Comparative evaluation of indirect-ELISA and DOT blot assay for serodetection of Mycoplasma gallisepticum and Mycoplasma synoviae antibodies in poultry.

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is an economically important disease of the poultry industry. The present study was aimed to develop whole cell based indirect-ELISA (i-ELISA) and DOT blot assay (DOT-ELISA) as rapid, sensitive, specific and economical sero-detection tests for MG and MS. A total of 306 blood samples were collected from birds slaughtered at local meat shops of different districts of Haryana, India to detect MG and MS antibodies. Sonicated antigens prepared from freshly grown culture of MG and MS were used to develop i-ELISA and DOT blot assay. In i-ELISA, 50.32% and 61.76% serum samples were found to be positive for MG and MS antibodies, respectively. However in DOT blot assay, 41.83% and 53.92% serum samples were found positive for MG and MS antibodies, respectively. The relative diagnostic sensitivity and specificity of DOT-ELISA were measured considering i-ELISA as a reference test. The relative diagnostic sensitivity of the DOT blot assay was found to be 69.48% and 82.01%; whereas relative diagnostic specificity was 86.18% and 91.45% for the detection of MG and MS antibodies, respectively. The developed serological assays may be used as rapid and economical diagnostic tools for large scale screening of poultry sera for MG and MS antibodies.

Evaluation of protective efficacy of inactivated Mycoplasma synoviae vaccine with different adjuvants.

Mycoplasma synoviae (MS) is a poultry pathogen with a reported distribution throughout the world. Vaccination is utilized as an important component of MS control programs for MS infection. The aim of this study was to evaluate protection efficacy of an inactivated MS vaccine (MS bacterin) with different adjuvants in broilers against a Chinese field isolate (CHN-BZJ2-2015). Vaccination with adjuvants ISA 71 VG and chitosan, respectively, enhanced specific lymphocyte proliferation responses and upregulated the expression of IL-1ß, IL-6, IL-2 and IFN-γ prior to challenge. Furthermore, vaccination with adjuvant ISA 71 VG elicited the highest antibody titers, exhibited significantly lower air sac, foot pad and tracheal lesions than the other groups (P < 0.05), and decreased MS colonization. These results demonstrated that inactivated MS vaccine with ISA 71 VG is able to induce both cellular and humoral immune response in broilers and confers a high level of protection upon challenge, demonstrating a potential application in the field.

Mutation of oppF gene in the Mycoplasma synoviae MS-H vaccine strain and its implication for differential serological responses to vaccination versus field challenge.

Mycoplasma synoviae (MS) is a major pathogen of poultry globally, causing chronic respiratory disease and arthritis. Vaccination is an effective means for the control of the disease. The MS-H vaccine is an attenuated strain developed through chemical mutagenesis of an Australian field strain, 86079/7NS. Analysis of whole genome of MS-H and its comparison with that of 86079/7NS has revealed a frameshift mutation early in a gene (oppF) that codes for an oligopeptide transporter permease, OppF. Monospecific antibodies raised against peptides upstream and downstream of the mutation in OppF revealed that only N-terminus of the OppF was expressed in MS-H while the full version was expressed in 86079/7NS. Also, examination of the recombinant N- (OppF-N) and C termini (OppF-C) of OppF, upstream and downstream of the mutation site respectively, as well as the full length OppF in Western immunoblotting experiments showed that serum from MS-H vaccinated chicken strongly bound OppF-N while serum from 86079/7NS challenged chicken detected OppF, OppF-N and OppF-C. The potential of the recombinant OppF, OppF-N and OppF-C to discriminate antibody responses to MS-H reisolates with wild or vaccine type OppF was assessed against 88 chicken sera in indirect ELISA and ratios were calculated between optical densities (OD) over those obtained in MS major membrane protein MSPB ELISA. Comparison of the OD ratios revealed that the MSPB/OppF and MSPB/OppF-C OD ratios of the sera against isolates with vaccine type OppF were significantly higher than those against isolates with wild type OppF. These results are in accordance with oppF gene mutation in MS-H and confirms that MS-H does not express OppF beyond the frame shift mutation found in its oppF gene. Also, the indirect ELISA based on OppF-C in combination with the MSPB has the potential to differentiate between MS-H and field strain antibody responses.

