Multilocus sequence analysis for Mycoplasma synoviae molecular genotyping.

The aim of this study was to identify molecular techniques which enable clear discrimination between Mycoplasma synoviae isolates for improved epidemiology. Multilocus sequence analysis (MLSA) of 6 M. synoviae loci was conducted for genotyping of isolates with previously determined 5'-vlhA sequences. Sequencing of three polymorphic genes (5'-vlhA, cysP and nanH) enables good discrimination between isolates with different genotypes. Such a genotyping scheme revealed 10 distinct genotypes, which were confirmed by sequencing of an additional three loci of the M. synoviae genome. Epidemiologically linked strains formed clusters with the same genotypes which clearly differed between clusters. MLSA used in this study is a promising tool for epidemiology of M. synoviae isolates, but it should be evaluated by further investigations using a much higher number of M. synoviae strains.

Distribution of chicken cathepsins B and L, cystatin and ovalbumin in extra-embryonic fluids during embryogenesis.

1. Concentrations of chicken cathepsin B, cathepsin L, cystatin and ovalbumin were determined in the allantoic fluid, amniotic fluid and extracts of chorioallantoic membranes during days 6 to 12 of embryogenesis. 2. Similar trends for cystatin and ovalbumin were observed in the allantoic fluid with maximum concentrations of cystatin on day 7 (12 ± 4 µg/ml) and ovalbumin on day 8 (∼19 ± 2.5 µg/ml) of embryonic development. The highest concentrations of cathepsin B was found on day 7 and of cathepsin L on day 10, but were significantly lower than those of cystatin and ovalbumin. 3. In the allantoic fluid, especially on day 7, considerable proportions of cystatin and ovalbumin were phosphorylated and contained phosphorylated serine. 4. Concentrations of cathepsin B and L, cystatin and ovalbumin in the amniotic fluid were variable but were comparable to those in allantoic fluid.

Characterisation of Mycoplasma gallisepticum strains involved in respiratory disease in pheasants and peafowl.

Two cases of Mycoplasma gallisepticum infection in different avian species in backyard gamebird operations in Slovenia were investigated. In the first case, M gallisepticum was associated with severe respiratory disease with almost 20 per cent mortality in pheasants, whereas the infection was less pathogenic for chickens and turkeys reared at the same site. The M gallisepticum isolates from pheasants had a unique pMGA gene sequence containing a repeat of 12 nucleotides, and they contained only small amounts of the cytadhesins MGC1 and MGC3 and no PvpA protein. However, they expressed some typical M gallisepticum proteins and several proteins which were immunogenic for pheasants, chickens and turkeys. A strain of M gallisepticum isolated from the sinus of a pheasant was highly pathogenic for chicken embryos. In the second case, the M gallisepticum strain that was associated with respiratory disease and mortality in peafowl also affected chickens. M gallisepticum strain ULB 992 was isolated from the infraorbital sinus of a dead peafowl. The ULB 992 strain synthesised a small amount of MGC3, a truncated form of MGC1 and lacked PvpA. However, it expressed several proteins which were immunogenic for the birds infected with M gallisepticum at both gamebird operations.

Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin.

Mycoplasma synoviae is a major avian pathogen that synthesizes hemagglutinin VlhA, an abundant immunodominant surface lipoprotein. In most M. synoviae strains, the VlhA protein cleaves into the N-terminal part, a lipoprotein MSPB, and a C-terminal part MSPA, which mediates binding to erythrocytes. VlhA is encoded by the vlhA gene of which the 5'-end is present in the genome as a single copy, which does not change its sequence during recombination of the vlhA gene with pseudogenes. In this study, sequence analyses of the 5'-end vlhA sequences of 30 M. synoviae isolates revealed a highly polymorphic region encoding the proline-rich repeats (PRR) in the N-terminal part of MSPB. Pathogenic strain K1968 had an insertion encoding sequence DNPQNPN in PRR, whereas strains F10-2AS, K2581, K3344 and five strains belonging to two related clusters of strains isolated recently from chickens in Slovenia lacked one PRR repeat of 19 amino acids. The predicted length variations correlated well with the lengths of the corresponding MSPB proteins detected in immunoblots with specific antibodies. Comparison of the 5'-end vlhA sequences of 30 M. synoviae strains showed 11 different types of vlhA sequences indicating that the analysis of this vlhA part is useful for strain differentiation. Distinct sequence motifs seem to be characteristic for vlhA genes of individual M. synoviae strains or clusters of strains and can be used as markers for tracing their spreading between poultry farms.

