; 67: 168-74).
• ts-11 Reversion to virulence of ts-11 has also been reported (Armour et al., 2015, Avian Pathol; 44:296-304). Recently, ts-304, a GapA+ ts-11 vaccine, has been developed. While the persistence of the vaccine is still similar to ts-11 (Condello et al., 2020, Veterinary Microbiology; 251 : 108883), it is reported to be safe and efficacious to use in turkeys (Kanci et al., 2018, Vaccine; 36(18):2487-2493).
• the present invention includes compositions and vaccines of the M. gallisepticum isolates and progeny and derivatives thereof as described herein.
• the M. gallisepticum isolate is live.
• the M. gallisepticum isolate may be inactivated or killed.
• a M. gallisepticum strain 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.
• a AL gallisepticum strain or composition or vaccine thereof may be frozen.
• a M.. gallisepticum isolate or composition or vaccine thereof may be formulated as an effervescent table.
• effervescent tablets may, for example, be packaged in lightweight aluminum blisters.
• the table may be dissolved in water and administered, for example, orally, nasally, or by aerosol spray, whereby droplets enter via the mucus membranes of the birds.
• compositions and vaccines of the present invention may include, for example, water or culture medium.
• Such compositions and vaccines 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).
• a stabilizer is particularly advantageous when a 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 buffer (such as buffered saline), alcohols, and polyols (such as glycerol).
• a composition or vaccine of the present invention may also include one or more compounds with adjuvant activity.
• Suitable compounds or compositions for this purpose include aluminum hydroxide, aluminum phosphate, aluminum oxide, plant oils, animal oils, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol FTM or Marcol 52TM , Complete Freund's adjuvant, incomplete Freund's adjuvant, or a vegetable oil such as vitamin E acetate, and saponins.
• a composition or vaccine of the present invention may further include one or more immunogens derived from other pathogens infectious to poultry.
• 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).
• compositions and vaccines of the present invention may be substantially pure.
• substantially pure will mean material essentially free of macromolecules or other biological entities that would normally be found with it in nature.
• Compositions and vaccines of the present invention may be administered to birds of any of a variety of avian species that are susceptible to Mycoplasma gallisepticum infection, including, but not limited to, poultry, birds of the order GaHiformes. and exotic bird species.
• Birds of the order Galliformes include, but are not limited to, chickens, turkeys, grouse, quails, and pheasants.
• poultry includes domesticated birds that are kept for the purpose of collecting their eggs or killing for their meat and/or feathers.
• Poultry may also include other birds which are killed for their meat, such as pigeons or doves or birds considered to be game, like pheasants.
• Chickens include, but are not limited to, hens, roosters, broilers, roasters, layers, breeders, the offspring of breeder hens, and layers.
• the term “susceptible to” means the possibility or actuality of a detrimental response to the referenced microorganism, such as, for example, reduced vigor or a failure to thrive, when compared to a non-susceptible individuals or groups, and/or one or more pathological state(s) indicative of Mycoplasma gallisepticum infection.
• compositions and vaccines of the present invention may be formulated for delivery by any of a variety of routes known in the veterinary arts, including, but not limited to, for example, mucosal, intranasal, intraocular, or oral administration.
• Compositions and vaccines of the present invention may be formulated for delivery to the respiratory mucosa and may be administered such that it is immediately or eventually brought into contact with the bird's respiratory mucosal membranes.
• a composition or vaccine of the present invention may be administered by any suitable known method of inoculating poultry including, but not limited to, nasally, ocularly, by eye drop, by injection, in drinking water, in the feed, in ovo, maternally, by respiratory inhalation, and the like.
• the immunogenic composition or vaccine may be administered parenterally.
• Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.
• a composition or vaccine may be formulated for administered by mass administration techniques such as by placing the vaccine in drinking water or by spraying or aerosolizing.
• a composition may be administered by spraying an individual or the flock with a solution, such aerosol delivery may involve the administration of the composition incorporated in small liquid particles.
• Such spray -type particles may have a droplet size ranging from between about 10 to about 100 microns, more preferably, a droplet size from between about ⁇ 1 to about 50 microns.
• conventional spray-apparatus and aerosol generators may be used, such as the commercially available spray generators for knapsack spray, hatchery spray and atomist spray.