Application of different laboratory techniques to monitor the behaviour of a Mycoplasma synoviae vaccine (MS-H) in broiler breeders.

BACKGROUND: Mycoplasma synoviae (MS) is a major poultry pathogen which causes severe economic losses in all the productive sectors. The prevalence of MS in European countries has increased in the last few years, leading to greater attention to the available methods to prevent its spread. The main strategy currently applied for its containment is the development and maintenance of MS-free breeder flocks. A live MS vaccine (MS-H) obtained by mutagenizing an Australian field strain has recently been introduced in Italy. The aim of the present study was to evaluate the vaccine behaviour in broiler breeder groups at different production stages and the effectiveness of the available laboratory tests in discriminating the MS-H from a field strain. RESULTS: The vaccine diffused extensively through the population, shown by the wide serological response (over 80% of positive samples in RSA and 85% in ELISA), the high serological titres, the positivity of all the tracheal samples collected during the production phase by MS PCR and the positivity by cultivation from tracheal swabs at the end-point (55 weeks after vaccination). In contrast, only one swab from a sternal bursa was positive in MS PCR, while all the joint and oviduct samples were negative. There was no evidence of vertical transmission. Different genotyping techniques were used to achieve a clear classification of the MS positive samples. The vlhA and the obg gene analysis showed that most of the strains were homologous with the vaccine, but some ambiguous samples were further investigated with the multi locus sequence typing (MLST) scheme which confirmed the homology. CONCLUSIONS: The development of a multi-technique approach to monitor vaccinated avian flocks, based both on serological and biomolecular methods, is advised as well as the use of effective genotyping techniques to analyse the MS strains circulating in high densely populated poultry areas.

Identification of a new genetic marker in Mycoplasma synoviae vaccine strain MS-H and development of a strategy using polymerase chain reaction and high-resolution melting curve analysis for differentiating MS-H from field strains.

Mycoplasma synoviae (MS) is an economically important avian pathogen worldwide, causing subclinical respiratory tract infection and infectious synovitis in chickens and turkeys. A temperature-sensitive (ts+) live attenuated vaccine MS-H, derived from the Australian field strain 86079/7NS, is now widely used in many countries to control the disease induced by MS. Differentiation of MS-H vaccine from field strains is crucial for monitoring vaccination programs in commercial poultry. Comparison of genomic sequences of MS-H and its parent strain revealed an adenine deletion at nucleotide position 468 of the MS-H oppF-1 gene. This mutation was shown to be unique to MS-H in further comparative analyses of oppF-1 genes of MS-H re-isolates and field strains from Australia and other countries. Based on this single nucleotide, a combination of nested PCR and high-resolution melting (HRM) curve analysis was used to evaluate its potential for use in differentiation of MS-H from field strains. The mean genotype confidence percentages of 99.27 and 48.20 for MS-H and field strains, respectively, demonstrated the high discriminative power of the newly developed assay (oppF PCR-HRM). A set of 13 tracheal swab samples collected from MS-H vaccinated specific pathogen free birds and commercial chicken flocks infected with MS were tested using the oppF PCR-HRM test and results were totally consistent with those obtained using vlhA genotyping. The nested-PCR HRM method established in this study proved to be a rapid, simple and cost effective tool for discriminating the MS-H vaccine strain from Australian and international strains in pure cultures and on tracheal swabs.

Quantification of the horizontal transmission of Mycoplasma synoviae in non-vaccinated and MS-H-vaccinated layers.

The number of newly infected birds attributable to one infectious bird per day (= transmission rate ß) was assessed in non-vaccinated and MS-H-vaccinated experimental specified pathogen-free White Leghorns after Mycoplasma synoviae challenge. Furthermore, the effect of vaccination on the shedding of the challenge strain was determined. The following groups were made: a negative control group (n = 5), a vaccinated (MS-H vaccine by eye drop (>105.7 colour changing units/bird)) non-challenged group (n = 5), two non-vaccinated challenged groups (n = 18 each) and two vaccinated challenged groups (n = 18 each). In the challenged groups, six seeder birds were intratracheally inoculated with 105.4 colony forming units (CFUs)/bird. Trachea swabs were taken at day (D)2, D3, D4, D5, D7, D9, D11, D14, D17, D21, D25, D28, D32, D35, D42 and D46 after contact with seeders and analyzed with a quantitative PCR able to detect the vaccine and field strain separately. The transmission rate and shedding were estimated using the susceptible exposed infectious transmission model and a linear mixed model, respectively. The mean shedding of the challenge strain was 106.4 CFU equivalents M. synoviae/g trachea mucus in vaccinates shedding MS-H, while in the birds not shedding the vaccine (non-vaccinates and vaccinates not shedding MS-H) it was 106.9 CFU equivalents M. synoviae/g trachea mucus. In vaccinates shedding MS-H, ß was 0.0012 (95% C.I.: 0.00048 - 0.0024), while in birds not shedding vaccine (non-vaccinates and vaccinates not shedding MS-H) a significantly higher ß of 0.022 (95% C.I.: 0.015 - 0.031) was found.

Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates.

Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

High-resolution melting-curve analysis of obg gene to differentiate the temperature-sensitive Mycoplasma synoviae vaccine strain MS-H from non-temperature-sensitive strains.

Temperature-sensitive (ts+) vaccine strain MS-H is the only live attenuated M. synoviae vaccine commercially available for use in poultry. With increasing use of this vaccine to control M. synoviae infections, differentiation of MS-H from field M. synoviae strains and from rarely occurring non-temperature-sensitive (ts-) MS-H revertants has become important, especially in countries where local strains are indistinguishable from MS-H by sequence analysis of variable lipoprotein haemagglutinin (vlhA) gene. Single nucleotide polymorphisms (SNPs) in the obg of MS-H have been found to associate with ts phenotype. In this study, four PCRs followed by high-resolution melting (HRM)-curve analysis of the regions encompassing these SNPs were developed and evaluated for their potential to differentiate MS-H from 36 M. synoviae strains/isolates. The nested-obg PCR-HRM differentiated ts+ MS-H vaccine not only from field M. synoviae strains/isolates but also from ts- MS-H revertants. The mean genotype confidence percentages, 96.9±3.4 and 8.8±11.2 for ts+ and ts- strains, respectively, demonstrated high differentiating power of the nested-obg PCR-HRM. Using a combination of nested-obg and obg-F3R3 PCR-HRM, 97% of the isolates/strains were typed according to their ts phenotype with all MS-H isolates typed as MS-H. A set of respiratory swabs from MS-H vaccinated specific pathogen free chickens and M. synoviae infected commercial chicken flocks were tested using obg PCR-HRM system and results were consistent with those of vlhA genotyping. The PCR-HRM system developed in this study, proved to be a rapid and reliable tool using pure M. synoviae cultures as well as direct clinical specimens.

Molecular typing of Iranian field isolates Mycoplasma synoviae and their differentiation from the live commercial vaccine strain MS-H using vlhA gene.

1. The single-copy domain of the N-terminal region of the vlhA gene of Mycoplasma synoviae was sequenced, analysed and verified and used to type Iranian field isolates of M. synoviae and the MS-H live vaccine strain. In addition, a restriction fragment length polymorphism (RFLP) method was developed to differentiate between field isolates of Iranian and MS-H vaccine strains. 2. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and dendrograms were constructed. Based on single nucleotide polymorphism (SNP) that existed in all field isolates in Iran, the PCR-RFLP method allowed the differentiation of all M. synoviae field isolates from the vaccine strain. 3. Using phylogenetic analysis, the isolates were assigned to 8 unique genotypes and, within each group, DNA had a high level of similarity. 4. DNA sequence analysis and PCR-RFLP of the amplicon based on percent similarity and evolutionary relationship appeared to be useful tools for strain differentiation whether M. synoviae clinical isolates from infected chickens were derived from the vaccine strain or wild-type strains. 5. This study confirms the potential value of strain typing for epidemiological purposes and suggests that phylogenetic studies are essential to understand the true relationships between strains.

Mutations in GTP binding protein Obg of Mycoplasma synoviae vaccine strain MS-H: implications in temperature-sensitivity phenotype.