Neurological symptoms in patients whose cerebrospinal fluid is culture- and/or polymerase chain reaction-positive for Mycoplasma pneumoniae.

We describe 13 patients with neurological signs and symptoms associated with Mycoplasma pneumoniae infection. M. pneumoniae was isolated from the cerebrospinal fluid (CSF) of 9 patients: 5 with meningoencephalitis, 2 with meningitis, and 1 with cerebrovascular infarction. One patient had headache and difficulties with concentration and thinking for 1 month after the acute infection. M. pneumoniae was detected, by means of PCR, in the CSF of 4 patients with negative culture results. Two had epileptic seizures, 1 had blurred vision as a consequence of edema of the optic disk, and 1 had peripheral nerve neuropathy.

Intrathecal synthesis of specific antibodies in patients with invasion of the central nervous system by Mycoplasma pneumoniae.

Mycoplasma pneumoniae commonly causes respiratory tract infections in humans, but it may also be associated with central nervous system manifestations. The aim of the present study was to determine whether the cerebrospinal fluid taken from patients with neurologic symptoms due to Mycoplasma pneumoniae infection contains specific antibodies and whether the detection of these antibodies can be used for diagnosis. Mycoplasma pneumoniae was isolated from the cerebrospinal fluid taken from nine patients with central nervous system symptoms on admission to the hospital. In addition, Mycoplasma pneumoniae was detected in cerebrospinal fluid using polymerase chain reaction in four other patients. Antibodies to Mycoplasma pneumoniae were detected using the enzyme immunosorbent assay, indirect immunoperoxidase assay and immunoblotting in cerebrospinal fluid samples from 14 of 19 patients included in the study. The indirect immunoperoxidase assay showed high titers of Mycoplasma pneumoniae immunoglobulin G1 (IgG1) and IgM antibodies in cerebrospinal fluid samples of some patients with meningoencephalitis or meningitis. Titers of specific IgA, IgG2 and IgG3 antibodies were lower, while specific IgG4 was not detectable. Cerebrospinal fluid samples with higher antibody titers also contained IgA, IgG1, IgG2, IgG3 and IgM antibodies that recognized the P1 adhesin (170 kDa protein) of Mycoplasma pneumoniae. A comparison of antibody titers of concomitant serum/cerebrospinal fluid samples to Mycoplasma pneumoniae and those to measles virus by enzyme immunosorbent assay suggested the intrathecal synthesis of IgG and IgM antibodies to Mycoplasma pneumoniae in patients with acute meningoencephalitis. Data from this study clearly reinforce previous findings that Mycoplasma pneumoniae is an etiologic agent of central nervous system infections in humans.

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum.

Antigenic variants of Mycoplasma gallisepticum major surface lipoprotein, pMGA, are encoded by a large gene family. In this study sequence analyses of the PCR-amplified pMGA genes showed two types of sequences similar to the pMGA1.2 gene in M. gallisepticum strains. They differed in the sequence encoding a proline-rich region (PRR) at the N-terminus of the pMGA protein. The type A genes had sequences similar to the published pMGA1.2 gene sequence of strain S6, whereas the type B genes lacked the second repetitive segment encoding PTPN sequence within PRR and were similar to the published sequence of PG31 strain. Low in vitro passages of M. gallisepticum strains isolated recently in Slovenia from four avian species showed very different expression patterns of pMGA1.2 and pMGA1.9 genes. Among isogenic populations of S6(B) and IHB1 strains a high frequency of pMGA antigenic variants lacking an epitope for monoclonal antibody (mAb) 71 was found. Strain IHB1 clones, which synthesized pMGA recognized by mAb 71, transcribed pMGA genes whose partial sequence encoded the amino acid sequence (262)TNGDEPRSVS of the mAb 71 epitope. Other IHB1 clones synthesized pMGA variants with different isoelectric points, lacking the epitope for mAb 71, but expressing downstream epitopes for other mAbs. Our study suggests that a molecular basis for pMGA antigenic variation lies in the corresponding changes at the DNA level.

