Development and evaluation of temperature-sensitive Mycoplasma anserisalpingitidis clones as vaccine candidates.

Mycoplasma anserisalpingitidis is economically the most important pathogenic Mycoplasma species of waterfowl in Europe and Asia. The lack of commercially available vaccines against M. anserisalpingitidis had prompted this study with the aim to produce temperature-sensitive (ts+) clones as candidates for an attenuated live vaccine. The production of ts+ clones was performed by N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis of Hungarian M. anserisalpingitidis field isolates. The clones were administered via eye-drop and intracloacally to 33-day-old geese. Colonization ability was examined by PCR and isolation from the trachea and cloaca, while the serological response of the birds was tested by ELISA. Pathological and histopathological examinations were performed in the eighth week after inoculation. Whole-genome sequence (WGS) analysis of the selected clone and its parent strain was also performed. NTG-treatment provided three ts+ mutants (MA177/1/11, MA177/1/12, MA271). MA271 was detected at the highest rate from cloacal (86.25%) and tracheal (30%) samples, while MA177/1/12 and MA271 elicited remarkable serological responses with 90% of the birds showing seroconversion. Re-isolates of MA271 remained ts+ throughout the experiment. Based on these properties, clone MA271 was found to be the most promising vaccine candidate. WGS analysis revealed 59 mutations in the genome of MA271 when compared to its parent strain, affecting both polypeptides involved in different cellular processes and proteins previously linked to bacterial fitness and virulence. Although further studies are needed to prove that MA271 is in all aspects a suitable vaccine strain, it is expected that this ts+ clone will contribute to the control of M. anserisalpingitidis infection.RESEARCH HIGHLIGHTS Three M. anserisalpingitidis ts+ vaccine candidates were produced by NTG-mutagenesis.Clone MA271 was able to colonize geese and induce a serological response.MA271 re-isolates remained ts+ during the 8-week-long experiment.WGS analysis revealed 59 mutations in the genome of MA271.

DF-1 cells prevent MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis.

Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in the poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed gga-miR-24-3p expression level was significantly increased in MG-infected chicken lungs. The aim of this study is to reveal the cellular mechanism behind the MG-HS infection. We found that gga-miR-24-3p was significantly upregulated and Ras-related protein-B (RAP1B) was downregulated in chicken fibroblast cells (DF-1) with MG infection. Dual luciferase reporting assay and rescue assay confirmed that RAP1B was the target gene of gga-miR-24-3p. Meanwhile, overexpressed gga-miR-24-3p increased the levels of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), and significantly inhibited cell proliferation as well as promoted MG-infected DF-1 cell apoptosis, whereas inhibition of gga-miR-24-3p had the opposite effect. More importantly, the results of overexpression and knockdown of target gene RAP1B demonstrated that the presence of RAP1B promoted cell proliferation and it saved the reduced or increased cell proliferation caused by overexpression or inhibition of gga-miR-24-3p. Furthermore, the overexpression of gga-miR-24-3p could significantly inhibit the expression of MG-HS adhesion protein. Taken together, these findings demonstrate that DF-1 cells can resist MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis, which provides a new mechanism of resistance to MG infection in vitro.

Call to action for health systems integration of point-of-care testing to mitigate the transmission and burden of sexually transmitted infections.

OBJECTIVES: In 2016, WHO estimated 376 million new cases of the four main curable STIs: gonorrhoea, chlamydia, trichomoniasis and syphilis. Further, an estimated 290 million women are infected with human papillomavirus. STIs may lead to severe reproductive health sequelae. Low-income and middle-income countries carry the highest global burden of STIs. A large proportion of urogenital and the vast majority of extragenital non-viral STI cases are asymptomatic. Screening key populations and early and accurate diagnosis are important to provide correct treatment and to control the spread of STIs. This article paints a picture of the state of technology of STI point-of-care testing (POCT) and its implications for health system integration. METHODS: The material for the STI POCT landscape was gathered from publicly available information, published and unpublished reports and prospectuses, and interviews with developers and manufacturers. RESULTS: The development of STI POCT is moving rapidly, and there are much more tests in the pipeline than in 2014, when the first STI POCT landscape analysis was published on the website of WHO. Several of the available tests need to be evaluated independently both in the laboratory and, of particular importance, in different points of care. CONCLUSION: This article reiterates the importance of accurate, rapid and affordable POCT to reach universal health coverage. While highlighting the rapid technical advances in this area, we argue that insufficient attention is being paid to health systems capacity and conditions to ensure the swift and rapid integration of current and future STI POCT. Unless the complexity of health systems, including context, institutions, adoption systems and problem perception, are recognised and mapped, simplistic approaches to policy design and programme implementation will result in poor realisation of intended outcomes and impact.