Mycoplasma synoviae strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (ts (+)) vaccine used for control of M. synoviae infection in poultry worldwide. Genetic basis of temperature sensitivity and attenuation of MS-H has not been revealed thus far. Comparison of the complete genome sequence of MS-H, its parent strain 86079/7NS and two non-temperature sensitive (ts (-)) reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in ts (-) MS-H reisolates. Nucleotide change from G to A at position 369 of the obg gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete obg gene sequence in several MS-H reisolates revealed that a Gly123Arg substitution was associated with alteration in temperature sensitivity phenotype of MS-H. A second mutation, C to T at position 629, in obg gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. In silico analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions in vivo especially at non-permissive temperature. Findings of this study implicate Obg alteration (Gly123Arg) as one of the possible causes of MS-H attenuation/temperature sensitivity and warrant further investigations into exploring the role of Obg-like proteins, an evolutionarily conserved protein from human to bacteria, in the biology of mycoplasmas.

Prevalence of antibodies to Mycoplasma synoviae in laying hens and possible effects on egg shell quality.

Mycoplasma synoviae (M. synoviae) can cause respiratory disease, synovitis, or result in a silent infection in chickens and turkeys. The importance of M. synoviae is well established in broilers but only a few studies have been conducted in layers. In the present study, the prevalence of M. synoviae in commercial layer flocks was estimated using ELISA. For this study, 19 commercial layer flocks were selected randomly from New South Wales and Queensland region of Australia from producers who were willing to participate in the survey. Sixty eggs per flocks were randomly collected, out of these 30 eggs were used for ELISA and remaining 30 eggs were used to estimate various egg shell quality parameters. Subsequently, association between the serological status of eggs for M. synoviae and egg shell quality was studied. In the flocks under study, seroprevalence of M. synoviae was found to be high at 69% (95% confidence interval (CI)=41.3-89.0). Statistical analysis showed an association between serological status for M. synoviae and egg quality parameters such as translucency, shell breaking strength, % shell reflectivity and shell deformation. On the other hand, there was no significant association between serological status for M. synoviae and other egg quality parameters such egg weight, egg shell weight, % egg shell or shell thickness.

Characterization of the antigenic and functional domains of a Mycoplasma synoviae variant vlhA gene.

The Mycoplasma synoviae haemagglutinin gene, vlhA, encodes two major immunodominant and surface-exposed membrane proteins, MSPB and MSPA. Both products are antigenically variable but only MSPA mediates binding to erythrocytes. Previously we have shown that M. synoviae type strain WVU 1853 could express a variant vlhA gene, referred to as MS2/28.1, with a considerably shorter and divergent MSPA region. A finding that prompted detailed characterization of its antigenic and functional properties. Here we mutagenized each of the six opal codons of the variant MS2/28.1 vlhA member into tryptophan, thus allowing its expression in Escherichia coli as well as its cleavage products, MSPB and MSPA. In addition, we expressed 5 contiguous regions of MS2/28.1 extending from the last part of MSPB to the C-terminus of MSPA. Colony immunostaining with region-specific antisera mapped antigenic variation to the N-terminal half of MS2/28.1 MSPA. No haemagglutinating activity was observed for MSPB, but consistent haemadsorption inhibition was mapped to the region extending from amino acid 325 to 344. Inhibition of both haemagglutination and haemadsorption activities were obtained with sera directed against the C-terminal region of MSPA, with the highest titers (1/320 and 1/160, respectively) being recorded for its last 60 residues. Importantly, antibodies to this region also yielded the highest metabolic inhibition titer of 1/1280. Overall, aside from mapping the functional domains of a M. synoviae highly divergent haemagglutinin gene, this study shows that the C-terminal half of its MSPA region induced the highest titers of antibodies inhibiting haemagglutination, haemadsorption, and metabolism.

Rapid serodiagnosis with the use of surface plasmon resonance imaging for the detection of antibodies against major surface protein A of Mycoplasma synoviae in chickens.

Mycoplasma synoviae, a major worldwide pathogen in poultry, causes respiratory tract infection and arthritis in chickens and turkeys. Two major surface antigens of M. synoviae are encoded by a single gene, vlhA (variably expressed lipoprotein and hemagglutinin). The gene product is cleaved post-translationally to yield the lipoprotein major surface protein (MSP) B (MSPB) and the hemagglutinin MSPA. The availability of MSPA as an antigen for serodiagnosis was studied by means of a protein chip based on surface plasmon resonance imaging (SPRi). The diagnostic potential of SPRi for measurement of levels of antibody to MSPA was compared with that of a conventional enzyme-linked immunosorbent assay (ELISA) kit. The results from SPRi, a process that took only 1 h, were similar to those from ELISA. Therefore, MSPA can be used as an antigen for serologic studies, and SPRi, a label-free and high-throughput method, may be a valuable tool in avian serodiagnostic studies.