• Administration through drinking water may can be carried out using conventional apparatus.
• a composition or vaccine of the present invention may be administered to poultry before or after hatching.
• ovo vaccination may take place, 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 at any range thereof.
• laying stock or reproduction stock may be vaccinated, for example, at about 6-12 weeks of age and boosted at about 16-20 weeks of age.
• Such laying stock or reproduction stock may be vaccinated at about 6, at about 7, at about 8, at about 9, at about 10, at about 11, or at about 12 weeks of age.
• such laying stock or reproduction stock may be vaccinated within about the first two weeks of age.
• Such laying stock or reproduction stock may be boosted at about 16, at about 17, at about 18, at about 19, or at about 20 weeks of age.
• the offspring of such laying stock or reproduction stock may demonstrate an antibody titer o Mycoplasma gallisepticum, which may prevent or mitigate the symptoms of a Mycoplasma gallisepticum infection in the offspring
• Poultry may receive a composition or vaccine as described herein at a variety of ages.
• materials may 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.
• the chickens may be vaccinated only once. Or, if two doses of vaccine are used, the first is given, for example, when the chickens are 3 days to a week old and subsequently after a further 1-10 weeks.
• compositions can be administered throughout the life of the chicken.
• maternal immunity is a primary source of providing protection to broiler progeny
• breeder chickens are typically vaccinated, although broiler chickens can be vaccinated if so desired.
• compositions and vaccines of the present invention may be adjusted to include a designated concentration of Mycoplasma gallisepticum.
• Organisms may be measured as color changing units. Color changing units, also referred to herein as “ecu,” of Mycoplasma gallisepticum can be quantified using established standard methodology, including, for example, protocols set forth in Rodwell and Whitcomb (In “Methods in Mycoplasmology,” Eds. Razin and Tully, 1993).
• compositions or vaccines may have concentration of about 50, about 100, about 1x10 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,
• an effective amount may be administered to a single bird at one drop per eye per bird.
• One drop may be approximately 0.05 ml to 0.1 ml.
• Mycoplasma gallisepticum strains of the present invention may be administered to birds to reduce susceptibility to Mycoplasma gallisepticum infection. With such administration, the materials do not result in significant clinical signs or lesions indicative of Mycoplasma gallisepticum. Accordingly, it is an object of the present invention to provide immunological materials that with administration do not result in significant clinical signs or lesions indicative of MS disease. It is another object to provide immunological materials of low virulence.
• the present invention includes a method of producing an anti-MG immune response in poultry, the method including administering a Mycoplasma gallisepticum strain, composition, or vaccine as described herein.
• immunity includes humoral and/or cellular immunity.
• anti-MS antibodies may be measured, for example, by the serum plate agglutination (SPA) test (using for example, commercial antigen (Charles River Laboratories International, Inc., Wilmington, MA)); the hemagglutination inhibition (HI) test; and the enzyme-linked immunosorbent assay (ELISA) test (using for example, a commercial kit (IDEXX, Westbrook, Maine)).
• SPA serum plate agglutination
• HI hemagglutination inhibition
• ELISA enzyme-linked immunosorbent assay
• immunity includes mucosal immunity.
• the present invention includes a method of reducing, inhibiting, or preventing an MG infection in poultry, the method including 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 the reduction, inhibition, or prevention of one or more of the disease manifestations of challenge with a further infection with MG, including one or more of the disease manifestations of infectious MG.
• Such clinical signs and symptoms may include, for example, one or more of respiratory distress, tracheal rales, difficulty breathing, coughing, sneezing, nasal discharge, swollen eyelids, ocular discharge, impaired vision, conjunctivitis, frothiness about the eyes, swelling of the infraorbital sinuses, sinusitis, catarrhal sinusitis, tracheitis, airsacculitis, pneumonia, leg problems, stunting, depression, weight loss, inappetence, slow growth, reduced hatchability, reduced chick viability, abnormal feathers, poor productivity, reduction in feed efficiency, reduction in weight gain, drop in egg production, condemnations at processing and/or increased mortality.
• the invention also provides a kit including Mycoplasma gallisepticum strain K6067 and/or a progeny or derivative thereof as described herein.
• the kit may include one or more containers filled with a Mycoplasma gallisepticum of the present invention.