Chitosan-adjuvanted Mycoplasma gallisepticum bacterin via intraocular administration enhances Mycoplasma gallisepticum protection in commercial layers.

Mycoplasma gallisepticum (MG) causes respiratory signs and economic losses in the poultry industry. MG vaccination is one of the effective prevention and control measures that have been used around the world. Our previous study demonstrated that chitosan-adjuvanted MG bacterin could effectively reduce pathological lesions induced by MG and that chitosan could be used as an adjuvant in MG bacterin. The present study determining the efficacy of MG bacterins against the Thai MG strain was based on vaccine programs. Seven groups (25 layers/group) were received MG bacterins containing 0.5% chitosan or a commercial bacterin via intramuscular (IM) or intraocular (IO) route at 6 and 10 wk of age. Sham-negative and sham-positive controls were groups 1 and 2, respectively. Group 3: IM route of chitosan bacterin followed by IM route of chitosan bacterin; group 4: commercial bacterin via IM route followed by chitosan bacterin via IO route; group 5: commercial bacterin via IM route followed by commercial bacterin via IM route; group 6: chitosan bacterin via IM followed by chitosan bacterin via IO route; and group 7: chitosan bacterin via IO route followed by chitosan bacterin via IO route were determined. At 16 wk of age, all groups, excluding group 1, were challenged intratracheally with 0.1 mL containing Thai MG strain 107 colony-forming unit. At 17, 18, and 20 wk of age, 5 birds in each group were bled for serological testing and swabbed at the choanal cleft for the quantitative real-time PCR assay, the euthanized and necropsied. The results showed that birds vaccinated with a commercial intramuscular bacterin followed by an intraocularly chitosan adjuvant bacterin showed the best protection against the MG challenge. The study indicated that chitosan could be the effective mucosal adjuvant and increased the effectiveness of MG bacterin.

Development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and Mycoplasma gallisepticum in chickens.

Avian infectious bronchitis caused by the infectious bronchitis virus (IBV), and mycoplasmosis caused by Mycoplasma gallisepticum (MG) are two major respiratory diseases in chickens that have resulted in severe economic losses in the poultry industry. We constructed a recombinant adenovirus that simultaneously expresses the S1 spike glycoprotein of IBV and the TM-1 protein of MG (pBH-S1-TM-1-EGFP). For comparison, we constructed two recombinant adenoviruses (pBH-S1-EGFP and pBH-TM-1-EGFP) that express either the S1 spike glycoprotein or the TM-1 protein alone. The protective efficacy of these three vaccine constructs against challenge with IBV and/or MG was evaluated in specific pathogen free chickens. Groups of seven-day-old specific pathogen free chicks were immunized twice, two weeks apart, via the oculonasal route with the pBH-S1-TM-1-EGFP, pBH-S1-EGFP, or pBH-TM-1-EGFP vaccine candidates or the commercial attenuated infectious bronchitis vaccine strain H52 and MG vaccine strain F-36 (positive controls), and challenged with virulent IBV or MG two weeks later. Interestingly, by days 7 and 14 after the booster immunization, pBH-S1-TM-1-EGFP-induced antibody titre was significantly higher (P < 0.01) compared to attenuated commercial IBV vaccine; however, there was no significant difference between the pBH-S1-TM-1-EGFP and attenuated commercial MG vaccine groups (P > 0.05). The clinical signs, the gross, and histopathological lesions scores of the adenovirus vaccine constructs were not significantly different from that of the attenuated commercial IBV or MG vaccines (positive controls) (P > 0.05). These results demonstrate the potential of the bivalent pBH-S1-TM-1-EGFP adenovirus construct as a combination vaccine against IB and mycoplasmosis.

Immune responses to vaccination and infection with Mycoplasma gallisepticum in turkeys.

Infection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log10 antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose-response study, there was a significant difference in the mean log10 antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4+ and CD8+ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA+ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA+ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.

Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection.

The role of Mycoplasma and Ureaplasma in adverse pregnancy outcomes.