Infection of turkeys with Ornithobacterium rhinotracheale and Mycoplasma synoviae.

Within 1 mo, two separate outbreaks of respiratory disease occurred in two flocks on the multiage market turkey farm in Slovenia. More severe dinical signs and higher mortality were observed in male birds. Ornithobacterium rhinotracheale (ORT) was isolated in pure culture from tracheas of the affected birds in both outbreaks. Commercial enzyme-linked immunosorbent assay test showed the presence of antibodies to ORT in sera of birds from both clinically affected flocks and also in two flocks of younger birds without clinical sings. Immunoblotting with ORT culture isolated during the outbreak as an antigen confirmed the presence of antibodies to ORT in sera of turkeys of all four flocks examined. In addition, three different serologic assays also detected antibodies to Mycoplasma synoviae (MS) in three out of four flocks. The concomitant infection with MS did not show an obvious effect on mortality rates nor on the antibody response against ORT. Younger birds appeared to be less susceptible to ORT pathogenicity because in those flocks the infection was subclinical.

The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences.

An abundant cytoplasmic 43-kDa protein from Mycoplasma synoviae, a major pathogen from poultry, was identified as elongation factor Tu. The N-terminal amino acid sequence (AKLDFDRSKEHVNVGTIGHV) has 90% identity with the sequence of the Mycoplasma hominis elongation factor Tu protein. Monoclonal antibodies reacting with the M. synoviae elongation factor Tu protein also reacted with 43-kDa proteins from the avian Mycoplasma species Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma pullorum, Mycoplasma cloacale, Mycoplasma iners and Mycoplasma meleagridis, but not with the proteins from Mycoplasma gallisepticum, Mycoplasma imitans or Mycoplasma iowae. In addition, two groups of phase variable integral membrane proteins, pMSA and pMSB, associated with hemadherence and pathogenicity of M. synoviae strains AAY-4 and ULB925 were identified. The cleavage of a larger hemagglutinating protein encoded by a gene homologous to the vlhA gene of M. synoviae generates pMSB1 and pMSA1 proteins defined by mAb 125 and by hemagglutination inhibiting mAb 3E10, respectively. The N-terminal amino acid sequences of pMSA proteins (SENKLI ... and SENETQ ...) probably indicate the cleavage site of the M. synoviae strain ULB 925 hemagglutinin.

The hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens more frequently than does the hemagglutination-negative phenotype.

Inoculation with hemagglutination-positive (HA+) cultures of Mycoplasma synoviae AAY-4 induced acute synovitis significantly more frequently (P = 0.001) in chicken tibiotarsal-tarsometatarsal joints than did inoculation with HA-negative (HA-) cultures derived from the same clone of AAY-4. Immunoblotting analyses showed that HA+ cultures abundantly expressed two phase-variable hemadherence-associated surface membrane proteins of 53 kDa and 48 to 50 kDa defined by monoclonal antibodies. HA- cultures lacked the 53-kDa proteins and synthesized truncated 27- to 30-kDa forms of the 48- to 50-kDa proteins. Inoculation of cyclosporin A (CsA) into infected joints significantly decreased the frequency of acute synovitis (P = 0.001). Moreover, repeated intra-articular inoculation of CsA (three doses of 1 mg at 2-day intervals) significantly reduced the local antibody response to M. synoviae in the joints treated with CsA.

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains.

Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by EcoRI, HindIII, BglII, RsaI and BamHI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by DdeI enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.

Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies.

Twelve monoclonal antibodies (Mabs) against Mycoplasma gallisepticum (Mg) strains F, R, S6(208) and PET2 were used for analysis of epitopes of 22 Mg strains. Six Mabs recognized surface epitopes in the majority of strains, but did not react with variant strains like K 503 and K 703. Two Mabs reacted with epitopes on about 56 kilodalton (kDa) proteins and showing consistent expression on Mg colonies. Three Mabs recognized three different variable surface epitopes associated with about 67 kDa proteins and one Mab variable epitope on about 33 and 80 kDa proteins. Two-dimensional immunoblotting showed considerable differences in the charge of proteins bearing variable surface epitopes in different Mg strains. Subcloning of four low passage Mg strains using Mabs for screening populations that derived from a single colony with defined surface epitopes showed that some colonies may switch surface epitopes associated with 67 and 80 kDa proteins. This switching was reversible and generated subpopulations of Mg expressing different combinations of surface epitopes. Phenotypic switching of epitopes probably occurs also in vivo and may be the mechanism enabling Mg to evade the host immune response.