• the Mycoplasma gallisepticum strain K6067 may be lyophilized.
• the invention also provides a kit including Mycoplasma gallisepticum strain K4110 and/or a progeny or derivative thereof as described herein.
• the kit may include one or more containers filled with a Mycoplasma gallisepticum of the present invention.
• the Mycoplasma gallisepticum strain K4110 may be lyophilized.
• a kit may include additional, separate containers of other strains of Mycoplasma gallisepticum or other pathogens of poultry. Additionally, the kit may include other reagents such as buffers and solutions needed to practice the invention are also included. Optionally associated with such container(s) can be a notice or printed instructions.
• a kit of the present invention may include “packaging material.” As used herein, the term “packaging material” refers to one or more physical structures used to house the contents of the kit. Packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. Packaging material may be a solid matrix or a material such as glass, plastic, paper, foil, and the like.
• a package can be a glass or plastic vial used to contain ecu quantities of Mycoplasma gallisepticum strain K6067 or Mycoplasma gallisepticum strain K4110.
• Exemplary Embodiments of the present invention include, but are not limited to, the following.
• An isolated Mycoplasma gallisepticum strain wherein the isolated Mycoplasma gallisepticum strain is the K6067 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA-127168, or a progeny or derivative thereof.
• An isolated Mycoplasma gallisepticum strain wherein the isolated Mycoplasma gallisepticum strain is the K4110 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Designation PTA- 127282, or a progeny or derivative thereof.
• composition comprising the isolated Mycoplasma gallisepticum of any one of Embodiments 1 to 4.
• Embodiment 5 comprising water.
• composition of Embodiment 5 comprising a pharmaceutically acceptable carrier.
• composition of any one of Embodiments 5 to 7 comprising an adjuvant.
• 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.
• Embodiment 13 The vaccine of Embodiment 11 or 12, wherein the vaccine reduces the susceptibility of a birds of the order Galliformes to disease induced by Mycoplasma gallisepticum.
• a live vaccine for birds of the order Galliformes comprising an amount of the K6067 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Deposit Designation PTA-127168 or a progeny or derivative thereof, sufficient to protect the birds from disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.
• a live vaccine for birds of the order Galliformes comprising an amount of the K4110 Mycoplasma gallisepticum strain deposited at the ATCC under Patent Deposit Designation PTA-127282 or a progeny or derivative thereof, sufficient to protect the birds from disease induced by Mycoplasma gallisepticum, and a pharmaceutically acceptable carrier.
• a kit comprising the isolated Mycoplasma gallisepticum, the composition, or the vaccine of any one of Embodiments 1 to 16 and printed instructions, wherein the contents of the kit are contained within packaging material.
• An effervescent tablet comprising the isolated Mycoplasma gallisepticum, the composition, or the vaccine of any one of Embodiments 1 to 16.
• a method of producing an immune response to Mycoplasma gallisepticum in a bird comprising administering the isolated Mycoplasma gallisepticum, the composition, or the vaccine of any one of Embodiments 1 to 16 to the bird.
• a method for reducing susceptibility of a bird against disease induced by Mycoplasma gallisepticum comprising administering the isolated Mycoplasma gallisepticum, the composition, or the vaccine of any one of Embodiments 1 to 16 to the bird.
• a method for protecting a bird against Mycoplasma gallisepticum infection comprising administering the isolated Mycoplasma gallisepticum, the composition, or the vaccine of any one of Embodiments 1 to 16 to the bird.
• a method of reducing one or more clinical signs induced by a Mycoplasma gallisepticum infection in a bird comprising administering an effective amount of the isolated Mycoplasma gallisepticum, the composition, or the vaccine of any one of Embodiments 1 to 16 to the bird.
• I'm Mycoplasma gallisepticum (MG) isolates were selected based on case history and analysis of targeted sequencing results from the isolate reservoir at Poultry Diagnostic and Research Center (PDRC).
• PDRC Poultry Diagnostic and Research Center
• the safety of the isolates was evaluated in turkeys and two isolates (K4110A and K6067) was selected based on successful colonization of the trachea while showing no clinical signs or gross lesions at the necropsy (Trials 1 and 2).