Genital mycoplasmas are frequently found in the vaginal flora across socioeconomic and ethnic groups and have been demonstrated to be involved in adverse perinatal outcomes. Both Mycoplasma and Ureaplasma spp cause inflammation potentially leading to spontaneous preterm birth and PPROM as well as postdelivery infectious complications and neonatal infections. Herein we have provided an overview of the existing literature and supportive evidence for genital mycoplasma's role in perinatal complications. Future research will need to focus on clearly delineating the species, allowing for discrimination of their effects.

Attenuated Mycoplasma bovis strains provide protection against virulent infection in calves.

Mycoplasma bovis is a major etiological agent of pneumonia and arthritis in feedlot beef cattle. To develop a novel live vaccine against M. bovis, two attenuated M. bovis strains, P150 and P180, were tested in calves for protection against challenge with the virulent M. bovis parental strain. Twenty calves were divided into four groups of five calves each that were designated as the P150, P180, positive control (PC), and negative control (NC) groups. Each calf in the P150 and P180 groups was immunized with 10(9)CFU of P150 or P180, respectively, via the nasal cavity, and the PC and NC groups received the mock inoculation. Baseline data were collected for 46 days post-immunization. The clinical signs were scored, and rectal temperatures and daily weight gain were recorded. The blood leukocyte count, the neutrophil ratio, and the serum levels of IgG, IgA, IFN-ß, and TNF-α were quantified using laboratory tests, and the nasal shedding was evaluated using microbiological methods. The P150, P180, and PC calves were challenged with a dose of 10(10)CFU of virulent M. bovis by intratracheal injection on 3 consecutive days. The calves were monitored for 25 days post-challenge to observe changes in the baseline parameters. On day 25 post-challenge the calves were euthanized for necropsy and analysis of tissue samples. The P150 and P180 immunizations caused no clinical abnormality, and did not affect daily weight gain. The post-inoculation neutrophil ratio and serum levels of IgG and IFN-ß significantly increased in the P150, P180, and PC calves, whereas the serum levels of IgA and TNF-α did not. After challenge, the PC group developed the typical clinical signs and pathology associated with M. Bovis infection, whereas immunization with P150 or P180 provided efficacious protection. Based on the scores for gross pathology and lung pathology, the protection rates of the P150 and P180 immunizations were 87.7% and 70.8%, respectively. The P150 attenuated strain is a promising candidate for a live vaccine against M. bovis infection in cattle.

Impact of fowlpox-vectored Mycoplasma gallisepticum vaccine Vectormune FP MG on layer hen egg production and egg quality parameters.

This study was conducted to determine the impact of vaccination with Vectormune FP MG on egg production and egg quality characteristics of Single Comb White Leghorn hens. Due to questions of the efficacy of this vaccine in preventing Mycoplasma gallisepticum-mediated pathology, the ability of this vaccine to protect against postproduction-peak egg losses associated with F-strain M. gallisepticum (FMG) vaccination was also investigated. Vaccination with Vectormune FP MG did not result in any significant change in egg production or egg quality parameters compared with control (unvaccinated) hens. Subsequent revaccination with FMG at 45 wk of age (woa) yielded no impact on egg production or egg quality parameters of Vectormune FP MG vaccinated hens, unlike prior results for postproduction-peak vaccination of M. gallisepticum-clean hens with FMG, which exhibited a drop in egg production of approximately 6%. No difference in egg size distribution was observed for any of the treatment groups before or after FMG revaccination. These results suggest that hens can be safely vaccinated with Vectormune FP MG as pullets and can be revaccinated with a live M. gallisepticum vaccine such as FMG at a later date with no deleterious effects on egg production or egg or eggshell quality parameters.

AA amyloidosis in vaccinated growing chickens.

Systemic amyloid-A (AA) amyloidosis in birds occurs most frequently in waterfowl such as Pekin ducks. In chickens, AA amyloidosis is observed as amyloid arthropathy. Outbreaks of systemic amyloidosis in flocks of layers are known to be induced by repeated inflammatory stimulation, such as those resulting from multiple vaccinations with oil-emulsified bacterins. Outbreaks of fatal AA amyloidosis were observed in growing chickens in a large scale poultry farm within 3 weeks of vaccination with multiple co-administered vaccines. This study documents the histopathological changes in tissues from these birds. Amyloid deposits were also observed at a high rate in the tissues of apparently healthy chickens. Vaccination should therefore be considered as a potential risk factor for the development of AA amyloidosis in poultry.