Combination of immunofluorescence and immunoperoxidase techniques for serotyping mixtures of Mycoplasma species.

We describe a method for the simultaneous identification of up to three Mycoplasma species by the use of contrast-labeled fluorescent antibodies against two species and peroxidase-labeled antibody against a third species. The procedure enabled the rapid identification of colonies of three artificially mixed avian Mycoplasma species on agar blocks and also mixtures of species in cultures from naturally infected chickens. Furthermore, it was possible to quantitate the components of a mixture of Mycoplasma gallisepticum, M. synoviae, and M. gallinarum. This new procedure offers an improvement over existing methods in terms of both speed and analytical capability.

Genotypic heterogeneity among strains of Mycoplasma gallinarum.

Seven field strains of Mycoplasma gallinarum isolated from chickens on three local farms and the type strain PG 16 were compared by restriction endomiclease DNA analysis using EcoRI, HindIII, BamHI, PstI and Xhol. The restriction patterns generated by the first four enzymes indicated considerable DNA polymorphisms among M. gallinarum strains. Field strains revealed 3 distinct patterns of the genomic DNA which differed from that of the PG 16 strain. BamHI enabled easy distinguishing between related and unrelated strains due to the fragments being characteristic for certain strains. Interestingly, the characteristic patterns were associated with the corresponding chicken selection and this suggests that DNA analysis has a potential use for studying the epidemiology of M. gallinarum.

Reactions of chicken biliary immunoglobulin A with avian mycoplasmas.

Chicken bile was examined for mycoplasma by culture and for antibody against mycoplasma by indirect immunoperoxidase assay (IIPA) detecting chicken either IgA or IgG and IgM, as well as by rapid plate agglutination (RPA), haemagglutination-inhibition (HI) and double immuno-diffusion (DID). Cultures indicated the presence of M. gallisepticum (Mg) and Ai. synoviae (Ms) in bile and their isolations were positively correlated with those from upper respiratory tract. In 45 chickens from five flocks examination of biliary samples with IIPA detected considerably higher rates of Mg and Ms antibodies than the same assay performed with sera especially those of IgA class of antibody. In chickens with a strong serological response to Mg, titres of specific agglutinins and HI antibody in biliary samples were significantly higher than those found in sera. Only biliary fractions containing Ig exhibited antibody activity.

Humoral and local antibodies in chickens with mixed infection with three Mycoplasma species.

Eight one-year-old commercial layer hens with strong humoral antibody response to Mycoplasma synoviae were examined for mycoplasmas. Cultures from the respiratory tract, the Harderian gland and the oviduct showed the group to be infected with M. synoviae, M. gallisepticum, M. gallinarum and Bordetella avium. The humoral antibody response to M. synoviae was strongly positive and supported by high titre specific IgG and IgM class antibody, while that against M. gallisepticum and M. gallinarum was weak or undetectable. However, an antibody response to M. gallisepticum and M. gallinarum was demonstrated in the Harderian gland, respiratory secretions and oviduct of some birds along with antibody to M. synoviae. In contrast with serum, antibodies of IgA and IgG classes against M. gallisepticum were observed in the extracts of spleen of all birds. These data indicate that adult chickens are capable of mounting an antibody response against at least three Mycoplasma species during a mixed infection but it may be necessary to examine tissues as well as serum to demonstrate them.

Indirect immunoperoxidase assay for the detection of antibody in chicken Mycoplasma infections.