• Trial 3 the safety and efficacy of both isolates were evaluated in chickens; SPF chickens were vaccinated via eyedrop at 4 weeks of age and R strain was used to challenge the chickens via aerosol 4 weeks post vaccination.
• Ten vaccine candidates were selected from MG culture repository at Poultry Diagnostic and Research Center (University of Georgia, Athens, GA) based on criteria including indication that the naturally attenuated (no case history clinical signs or mortality), as well as rapid and consistent in vitro growth rate.
• DNA was extracted following growth in Frey’s modified broth; the cells were centrifuged at 13,000 x g for 3 minutes, supernatant discarded, and the cell pellets were reconstituted in 200 ⁇ l of phosphate buffered saline (pH 7).
• Illumina based sequencing (Illumina, San Diego, California) was conducted at Novogene (Sacramento, CA).
• K4110A and K6067 were further assessed regarding safety and efficacy in chickens (Trial 3), the most protective and effective dose and route of administration of each (Trials 4 and 5) and potential vertical transmission of each (Trial 6).
• R-strain a well described pathogenic strain of MG (Rodriguez and KI even, 1980, Avian Dis 24:800-7) was used to challenge vaccinated or naive birds as a positive control group.
• Aerosol and eye drop routes were the two routes of administration used to inoculate the birds; with the former, 1 mL of actively growing culture of the strain was aerosolized using a commercial paint sprayer (PREVAL® Sprayer Division, Precision Valve Corporation, Yonkers, NY) and with the latter, 100 pl of the actively growing culture was administered into the eye.
• a commercial paint sprayer PREVAL® Sprayer Division, Precision Valve Corporation, Yonkers, NY
• Sera were analyzed to confirm the stimulation of the humoral immune system, the presence of anti-MG antibodies was evaluated by serum plate agglutination (SPA) using commercial antigen (Charles River Laboratories, North Franklin, CT), the hemagglutination (HI) test using antigen prepared from the A5969 strain and chicken red blood cells (RBCs), and enzyme-linked immunosorbent assay (ELISA) using a commercial kit (IDEXX, Westbrook, ME; Biochek, Scarborough, ME). Procedures described previously (Kleven SH, “Mycoplasmosis,” In: Dufour-Zavala L, Swayne DE, Glisson JR, Pearson JE, Reed WM, Jackwood MW. Woolcock PR, editors.
• SPA serum plate agglutination
• HI hemagglutination
• ELISA enzyme-linked immunosorbent assay
• Cotton swabs were taken from tracheas, choanal clefts and air sacs and inoculated in Frey’s modified broth and agar (Ferguson-Noel and Kleven, “Mycoplasma species,” In: Williams SM, Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E. Woolcock PR, editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. American Association of Avian Pathologists, p. 63-70; 2016).
• qPCR Real-time quantitative PCR
• Genomic DNA was extracted from 200 pl of the laryngeal wash and tracheal swabs using the MAG-BIND® Blood and Tissue DNA HDQ 96 kit (Omega Bio-tek, Inc., Norcross, GA) on the MAGMAXTM Express-96 Magnetic Particle Processors (Thermo Fisher Scientific) following 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) value ⁇ 39 was considered positive.
• plasmids were constructed containing the genome target as standard DNA controls.
• the procedures used in constructing the DNA controls and standard curves for quantitation have been described in detail elsewhere (Raviv et al., 2008, Vet Microbiol,' 129: 179-87).
• Gross air sac lesions were scored on a scale of 0 to 4 using scoring systems 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 evaluated microscopically by measuring the width of the tracheal mucosa. A section of the upper third of the trachea (approximately 1 inch distal from the larynx) was fixed in 10% neutral formalin. The tracheal mucosa thickness was measured at four equidistant points on histological slides of cross sections of tracheas (Whithear, 1996, Rev. Set. Tech. Off. Ini. Epiz. 15: 1527-1553). Any other necropsy findings were also recorded.
• Trials 1 and 2 had the same experimental design; in each trial 5 candidates were included for a total of 10 vaccine candidate evaluated over the two trials.
• 42 turkey poults were acquired at 1 day of age from a source known to be free of MG and MS and housed in 2 isolators.
• the turkeys were leg banded with random numbers (www.randomlists.com) and randomly assigned to 7 treatment groups (7 isolators) of 6 poults each.
