Rebeka Lucijana Berčič

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5+/-1.5%) and CEC-32 (RIF 7.0+/-0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R(low). Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2+/-0.3%) similar to that of R(low) (1.1+/-0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.

A survey of avian Mycoplasma species for neuraminidase enzymatic activity

Among 23 currently recognized avian Mycoplasma (AM) species only Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae cause disease and loss of production in chickens and/or turkeys. Because neuraminidases are considered virulence factors in many pathogenic microorganisms the aim of our study was to determine which AM species possess neuraminidase enzymatic activity (NEAC). Small samples of AM cells were assayed for NEAC using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-alpha-d-N-acetylneuraminic acid. In the case of positive NEAC reaction the substrate gave the insoluble indigoblue product what enabled simple test and easy estimation of NEAC. M. gallisepticum and M. synoviae which share sequences of the gene encoding neuraminidase (sialidase NanH) exhibited considerable levels of NEAC. However, NEAC levels differed among their strains, as well as among cultures of different strains. Only certain cultures of the type strain of M. meleagridis showed NEAC, whereas among six serovars of M. iowae only serovar I (type strain 695) showed NEAC. Weak NEAC was detectable in M. anseris, M. cloacale and M. pullorum, whereas the type strain of M. corogypsi (BV1) showed strong NEAC. Our study provides novel informations about NEAC in AM species and suggests that higher invasiveness and possibly, the pathological processes might be associated with their NEAC.

Neuraminidase of Mycoplasma synoviae desialylates heavy chain of the chicken immunoglobulin G and glycoproteins of chicken tracheal mucus

Major poultry pathogens, Mycoplasma gallisepticum and Mycoplasma synoviae share several genes, including nanH that encodes their sialidases (neuraminidases). Previous studies have shown considerable differences in neuraminidase enzymatic activity (NEAC) in M. synoviae strains and NEAC absence in individual cultures of two strains, ULB 925 and ULB 9122. The present study shows that the cultures lacking NEAC did not express NanH neuraminidase detectable by specific antibodies. In cultures of M. synoviae ULB 925 and ULB 9122, which lacked NEAC and detectable NanH, deletions of a single adenine in different nanH regions of each strain created translational frameshifts resulting in TAA (UAA) stop codons and premature termination of translation. ULB 925 and ULB 9122 with such nanH mutations did not desialylate reference fetuin and transferrin or chicken glycoproteins that M. synoviae strains with NEAC efficiently desialylated. They desialylated several chicken serum glycoproteins with SAα(2–6)gal moieties, including the immunoglobulin G heavy chain. Neuraminidase inhibitor 2,3-didehydro-2-deoxy-N-acetylneuraminic acid inhibited such desialylation otherwise caused by M. synoviae WVU 1853 neuraminidase. WVU 1853 also cleaved sialic acid from SAα(2–3)gal moieties from glycoproteins of mucus from chicken tracheas. This is the first demonstration that M. synoviae desialylates glycoproteins of its host.

Variation of vlhA gene in Mycoplasma synoviae clones isolated from chickens

Mycoplasma synoviae synthesizes haemagglutinin VlhA, which cleaves into the N-terminal part, a lipoprotein MSPB, and a C-terminal part MSPA. Previous studies have shown that the 3′-end of the expressed vlhA gene can recombine with vlhA pseudogenes in a process called gene conversion, but there have been no data about diversification of the expressed vlhA gene in M. synoviae populations replicating in chickens. Following intratracheal inoculation with the M. synoviae strain ULB 02/T6, which showed only minor vlhA gene variation prior to inoculation, we investigated temporal changes in MSPB epitopes defined by monoclonal antibodies (mAbs) 3B4 and 50, as well as diversification of the vlhA gene sequence in M. synoviae populations recovered from chicken tracheas. In cultures isolated 8 and 18 days post inoculation (p.i.), most colonies showed variation of MSPB epitopes for mAbs 3B4 and 50. They also changed 3′-end vlhA gene sequences. Further diversity of the vlhA gene occurred in cultures isolated 8 weeks and 5 months p.i. The vlhA gene sequences from isolated cultures shared only 65 to 80% sequence identity with vlhA gene of the inoculated ULB 02/T6 culture. Notably, in most of those cultures their vlhA gene sequences contained stop codons potentially causing premature terminations of translation. Interestingly, in one culture isolated 8 weeks p.i. (clone T6-8W/IT2A) the 3′-vlhA gene sequence was identical in the last 1140 bases to that of the first vlhA pseudogene positioned the most far (upstream) of the expressed vlhA gene. This is the first demonstration of temporal diversity of the vlhA gene in M. synoviae populations isolated from chicken tracheas.