[Mycoplasma synoviae-associated egg-pole shell defects in laying hens]. / Mycoplasma synoviae-assoziierte Eischalenpoldefekte bei Legehennen.

Hens laying eggs with egg-pole shell defects (EPS) were examined in a clinical prospective study. 86 hens with EPS and 72 hens without EPS from three flocks were selected for this study. It could be proven serologically that hens with EPS had significant (p < 0.05) higher titers against Mycoplasma (M.) synoviae then hens without EPS. PCR tested cloacal swabs for M. synoviae were more frequently positive from hens with EPS (87%; n=72) then from hens without EPS (18%; n=13). Furthermore, M. synoviae could be cultivated from the oviduct of five hens with EPS. Additionally, M. synoviae-DNA was detectable in the albumen of nearly all eggs with EPS (n=48; 98%), contrary to the eggs without EPS (n=11; 26%). Ultrastructural investigation revealed that eggs with EPS showed considerable differences of the egg shell structure as well as the cross section dimension according to eggs without EPS. Due to the significantly more frequent detection of M. synoviae-DNA from the cloaca of chickens with EPS, is an involvement of M. synoviae in laying of EPS in the surveyed herds likely. Further infection experiments with the isolated M. synoviae were not perfomed, therefore about the causal pathogenic role of M. synoviae in the development of eggs with EPS in the surveyed herds can only be speculated.

Effect of a live Mycoplasma synoviae vaccine on the production of eggshell apex abnormalities induced by a M. synoviae infection preceded by an infection with infectious bronchitis virus D1466.

An experimental study was conducted to assess the effect of a live Mycoplasma synoviae vaccine (Vaxsafe MS; Bioproperties Pty Ltd, Ringwood, Victoria, Australia) on M. synoviae-induced eggshell apex abnormalities (EAA). Four experimental groups of specified-pathogen-free white laying hens were made. All groups were inoculated with infectious bronchitis virus D1466 at 18 weeks of age. One group did not receive further treatment (non-vaccinated non-challenged (NVNC)). Two groups were vaccinated at 14 weeks of age against M. synoviae, and one of these groups was also challenged with an EAA-inducing M. synoviae strain 5 days after infectious bronchitis virus challenge (vaccinated non-challenged (VNC) and vaccinated challenged group (VC), respectively). The fourth group was not vaccinated but was challenged with M. synoviae (non-vaccinated challenged (NVC)). Eggs with EAA eggs were produced only in the NVC and VC groups. However, the proportion of eggs with EAA and the mean daily production of eggs with EAA per chicken was significantly lower (P<0.05) in the VC group (88/741 (11.9%) and 0.09+/-0.01 eggs per hen) compared with the NVC group (148/646 (22.9%) and 0.14+/-0.01 eggs per hen). The mean daily egg production per chicken was significantly lower in the NVC group (0.48+/-0.03 eggs) compared with that of the NVNC group (0.60+/-0.03 eggs), but not significantly different from other groups. The eggshell strength of eggs with EAA (22.8 N) was significantly lower (P<0.05) than non-affected eggs from the other groups (33.7 to 39.5 N). Furthermore, the eggshell strength of non-affected eggs in the NVC group was significantly lower (P<0.05) compared with that of non-affected eggs from the flock of origin (33.7 versus 41.2 N), but not different from the other groups. It can be concluded from the present study that vaccination with a live M. synoviae vaccine reduces the occurrence of M. synoviae-induced EAA significantly.

Induction of eggshell apex abnormalities by Mycoplasma synoviae: field and experimental studies.

