Comparison of the short-term and long-term efficacies of the Mycoplasma gallisepticum vaccines ts-11 and 6/85.

In order to compare the short-term efficacies of the live attenuated Mycoplasma gallisepticum (MG) vaccine strains ts-11 and 6/85, four groups of SPF chickens were vaccinated with each of the vaccines using eye drop and aerosol inoculations, and were subsequently challenged with a wild-type MG strain. When administered by the recommended routes (eye drop for ts-11 and fine aerosol for 6/85), both vaccines induced substantial and comparable levels of protection against airsacculitis and tracheitis caused by wild-type MG. The long-term efficacies of the two vaccines administered by the recommended route were also assessed. Serum antibody responses and colonization of the vaccines in the upper respiratory system were monitored at different time points after vaccination, and protective efficacies of the vaccines were evaluated at 36 weeks post vaccination as above. Systemic antibody response following ts-11 eye drop vaccination was initially strong but reduced gradually over time while, in contrast, that to 6/85 spray vaccination was initially weak but increased over time. Kinetics of the antibody response to the vaccines appeared to be correlated with the number of birds harbouring each vaccine in their upper respiratory system throughout the sampling timepoints. Regardless of the levels of serum antibodies or number of birds harbouring the vaccine, both vaccines induced substantial and comparable levels of protection against airsacculitis and tracheitis caused by wild-type MG. Therefore, kinetics of systemic antibody response and persistence in the upper respiratory system varies between vaccine strains; however, the levels of protection may not, at least up to 36 weeks post vaccination. RESEARCH HIGHLIGHTS The kinetics of systemic antibody response and persistence of the vaccine in the upper respiratory system varies between vaccine strains ts-11 and 6/85. The levels of protection induced by the two vaccines against virulent MG strain challenge are comparable when they are administered by the route recommended by their manufacturers.

Safety and efficacy of the Mycoplasma synoviae MS-H vaccine in turkeys.

The live, attenuated, temperature-sensitive Mycoplasma synoviae (MS) vaccine strain MS-H is used to control virulent MS infection in commercial chicken flocks. However, the safety of this vaccine and its potential to prevent disease in turkeys have not been investigated. In this study, MS-H was shown to colonize the upper respiratory system and to induce an antibody response in turkeys but, even at the maximum release dose, was not found to cause air sac, joint, or tracheal lesions typical of wild-type MS infection. Histopathologic examinations of the vaccinated turkeys after exposure to a virulent MS challenge revealed that administration of the vaccine by aerosol, but not eye drop, at the dose recommended for chickens protected the birds against microscopic lesions and colonization of the virulent MS in trachea. It is concluded that MS-H vaccine is safe for use in turkeys and, when used as aerosol at the dose recommended for commercial chickens, can protect turkeys against tracheal lesions caused by a wild-type MS strain.

Duration of immunity with Mycoplasma synoviae: comparison of the live attenuated vaccine MS-H (Vaxsafe MS) with its wild-type parent strain, 86079/7NS.

The duration of protective immunity elicited by the MS-H vaccine was evaluated by experimental challenge of chickens at 15 and 40 wk after eyedrop vaccination. Immunity induced by the parent strain of the vaccine, 86079/7NS, was also investigated for comparison. A serological response to Mycoplasma synoviae was detected in 89% to 100% of MS-H vaccinates and 86079/7NS inoculates at 15, 27, 30, 35, and 40 wk after inoculation. A significantly lower incidence of air-sac lesions and lower air-sac lesion severity were observed in both the MS-H vaccinated and the 86079/7NS inoculated groups, as compared to the unvaccinated controls, after both challenge points. Tracheal mucosal thicknesses in MS-H vaccinates was significantly lower in the upper, lower, and total trachea at 40 wk after vaccination, as compared to the controls. It was demonstrated in this experiment that protective immunity, as determined by protection against experimental challenge, was maintained to at least 40 wk after vaccination.

Onset of immunity with Mycoplasma synoviae: comparison of the live attenuated vaccine MS-H (Vaxsafe MS) with its wild-type parent strain (86079/7NS).