• Ten turkeys, distributed among the treatment groups, were screened for the presence of Mycoplasma by culture, quantitative polymerase chain reaction (qPCR) of tracheal samples and of Mycoplasma antibodies in blood serum at this time.
• qPCR quantitative polymerase chain reaction
• One hundred-twenty-five day-old SPF chicks were acquired from a source known to be free MG and MS and housed in one pen (1.5x3 m 2 ) with pine shavings litter.
• the chicks were randomly wing banded and assigned to 4 treatment groups.
• Fifteen chickens were screened for the presence of Mycoplasma spp. by culture, MG and M. synoviae qPCR of tracheal samples, and serology.
• the chickens were inoculated via aerosol with their respective treatments (B, C, D, K6067, K4110A or R- strain) or left unvaccinated as the negative control group as detailed below in Table 4.
• Groups A and B were left unvaccinated as negative control and challenge only groups, respectively.
• choanal cleft and tracheal swabs were taken from all groups for culture and MG qPCR.
• all eighty -four chickens were bled and swabbed for MG serology, culture, and MG qPCR.
• Four weeks post vaccination and at 7W0A chickens of groups B to L were challenged with R-strain via aerosol.
• 9 WOA all birds were necropsied and evaluated by gross air sac lesion scoring, serology, choanal cleft and air sac culture, tracheal histopathology, and MG and strain-specific qPCR of tracheal washes.
• the minimum infective dose 50 (MID50) and minimum protective dose 50 (MPD50) were calculated (Cottey et al., 2001, Current Protocols in Immunology; 42: 19-11) based on sampling and necropsy results.
• the chicks were wing banded and randomly assigned to 7 treatment groups in nineteen isolators (Table 6).
• Ten chicks, distributed among the treatment groups, were screened for the presence of Mycoplasma spp. by culture, MG, and M. synoviae qPCR of tracheal samples and serology.
• the chickens of groups C through G were vaccinated via eye drop with different doses of the vaccine candidate K4110A.
• Groups A and B were left unvaccinated as negative control and challenge only groups, respectively.
• MID50 minimum infective dose 50
• MPD50 minimum protective dose 50
• Infertile eggs or early dead embryos were discarded the yolk sac of any dead embryonated eggs at 9, 11, 13 and 15 DOE were sampled and inoculated into modified Frey’s broth for culture. All remaining embryonated eggs were sampled for culture at 18 DOE.
• HI and ELISA (IDEXX, Westbrook, Marine) assays were performed on yolk sac samples of the last set of eggs (National Poultry Improvement Plan, “Procedures for preparing egg yolk samples for diagnostic tests,” National Poultry Improvement Plan Program Standards, 23-24; 2017).
• the standard procedure recommended by NPIP for egg yolks was used to prepare the samples and perform the tests (National Poultry Improvement Plan, “Procedures for preparing egg yolk samples for diagnostic tests,” National Poultry Improvement Plan Program Standards, 23-24; 2017).
• the HI test procedure were as described by Ferguson-Noel et al.
• the number of eggs laid were recorded from 21 to 31 WOA and hen-week egg production rates were also calculated by dividing total number of eggs laid on the week by the total number of hens times 100.
• 31 WOA all birds were necropsied and evaluated by gross air sac lesion scoring, serology, choanal cleft and air sac culture, tracheal histopathology, and MG qPCR of tracheal washes and oviduct swabs.
• the birds tested pre-inoculation at 2 WOA were all negative for MG, MM and MS antibodies and were also negative for MG, MS, MM and MI by qPCR. All cultures were also negative for Mycoplasma spp. at that time.
• MG antibodies were detected in all of the inoculated groups; the serology results of Trial 1 are summarized in Table 8.
• the weakest antibody response was in Group E, which was inoculated with isolate K6837.
• the only group in which no air sac lesions were observed was the negative control group, however, the groups inoculated with K6067 (Group B) and K6837 (Group E) had significantly lower mean air sac lesion scores than the positive controls (Group G) (P ⁇ 0.05).
• the 10 birds tested pre-inoculation at 3 WOA were negative for MG, MM and MS antibodies and were also negative for MG, MS, MI, and MM by qPCR. All cultures were also negative for Mycoplasma spp. MG antibodies were detected in all of the inoculated groups and the weakest antibody response was in Group C, which was inoculated 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 groups inoculated with K4110A (Group C) and K4179 (Group B) had lower mean air sac lesion scores than the positive controls (Group G).