A novel eggshell pathology, characterized by an altered shell surface, thinning, increased translucency, and cracks and breaks in the eggshell apex, has become increasingly common in layer flocks of various breeds in The Netherlands. Two field studies found an association between the eggshell apex abnormalities (EAA) and infection with Mycoplasma synoviae. M. synoviae was isolated from the oviduct of birds that produced abnormal eggs, but not from birds in control flocks, although both affected and control birds had agglutinating antibodies against M. synoviae. After a single injection with long-acting oxytetracycline, the production of abnormal eggs ceased, but then reoccurred about 12 days later. A causal relationship between EAA and M. synoviae infection was subsequently demonstrated experimentally. EAA occurred after intratracheal inoculation of birds with M. synoviae, and even more frequently in birds infected with infectious bronchitis virus 5 days before inoculation of M. synoviae. EAA also occurred, although less frequently, in birds inoculated intravenously with M. synoviae and infected with infectious bronchitis virus. EAA did not occur in birds only inoculated intravenously with M. synoviae. M. synoviae was only isolated from the oviducts of birds producing abnormal eggs. The mean daily egg production was reduced in all groups infected with M. synoviae. Examination of the eggshells by scanning electron microscopy revealed that the mammillary layer of the calcified zone was absent and that the inner eggshell membranes were thicker. Isolates of M. synoviae from the oviducts of birds from farms producing abnormal eggs were examined by amplified fragment-length polymorphism analysis and did not appear to be clonal.

A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis infections.

We have previously identified species-specific DNA fragments, referred to as MS2/28 and Mm14, of Mycoplasma synoviae and Mycoplasma meleagridis, respectively. In the present study, we extended our analysis of the MS2/28 fragment that was found to encode a species-specific antigenic site, and we demonstrated the specificity of the Mycoplasma gallisepticum hemagglutinin protein encoded by pMGA1.2 (a member of the vlhA gene family). Then, we combined the Escherichia coli-expressed products of MS2/28, Mm14, and pMGA1.2, to develop a recombinant antigen-based enzyme-linked immunosorbent assay (recELISA), for the simultaneous and specific detection of antibodies to the three aforementioned major avian mycoplasma species. For comparative purposes, a novel in-house crude antigen capture ELISA (capELISA) was developed in parallel. In the latter protocol, the microtiter wells were enriched in species-specific antigens by capturing sonicated crude antigens on coated rabbit polyclonal antibodies that had been extensively adsorbed with the whole antigen of the heterologous species. With regard to rapid serum agglutination, both ELISA tests were highly specific, and they showed a significant correlation when field sera from naturally infected birds were tested. recELISA proved to be highly specific because absorbance values, with the heterologous species, were significantly lower (P < 0.001) than those obtained with capELISA. Given its cost-effectiveness and simplicity, the recombinant antigen-based ELISA seems to represent a valid tool for the specific screening of the three major avian mycoplasma species. recELISA will be particularly useful with regard to trade control because a large number of samples from various fields could be rapidly processed.

Identification of major immunogenic proteins of Mycoplasma synoviae isolates.

Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively.

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.

Mycoplasma synoviae lipoprotein MSPB, the N-terminal part of VlhA haemagglutinin, induces secretion of nitric oxide, IL-6 and IL-1beta in chicken macrophages.

Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing respiratory disease and infectious synovitis. M. synoviae haemagglutinin VlhA is an abundant surface-exposed lipoprotein and immunodominant antigen. Post-translational cleavage of VlhA generates two proteins, MSPB and MSPA. MSPB, the amino-terminal end of VlhA, is a lipoprotein of about 40-50 kDa but can appear in truncated forms (tMSPB) of about 20-30 kDa. The aim of this study was to determine whether MSPB and tMSPB can stimulate chicken macrophages to secrete NO and cytokines. Macrophages derived from chicken monocytes (MDM) or the MQ-NCSU macrophage cell line were stimulated with M. synoviae protein extracts containing MSPB or tMSPB, as well as with purified MSPB and tMSPB. Proteins from detergent extractions induced IL-6 secretion in MDM and NO secretion in MQ-NCSU. Both MSPB and tMSPB were capable of inducing NO secretion in MQ-NCSU, as well as IL-6 and IL-1beta in MDM. The activity of IL-6 induced by purified tMSPB was similar to the effect of 60 pg/ml of recombinant chicken IL-6. The effect of IL-1beta induced by tMSPB was comparable to the effect of 10 ng/ml of recombinant IL-1beta. Whereas all samples containing MSPB were able to induce NO, IL-6 and IL-1beta, it seemed that the purified tMSPB of about 20 kDa was the most potent in its ability to induce IL-6 and IL-1beta in MDM. Compared to MSPB, tMSPB lacks about 200 amino acids in its carboxyl-terminal part. Therefore, our results suggest that the major part of the stimulating activity is associated with the amino-terminal part of MSPB, most likely with its lipid moiety.