Experimental induction and oral transmission of avian AA amyloidosis in vaccinated white hens.

Avian AA amyloidosis is commonly observed in adult birds afflicted with bacterial infections or chronic inflammatory disorders. Experimental AA amyloidosis in birds can be induced by repeated inflammatory stimulation, such as injection with casein or vaccination with oil-emulsified bacterins. However, the transmission of amyloidosis among avian species has not been studied well to date. In the present study, we confirm the potential induction of avian AA amyloidosis by inoculation of Salmonella enteritidis (SE) vaccine or Mycoplasma gallisepticum vaccine. To determine the transmission of chicken AA amyloidosis among white hens, we induced experimental AA amyloidosis in vaccinated chickens by intravenous or oral administration of chicken AA fibrils. Amyloid deposits were observed in chickens injected with SE and inoculated with chicken AA fibrils intravenously (21/26: 81%) and orally (8/12: 67%). These results suggest that chicken AA amyloidosis can be induced by vaccinations, and may be transmitted among like species by oral administration.

Long-term immunogenicity and protection against Mycoplasma agalactiae induced by an oil adjuvant vaccine in sheep.

The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vaccine was evaluated in sheep. The antigen suspension was emulsified with a mixture of three mineral oils (Montanide ISA-563, Marcol-52, Montane-80 at the ratio of 30%, 63%, and 7%, respectively). Twenty-two animals were divided in 2 groups (A and B) and immunised with two doses of the vaccine (group A, n=14) or used as unvaccinated control (group B, n=8). Five months after the second vaccination, seven animals of group A and four animals of group B were challenged by nasal route with M. agalactiae. The remaining seven vaccinated and four control animals were challenged intranasally eight months after vaccination. The vaccine was able to induce a full-protective immunity preventing the clinical signs of contagious agalactia and the infection by M. agalactiae in all groups of animals irrespective of the time of challenge after booster administration.

Development and immunogenicity of recombinant Mycoplasma gallisepticum vaccine strain ts-11 expressing chicken IFN-gamma.

Mycoplasma gallisepticum (MG) is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. To improve the functionality of the vaccine and investigate its potential as a delivery vector for host immune molecules and foreign antigens, we have developed ts-11 as a vector to express and secrete chicken IFN-gamma (ts-11 C3) using a transposon-based delivery vector. Following administration of ts-11 C3 in chickens by eye drop, up to 2 weeks post-vaccination, neither significant systemic IFN-gamma expression nor an antibody response as determined by the rapid serum agglutination (RSA) could be detected, while moderate RSA scores were detected in birds vaccinated with ts-11. However, the MG-specific IFN-gamma response in spleen cultures was significantly enhanced in ts-11 C3 vaccinated chickens and, more interestingly, significant heterophil infiltration was detected in the tracheal epithelium in ts-11 C3 vaccinated birds, but not in ts-11 vaccinated birds. These results indicate that the IFN-gamma expressed by ts-11 C3 enhanced host cellular immunity rather than humoral immunity and may also have stimulated mucosal heterophil infiltration. These results also suggest that ts-11 is a promising vector for protective antigens of other chicken respiratory pathogens.

Mycoplasma genitalium detected by transcription-mediated amplification is associated with Chlamydia trachomatis in adolescent women.

OBJECTIVES: The clinical significance of Mycoplasma genitalium (MG) infection in adolescent women is poorly understood. We compared the prevalence of MG with that of other sexually transmitted organisms such as Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) and assessed the associations of MG with sexual behaviors, genitourinary symptoms, physical and laboratory findings. STUDY DESIGN: Women aged 14 to 21 years (n = 331) were recruited from an urban medical center. The subjects' sexual behaviors, genitourinary symptoms, and physical findings were recorded. Endocervical swabs were collected for CT and NG testing and vaginal swabs for wet mount, Gram stain, TV and MG testing. MG infection was identified by nucleic acid amplification using a transcription-mediated amplification assay. RESULTS: MG was detected in 74 (22.4%), CT in 79 (24.4%), TV in 60 (18.2%), and NG in 35 (10.7%) subjects. MG infection was not associated with vaginal symptoms, physical evidence of cervicitis, or findings on wet mount or Gram stain. In logistic regression, variables positively associated with MG were current CT [odds ratio (OR), 2.3; 95% confidence interval (CI), 1.4-4.4] and recent sexual contact (< or =7 days) (OR, 2.0; CI, 1.1-3.2). Dysuria (OR, 0.44; CI, 0.2-0.96) and use of hormonal contraception (OR, 0.55; CI, 0.3-1.0) were negatively associated with MG infection. CONCLUSION: In adolescent women, MG infection was as common as chlamydial infection and trichomoniasis and more common than gonorrhea. MG was associated with CT and recent sexual contact but not with vaginal symptoms or signs of cervicitis.