The onset of protective immunity with MS-H was determined through experimental challenge and compared with the parent strain 86079/7NS. MS-H vaccinates and 86079/7NS inoculates were challenged at 1, 2, 3, 4, 5, and 6 wk after vaccination, then examined 2 wk after challenge for signs of respiratory disease. Serologic results indicated that 100% of MS-H vaccinates had antibodies to MS by 3 wk after vaccination and 100% of 86079/7NS inoculates were positive by 2 wk after inoculation. From 3 wk after vaccination, MS-H vaccinates had a significantly lower incidence of air sac lesions and, from 4 wk after vaccination, a significantly lower air sac lesion severity. In 86079/7NS-inoculated birds, a significantly lower incidence of air sac lesions was observed from 1 wk after inoculation, and air sac lesion severity was significantly lower than the unvaccinated controls at 3 wk after inoculation. It would appear that, under the conditions of this experiment, protective immunity elicited by MS-H appeared at 4 wk after vaccination, slightly later than the appearance of serum antibody. Although the MS-H vaccine was slower to establish protective immunity than 86079/7NS, there was no significant difference between the two strains by 4 wk after vaccination or inoculation.

Determination of the effective dose of the live Mycoplasma synoviae vaccine, Vaxsafe MS (strain MS-H) by protection against experimental challenge.

The minimum effective dose of the Mycoplasma synoviae-H (MS-H) vaccine was determined through protection against experimental challenge. Chickens were vaccinated by eyedrop with the following doses of a vaccine: 1.2 x 10(5), 2.4 x 10(5) 4.8 x 10(5), 9.6 x 10(5), 1.92 X 10(6), and 3.84 X 10(6) color change units (CCU), then challenged 6 wk after vaccination. Rapid serum agglutination results indicated that 100% of birds receiving an MS-H dose of > or = 4.8 x 10(5) CCU had antibodies to MS and enzyme-linked immunosorbent assay results showed that 60% of birds receiving a dose of 4.8 x 10(5) or 9.6 x 10(5) CCU and 100% of birds receiving a dose of 1.92 x 10(6) or 3.84 x 10(6) had antibodies to MS. At postmortem after challenge, the following parameters were significantly lower in birds vaccinated with an MS-H dose of > or = 4.8 x 10(5) CCU: air sac (AS) lesion severity; incidence of AS lesions; mucosal thicknesses in the upper trachea, middle trachea, and lower trachea (LT); and MS colonization of the LT and AS. It was concluded that an MS-H dose of 4.8 x 10(5) CCU was sufficient to elicit an antibody response in birds, prevent MS colonization in the LT and AS, and protect against AS lesions caused by an experimental MS and infectious bronchitis virus challenge.

Evaluation of the non-temperature-sensitive field clonal isolates of the Mycoplasma synoviae vaccine strain MS-H.

The live attenuated temperature-sensitive (ts+) Mycoplasma synovia (MS) strain, MS-H, is used as a vaccine in a number of countries to control virulent MS infection in commercial chicken flocks. Nine out of 50 isolates made from flocks vaccinated with MS-H were found to have lost the ts+ phenotype of the original vaccine strain. In order to examine the influence of the ts- phenotype on virulence of the isolates, four of the ts- isolates, the MS-H vaccine, and the vaccine parent strain 86079/7NS were administered by aerosol in conjunction with infectious bronchitis virus to 3-wk-old specific-pathogen-free chickens. The four ts- clones induced only minimal air sac lesions that were not different in severity from those caused by MS-H vaccine; however, the vaccine parent strain 86079/7NS caused air sac lesions that were significantly greater than those of MS-H and all ts- clones. The vaccine parent strain 86079/7NS and two of the ts- clones were recovered from the air sacs of the respectively infected chickens whereas the MS-H vaccine and two other ts- clones were not. Three of the ts- isolates caused increased tracheal mucosal thicknesses that were significantly greater than those from birds inoculated with MS-H, and one caused increased tracheal mucosal thicknesses that were significantly less than those from birds inoculated with 86079/7NS. In conclusion, unlike the MS-H vaccine, the MS-H ts- clones were associated with minor changes in tracheal mucosa; however, unlike the vaccine parent strain, they did not induce lesions in the air sacs. These results suggest that factors other than ts+ phenotype are involved in the attenuation of the MS-H vaccine.

Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay.

Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.

Improved detection of antibodies to Mycoplasma synoviae vaccine MS-H using an autologous recombinant MSPB enzyme-linked immunosorbent assay.

Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis. An indirect enzyme-linked immunosorbent assay (ELISA) has previously been devised in our laboratory using the major membrane antigen MSPB of M. synoviae strain WVU 1853 as antigen. However, sera from chickens inoculated with the M. synoviae vaccine strain MS-H showed lower optical densities in the assay than chickens infected with wild-type strains. In the present study, we investigate whether a low level of antibodies detected in MS-H-vaccinated birds is due to the limited ability of the vaccine to elicit antibodies, or to the reduced capacity of the antigen to specifically detect antibodies to this strain. Preliminary immunostaining experiments using native MSPBs from M. synoviae MS-H and WVU 1853 suggested that they were antigenically related but differed in at least some epitopes. Using a combination of polymerase chain reaction (PCR) and cloning, the gene encoding MSPB (vlhA) was cloned from strain MS-H, and its nucleotide sequence was partially determined. Analysis of the partial nucleotide sequence of the cloned vlhA gene revealed that it had a high identity (86%) with the previously published vlhA sequence from strain WVU 1853, but differed from it in several regions. Also, several nucleotide substitutions/deletions were detected in the conserved region (nucleotides 1 to 700) of the MS-H vlhA gene. A polypeptide, containing amino acids 27 to 299 of the MS-H MSPB, was expressed as a fusion protein in Escherichia coli and purified by affinity chromatography. An indirect ELISA was developed using the MS-H MSPB as coating antigen and compared with that of WVU 1853 MSPB and the commercial rapid serum agglutination test using a panel of sera from MS-vaccinated and/or challenged or unvaccinated specific pathogen free and commercial field chickens. Analysis of the absorbance values from specific pathogen free and field chicken sera showed that MS-H MSPB was species specific and more sensitive than the WVU-MSPB ELISA or the rapid serum agglutination test in detecting antibodies to the MS-H vaccine strain. These results emphasize the importance of using appropriate diagnostic antigens for sensitive detection of antibodies following vaccination or challenge with a M. synoviae strain.

Size and genomic location of the pMGA multigene family of Mycoplasma gallisepticum.

The pMGA multigene family encodes variant copies of the cell surface haemagglutinin of Mycoplasma gallisepticum. Quantitative Southern blotting, using an oligonucleotide probe complementary to a region conserved in the leader sequence of all known pMGA genes, was used to estimate the number of members of the family in the genome of seven strains of M. gallisepticum. The number of copies estimated to be present in the genome varied from 32 in strain F to 70 in strain R, indicating that the pMGA gene family may be second in size only to the tRNA family among prokaryotes. If all members of the pMGA family are of similar length to those which have been characterized, a minimum of 79 kb (7.7%) of the genome of strain S6, 82 kb (8.2%) of PG31 and 168 kb (16%) of the genome of strain R is dedicated to encoding variants of the same haemagglutinin. The GAA repeat motif identified in the intergenic region between all characterized pMGA genes appeared to be a feature common to most, if not all, pMGA genes, and furthermore probably exclusive to them. The genomic locations of members of the pMGA family were determined by PFGE and Southern blot hybridization of M. gallisepticum strain S6. The hybridizing regions were localized to four separate regions on the chromosome. The pMGA genes are likely to be predominantly arranged as tandem repeats within these regions, similar to the restricted regions for which the genomic sequence has been determined.

Mycoplasma synoviae surface protein MSPB as a recombinant antigen in an indirect ELISA.

Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis. A number of serological assays have been developed for diagnosis of M. synoviae infection; however, they lack sensitivity and/or are prone to false-positive reactions. Using a combination of PCR and expression cloning, four overlapping regions (regions 1-4) of the surface antigen MSPB of M. synoviae WVU-1853 were expressed in a bacterial expression system. Immunostaining of the resultant polypeptides with chicken sera raised against different M. synoviae strains, or Mycoplasma gallisepticum S6, suggested that region 4 contained a highly antigenic and species-specific domain (amino acids 212-317) [corrected] of MSPB. A fusion protein of region 4 was expressed in the pMAL expression system and purified from cold-osmotic-shock fluids of Escherichia coli cells for use in an indirect ELISA. The potential of the purified antigen for detection of M. synoviae antibodies was assessed with sera obtained from chickens experimentally infected with different strains of M. synoviae or M. gallisepticum, or from uninoculated chickens. All the sera from M. synoviae-inoculated chickens provided higher absorbance values than those from M. gallisepticum-inoculated or uninoculated chickens. Chickens inoculated with M. synoviae 86079/7NS had detectable increases of serum anti-MSPB immunoglobulins at day 7 after inoculation. These studies have identified the most antigenic region of one of the major species-specific surface proteins of M. synoviae, and shown the potential of this protein as a serodiagnostic reagent.