• the 15 birds tested pre-inoculation at 2 WOA were negative for MG and MS antibodies and were also negative for MG and MS by qPCR. All cultures were also negative for Mycoplasma spp. pre-inoculation.
• Three necropsies were performed in this trial with the aim of evaluating the safety of the vaccine candidates in chickens (Necropsy 1), evaluating the horizontal transmission possibility of the vaccine candidates (Necropsy 2), and evaluating the efficacy of the vaccine candidates post challenge (Necropsy 3).
• Necropsy 1 (Safety). MG antibodies were detected in all inoculated groups although the antibody response in the groups inoculated with the vaccine candidates was weaker compared to the positive control (R strain). The mean SPA scores of groups B (K6067) and C (K4110A) were significantly lower than the scores of group D (R strain) (P ⁇ 0.05). A summary of these serology results is presented in Table 14. All groups except the positive control group had no air sac lesions at 14 days post inoculation and these differences were statistically significant (P ⁇ 0.05).
• Necropsy 2 Horizontal Transmission to Contacts. There was a low level of infection in birds placed in direct contact with vaccinated birds at 19 days post commingling (2 of 5 for K6067 and 1 of 5 for K4110A detected by isolation from the trachea). There were no significant differences in serological response (Table 16), air sac lesions or tracheal mucosa thickness between the groups (Table 17).
• Necropsy 3 (Efficacy - Aerosol and Contact Challenge). Groups inoculated with the vaccine candidates showed a strong serological response at 14 days post challenge with R-strain (presented in Table 18). As can be seen in Table 19, the vaccinated groups also had significantly lower mean air sac lesion scores after challenge (P ⁇ 0.05). There were no significant differences with respect to tracheal measurements (Table 21), even between the negative and positive controls (P ⁇ 0.05); however, the positive control resulted in the highest mean measurement. The qPCR resulted are summarized in Table 20. The qPCR results show that the K6067 group had higher mean genome copy numbers log 10 in their tracheas compared to the K4110A groups.
• SPA serum plate agglutination
• HI hemagglutinin inhibition
• ELISA enzyme-linked immunosorbent assay
• group A All groups except the negative control group, (group A), had some level of air sac lesions after virulent challenge but the mean air sac lesion scores of groups D, E, F, G, J, K and L were lower than challenge only group (Group B). Of those, the mean scores of groups E, F and G were significantly lower compared to the other vaccinated and challenged groups (P ⁇ 0.05). The mean tracheal mucosal thickness measurements of groups D, E, F, G, J and K were lower compared to the challenge only group and the difference was significant with groups E, F and G (P ⁇ 0.05) (Table 27).
• Mycoplasma was recovered from the air sacs of all groups vaccinated via aerosol, (groups H to L). With groups vaccinated via eye drop, Mycoplasma was isolated from all birds of groups C, D and G, three birds of group E and five birds of group F (Table 27).
• the MID50 for K6067 was calculated based on the qPCR and culture results of 1- and 3-weeks post vaccination sampling for all doses and both routes of administration. While no MID50 could be calculated for qPCR results, the MID 50 for the culture results of groups vaccinated via eye drop route was 10 3.8 ccu/mL one week post vaccination and 10 3.53 CCU/mL at three weeks post vaccination.
• the MG qPCR and strain specific qPCR results are summarized in Table 36.
• the R strain mean genome copy number (MCNlog10) detected in the trachea was significantly higher in the challenge-only group compared to the vaccinated groups (P ⁇ 0.05).
• K4110A specific qPCR showed that the vaccine candidate colonized the trachea of all K4110A-vaccinated groups although the MCNlog10 of the vaccine strain in Group C (lowest vaccination dose) was significantly lower than the other vaccinated groups. Except for Group G, K4110A was not detected in the air sac cultures from the vaccinated groups (Table 36). Based on qPCR and culture results of 1 and 3 weeks post vaccination for all doses the MID50 for K4110A was calculated (Table 37).
• the MID 50 for the qPCR results of groups vaccinated via eye drop route was 10 5.3 CCU/mL one week post vaccination and 10 5.2 CCU/mL at three weeks post vaccination.