Shared epitopes of avian immunoglobulin light chains

Like all jawed vertebrates, birds (Aves) also produce antibodies i.e. immunoglobulins (Igs) as a defence mechanism against pathogens. Their Igs are composed of two identical heavy (H) and light (L) chains which are of lambda isotype. The L chain consists of variable (VL), joining (JL) and constant (CL) region. Using enzyme immunoassays (EIA) and two monoclonal antibodies (mAbs) (3C10 and CH31) to chicken L chain, we analysed their cross-reactivity with sera from 33 avian species belonging to nine different orders. Among Galliformes tested, mAbs 3C10 and CH31 reacted with L chains of chicken, turkey, four genera of pheasants, tragopan and peafowl, but not with sera of grey partridge, quail and Japanese quail. Immunoglobulins of guinea-fowl reacted only with mAb 3C10. Both mAbs reacted also with the L chain of Eurasian griffon (order Falconiformes) and domestic sparrow (order Passeriformes). Sera from six other orders of Aves did not react with either of the two mAbs. EIA using mAbs 3C10 and CH31 enabled detection of antibodies to major avian pathogens in sera of chickens, turkeys, pheasants, peafowl, Eurasian griffon and guinea-fowl (only with mAb 3C10). The N-terminal amino acid sequence of pheasant L chain (19 residues) was identical to that of chicken. Sequences of genes encoding the L chain constant regions of pheasants, turkey and partridge were determined and deposited in the public database (GenBank accession numbers: FJ 649651, FJ 649652 and FJ 649653, respectively). Among them, amino acid sequence of pheasants is the most similar to that of chicken (97% similarity), whereas those of turkey and partridge have greater similarity to each other (89%) than to any other avian L chain sequence. The characteristic deletion of two amino acids which is present in the L chain constant region in Galliformes has been most likely introduced to their L chain after their divergence from Anseriformes.

Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species.

Neuraminidases are virulence factors in many pathogenic microorganisms. They are present also in some Mycoplasma species that cause disease in birds, dogs and alligators. Thirty-seven Mycoplasma species have been examined previously for neuraminidase (sialidase) activity, whereas many of the species causing disease in man, ruminants, pigs, rodents and other animals have not. In this study neuraminidase enzymatic activity (NEAC) was examined in 45 previously untested Mycoplasma species, including those causing diseases in man, farm animals and laboratory animals. The only species in which NEAC was found was Mycoplasma neurolyticum, specifically, its type strain (Type A(T)) which is capable of inducing neurologic signs in inoculated young mice and rats. The NEAC of washed cells was relatively weak, but it differed even more than 10-fold among cells of cultures derived from individual colonies of M. neurolyticum. A weak NEAC was also detected in the supernatant of the M. neurolyticum broth culture. Canine Mycoplasma spp. with high sialidase activity reported previously, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare had 100-fold more NEAC than M. neurolyticum, but apparent differences in NEAC levels existed among strains of M. canis and of M. cynos. Zymograms using neuraminidase-specific chromogenic substrate were used to show proteins having NEAC. In M. canis (a field isolate Larissa and the type strain PG14(T)), M. cynos (isolate 896) and M. molare (type strain H542(T)) proteins with NEAC had molecular masses of ∼130kDa, 105kDa and 110kDa, respectively. Identification of these neuraminidases could provide the basis for their molecular characterization.

Multilocus sequence analysis for Mycoplasma synoviae molecular genotyping

Abstract 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 5ʹ-vlhA sequences. Sequencing of three polymorphic genes (5ʹ-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.