The indirect immunoperoxidase assay (IIPA) was used to examine chicken sera, egg yolks, respiratory secretions and synovial fluids for antibodies to M. gallisepticum and M. synoviae. IIPA was more sensitive than rapid serum agglutination (RSA) and haemagglutination inhibition (HI) for detecting serum antibodies in chickens infected with M. gallisepticum. It was also more sensitive than culture. In chickens infected with M. synoviae there was very good correlation between IIPA and RSA and between IIPA and HI, but culture was a more sensitive means of detecting infection than any of these tests. IIPA of yolk or respiratory secretions appeared to be a suitable means of detecting M. gallisepticum infection but, again, culture was more sensitive for M. synoviae detection. IIPA detected antibodies in the synovial fluid of a few infected chickens but the technique needs further evaluation. It was not successful in detecting antibodies to certain of the nonpathogenic avian mycoplasmas.

Natural infection of ducks with Mycoplasma synoviae and Mycoplasma gallisepticum and Mycoplasma egg transmission.

Mycoplasma gallisepticum (MG), M. synoviae (MS), M. cloacale (MC) and M. anatis were isolated from ducks kept in a yard in close contact with chickens that were infected with MG, MS and some other avian Mycoplasma species. MG, MS and MC were isolated also from embryonated duck eggs and from infertile duck eggs laid during the first four weeks of egg production. Infected ducks did not show clinical signs of MG or MS infection in chicken. Detectable MG and MS agglutinating antibodies were not present in duck sera. However, they were found in two yolks of 10 tested from embryonated eggs. In the haemagglutination - inhibition (HI) tests yolks from embryonated eggs yielded significantly higher (P<0.01) titres of MS antibodies than duck sera. Geometric mean value of MS HI titres in tested duck sera was 20, while those of yolks from embryonated eggs was 333. It is probably the first report concerning isolation of MS from the naturally infected ducks and furthermore, concerning isolation of MG, MS and MC from naturally infected embryonated eggs.

Mycoplasma species in chicken flocks with different management systems.

Chicken flocks hatched together but reared under different management systems were examined for mycoplasmas over a two-year period. On the farm A multiple-age flocks were reared in close contact. This farm had not been depopulated for over 15 years. On farms B, C and D single-age flocks were reared and the farms were depopulated every year. On farm A 218 birds from 20 flocks were tested: mycoplasmas were isolated from 202 (92.7%). On farms B, C and D 289 birds from seven flocks were tested: mycoplasmas were recovered from 55 (19.0%). On farm A the following mycoplasmas were identified: M. gallisepticum (46.9% of isolates), M. gallinarum (47.8%), M. pullorum (46.3%), M. gallinaceum (43.2%), M. iners (10.9%), M. iowae (8.3%),M. synoviae (51.7%), M. lipofaciens (23.8%) and M. glycophilum (16.7%). Furthermore, mycoplasma strains which do not belong to recognised species of avian mycoplasmas, Acholeplasma laidlawii and an unidentified Acholeplasma strain were also isolated. On farms B, C and D the isolates were identified as M. gallisepticum or M. synoviae. The only exception was one culture from which M. gallinarum was recovered. The differences between farm A and farms B, C and D regarding total mycoplasma isolation yields and incidence of their species are significant (/K0.01) and are attributed to the different management systems.

Mycoplasma species isolated from six avian species.

Mycoplasmas were isolated from chickens, chicken embryos, turkeys, ducks, geese, pigeons and Japanese quail and their embryos. Altogether 792 birds and embryos were examined and 411 of them (52%) were infected with mycoplasmas. Isolates were identified by indirect immunofluorescence using monospecific rabbit antisera against 16 recognised species of avian Mycoplasma (AM) and two species of Acholeplasma. In all 633 strains of Mycoplasma were detected and most of these belonged to recognised AM species. M. anatis was found only in ducks and geese; M. columbinasale, M. columbinum and M. columborale only in pigeons, while M. meleagridis and M. gallopavonis isolates were exclusive to the turkey. M. synoviae was the most frequently isolated species (172 isolates). The host range of M. gallisepticum was the same as M. synoviae but with slightly fewer isolations. M. gallinarum, M. gallinaceum, M. pullorum, M. glycophilum and M. lipofaciens were not uncommon but were mainly confined to the chicken. The exceptions were isolates of M. lipofaciens from a turkey and a duck and an isolate of M. gallinarum from a turkey. M. cloacale was isolated from a chicken, a turkey and a duck. Acholeplasmas and untyped strains were isolated from all the host species except Japanese quail. Isolations of M. synoviae from a pigeon and Japanese quail and M. cloacale from a chicken, are thought to be new findings.