• the MPD50 for K4110A was also calculated based on mean air sac scores necropsy and was 10 3.7 CCU/mL. (Table 38). No MPD50 could be calculated the tracheal thickness measurements of vaccinated groups.
• Trial 6 (Vertical Transmission) All 16 birds tested pre-inoculation at 23 WOA were negative for MG and MS antibodies and were also negative for MG and MS by qPCR. All cultures were also negative for Mycoplasma spp. at that time.
• Trial 1 The results of Trial 1 indicate that although K6067 inoculation did not result in significant lesions the turkeys were infected and replicated the MG (a requirement to generate a protective immune response). It has been shown that 6/85 poorly colonizes the trachea and this may explain the compared to F-strain and ts-11 (16, 29). The air sac culture results also indicated that K6067 may not be as invasive as the other MG isolates in this trial. Similarly, in Trial (Turkey Safety 2) K4110A was capable of infecting turkeys but did not result in severe clinical signs or lesions. All of the remaining trials were conducted in chickens although further research in turkeys is necessary to investigate efficacy and other parameters in this poultry species.
• the two vaccine candidates invoked a detectable and consistent serological response; although serum antibody levels do not appear to correlate with protection for MG live attenuated vaccines it is useful to be able to monitor vaccination using widely available and inexpensive serological tests.
• Table 1 The history of vaccine candidates selected for preliminary screening (Trials 1 and 2).
• Trial 1 (Turkey Safety 1). Lesions scores, tracheal mucosal thickness, genome copy numbers log10 and MG isolation from tracheas and airsacs of turkeys at 2 weeks post inoculation with K6067, K6524, K6694, K6837, K6813 or R strain. A different (P ⁇ 0.05) B No. of positive samples/No. of tested samples (Air sac score ⁇ 1) C Mean score (Macroscopically scored from 0 to 4) D Mean thickness for the group ⁇ SD ( ⁇ m) E Mean (genome) copy number log10 ⁇ SD
• Trial 1 (Turkey Safety 1). Clinical signs in turkeys at 2 weeks post inoculation with K6067, K6524, K6694, K6837, K6813 or R strain.
• Trial 2 (Turkey Safety 2). Lesion scores, tracheal mucosal thickness, MG isolation from tracheas and air sacs and qPCR from turkeys at two weeks post inoculation with K4179, K4110A, K5058E, K5792A, K6836A or R strain A
• Trial 2 (Turkey Safety 2). Clinical signs in turkeys at two weeks post inoculation with K4179, K4110A, K5058E, K5792A, K6836A or R strain. Group Vaccine/Challenge Clinical Picture A N N i t li i l i b d Table 14.
• Trial 3 (Chicken Safety and Efficacy). MG serological response, of chickens 14 days post inoculation with K6067, K4110A or R strain (Necropsy 1). A A Va p p g ificantly different (P ⁇ 0.05) B No. of positive samples/No.
• Trial 3 (Chicken Safety and Efficacy). MG serological response, of chickens 19 days post mingling with vaccinated groups (groups B & C) (Necropsy 2). A A Valu p p g ficantly different (P ⁇ 0.05) B No. of positive samples/No. of tested samples (SPA: ⁇ 1, HI: ⁇ 20, ELISA: ⁇ 0.5, Air sac score ⁇ 1) C Mean agglutination grade (from 0 to 4). D Mean titer log10 E Mean sample/positive ratio Table 17. Trial 3 (Chicken Safety and Efficacy).
• Trial 3 Chocken Safety and Efficacy. Tracheal mucosal thickness of chickens 28 days post inoculation with vaccine candidates or R strain and 14 days post challenge with R strain or mingling with R strain-challenged chickens (Necropsy 3).
• Trial 4 Dose Response 1). Minimum protective dose 50% (MPD50) of K6067 based on air sac lesion scores and tracheal thickness measurements at necropsy.
• Trial 5 Dose Response 2.
• B 1st sampling was performed 1 week post inoculation at 4 WOA.
• c 2nd sampling was performed 3 weeks post inoculation at 6 WOA.
• Trial 5 Dose Response 2.
• Minimum protective dose 50% (MPD50) of K4110A based on airsac scores and tracheal thickness measurements at necropsy.

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