An oil-emulsion vaccine induces full-protection against Mycoplasma agalactiae infection in sheep.

The immunogenicity and efficacy of three inactivated vaccines (A, B, C) prepared with Mycoplasma agalactiae (M. agalactiae) and with different oil-emulsion adjuvants were evaluated in sheep. Twenty-eight animals were used, divided into four groups (a, b, c, d) of seven animals each. Three groups were immunized with the same vaccine, but using different adjuvants, while one group (d) was used as an unvaccinated control group. All the vaccine formulations were able to induce clinical protection of animals after challenge with M. agalactiae, but only vaccine C, emulsioned with Montanide ISA-563, Marcol-52 and Montane-80 (ratio: 30%, 63%, 7% respectively), was able to induce full protection in challenged animals, preventing both the onset of clinical signs and infection by M. agalactiae.

Mycoplasma genitalium infection and persistence in a cohort of female sex workers in Nairobi, Kenya.

OBJECTIVE: The objective of this study was to assess the risk factors for and persistence of Mycoplasma genitalium (MG) in a highly exposed female population in Kenya. STUDY DESIGN: Two hundred fifty-eight sex workers in Nairobi, Kenya, 18 to 35 years of age, were enrolled. Every 2 months, cervical samples were collected for MG, Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (GC) testing by polymerase chain reaction. RESULTS: At enrollment, 16% were infected with MG. Seventy-seven subjects acquired 107 MG infections, giving an incidence of 22.7 per 100 women-years. Incident CT (adjusted hazard ratio [HR] = 2.4; 95% confidence interval [CI] = 1.5-4.0), GC (HR = 2.0; 95% CI = 1.2-3.5), and HIV infection (adjusted HR = 2.2; 95% CI = 1.3-3.7) were associated with an increased risk of MG. Seventeen percent, 9%, and 21% of MG infections persisted 3, 5, and >or=7 months, respectively. CONCLUSION: The high incidence of MG, greater than that for both CT (14.0%) and GC (8%), association with common sexually transmitted infection risk factors, and persistence in the female genital tract supports its role as a common sexually transmitted infection in Kenyan women.

The cellular immune response in the tracheal mucosa to Mycoplasma gallisepticum in vaccinated and unvaccinated chickens in the acute and chronic stages of disease.

Mycoplasma gallisepticum causes a lymphoproliferative response in the tracheal mucosa of infected birds. The studies reported here aimed to determine, using immunohistochemical and immunofluorescent staining, which lymphocyte subsets were infiltrating the mucosa during the acute and chronic phases of disease and to determine whether these subsets differed in birds that had been vaccinated with strain ts-11. In vaccinates there was no detectable infiltration of T or B lymphocytes between 1 and 6 weeks after infection with a virulent strain. Unvaccinated birds had an initial influx of CD8+TCR- lymphocytes at 1 week, with the numbers decreasing over the next 5 weeks. CD8+TCR+ cells increased over the 6 weeks. The proportion of CD4+TCRalphabeta2+ cells also increased, whilst there was an increase in CD4+TCRalphabeta1+ cells at 3 weeks and then a decrease by 6 weeks. B lymphocytes were not detected until 3 weeks after infection, and their appearance coincided with a decrease in the concentration of mycoplasma DNA detectable in the trachea. The formation of clusters of CD8+TCR- lymphocytes was a prominent feature of the early response, while at 3 and 6 weeks after infection clusters of B cells became prominent, although in some cases they surrounded a cluster of CD8+ cells. These observations suggest a primary role for local antibody mediated responses in controlling M. gallisepticum infection, but also show that there are probably significant natural killer and cytotoxic T cell responses to infection, although the efficacy of these in controlling infection was not able to be determined.

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.