Diversity and variation in antimicrobial susceptibility patterns over time in Mycoplasma agalactiae isolates collected from sheep and goats in France.

AIMS: Mycoplasma agalactiae is responsible for Contagious Agalactia, a severe syndrome affecting small ruminants worldwide and resulting in significant economic losses in countries with an important dairy industry. The aim of this study was to examine the antimicrobial susceptibility patterns of M. agalactiae isolates in France, their evolution over the last 25 years and their relationships with the genetic diversity of isolates and their origin (geographical and animal host). METHODS AND RESULTS: Susceptibility patterns were determined by measuring minimal inhibitory concentrations (MICs) of several antimicrobials used against mycoplasmas. Caprine M. agalactiae strains showed increased MICs over time for most of the antimicrobials tested, except fluoroquinolones. This susceptibility loss was homogeneous despite the considerable genetic and geographical heterogeneity of the isolates. In contrast, all the ovine isolates originating from a single clone and the same region showed increased MICs only to some macrolides. CONCLUSIONS: MICs have evolved differently depending on the origin of the isolates but the overall loss in susceptibility has remained far more moderate than that of Mycoplasma bovis, a cattle pathogen closely related to M. agalactiae. SIGNIFICANCE AND IMPACT OF THE STUDY: Several hypotheses are proposed to explain the differences in susceptibility patterns, such as local, specific, nonmycoplasma-targeting antibiotic treatments and the genetic background of isolates in connection with their animal host.

Response to somatic cell count-based selection for mastitis resistance in a divergent selection experiment in sheep.

A divergent selection experiment in sheep was implemented to study the consequences of log-transformed somatic cell score (SCS)-based selection on resistance to natural intramammary infections. Using dams and progeny-tested rams selected for extreme breeding values for SCS, we created 2 groups of ewes with a strong divergence in SCS of approximately 3 genetic standard deviations. A survey of 84 first-lactation ewes of both the High and Low SCS lines indicated favorable responses to SCS-based selection on resistance to both clinical and subclinical mastitis. All clinical cases (n = 5) occurred in the High SCS line. Additionally, the frequency of chronic clinical mastitis, as detected by the presence of parenchymal abscesses, was much greater in the High SCS line (n = 21) than in the Low SCS line (n = 1). According to monthly milk bacteriological examinations of udder halves, the prevalence of infection was significantly greater (odds ratio = 3.1) in the High SCS line than in the Low SCS line, with predicted probabilities of 37 and 16%, respectively. The most frequently isolated bacteria responsible for mastitis were staphylococci: Staphylococcus auricularis (42.6% of positive samples), Staphylococcus simulans, Staphylococcus haemoliticus, Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus lentus, Staphylococcus warneri, and Staphylococcus aureus. The incidence of positive bacteriology was greater in the High SCS line (39%) than in the Low SCS line (12%) at lambing, indicating that High SCS line ewes were especially susceptible to postpartum subclinical mastitis. Negativation of bacteriological results from one sampling time point to the next was markedly different between lines after weaning (e.g., 41 and 84% in the High and Low SCS lines, respectively). This result was consistent with differences in the duration of infection, which was much greater in the High SCS line compared with the Low SCS line. Finally, ewes from the High SCS line consistently had greater SCS in positive milk samples than did ewes from the Low SCS line (+2.04 SCS, on average), with an especially large difference between lines during the suckling period (+3.42 SCS). Altogether, the preliminary results suggest that the better resistance of Low SCS line ewes, compared with High SCS line ewes, was principally characterized by a better ability to limit infections during the peripartum period, to eliminate infections during lactation, and quantitatively to limit the inflammation process and its clinical consequences.

Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach.

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.

Characterization and analysis of a stable serotype-associated membrane protein (P30) of Mycoplasma agalactiae.

The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two -10 boxes and a possible -35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both -10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.

Characterization of a multigene family undergoing high-frequency DNA rearrangements and coding for abundant variable surface proteins in Mycoplasma agalactiae.

A family of abundant surface proteins (Vpmas [variable proteins of Mycoplasma agalactiae]) undergoing phase variation in M. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboring vpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes, vpmaX and vpmaY, were orientated divergently and shared highly homologous 5' untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. The vpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. The vpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by the vsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR.

The DNA repair genes uvrC from Mycoplasma bovis and Mycoplasma agalactiae type strains were cloned and their nucleotide sequences were established. These sequences were used to design polymerase chain reaction (PCR) primer pairs for M. bovis and M. agalactiae. Each primer pair amplified a 1-6 kb fragment of the uvrC gene in the respective species. The specificity of the primer pairs for the two species was demonstrated through the lack of cross-amplifications in heterologous PCR reactions and in reactions using DNA from other mycoplasma species. Subsequent restriction enzyme analysis of the amplified uvrC gene segments from type and field strains of M. bovis and M. agalactiae showed that the uvrC genes are well conserved in both species but differ significantly between the two species. The diagnostic PCR assay enabled unambiguous identification of M. bovis and M. agalactiae strains isolated from geographically diverse places, even in cases where 16S rRNA gene sequence analysis was unable to discriminate between the two species.

Contagious agalactia of small ruminants: current knowledge concerning epidemiology, diagnosis and control.

Contagious agalactia of small ruminants is a syndrome which principally affects the mammary glands, joints and eyes. The main causal agents are Mycoplasma agalactiae in sheep, and M. agalactiae, M. mycoides subsp. mycoides large colony type and M. capricolum subsp. capricolum in goats. In addition, M. putrefaciens can produce a similar clinical picture, particularly in goats. Contagious agalactia occurs on all five continents and is often enzootic. The evolution of the infection tends to be chronic in affected animals and herds. Symptomless shedding of mycoplasmas, mainly in the milk, may persist for a long time. These insidious infections, associated with carriage in the ears of healthy animals, are difficult to diagnose and to control. The main mode of transmission between flocks is related to the sale of carrier animals and contact during transhumance, whereas transmission within a flock occurs through contact, suckling and milking. This review discusses the clinical features, epidemiology, treatment, prevention and control of the disease.

[Contagious agalactia of small ruminants: epidemiology, diagnosis and control]. / Agalactie contagieuse des petits ruminants: épidémiologie, diagnostic et contrôle.

Contagious agalactia of small ruminants is a syndrome which affects mainly the mammary glands, joints and eyes. The principal causal agents are Mycoplasma agalactiae in sheep and M. agalactiae, M. mycoides subsp. mycoides large colony type and M. capricolum subsp. capricolum in goats. In addition, M. putrefaciens can produce a similar clinical picture, particularly in goats. Contagious agalactia occurs on all five continents and is often enzootic. These infections are chronic in animals and in flocks. Symptomless shedding of mycoplasmas, mainly in the milk, may persist for a long time. Associated with carriage in the ears of healthy animals, these insidious infections are difficult to diagnose and control. The sale of carrier animals and contact during transhumance are the main modes of transmission between flocks, while transmission within a flock occurs through contact, suckling and milking. This review discusses clinical features, epidemiology, treatment, prevention and control.

Variable expression and geographic distribution of Mycoplasma agalactiae surface epitopes demonstrated with monoclonal antibodies.

Species-specific monoclonal antibodies (MAbs) were developed against Mycoplasma agalactiae reference strain PG2 and French isolate P89 to study the in vitro expression of surface epitopes and to probe the antigenic profiles of 245 field isolates originating from 10 different countries. Colony immunostaining with MAbs on clonal lineage showed that 4 out of 9 species-specific epitopes exhibited a high rate of variation, demonstrating that M. agalactiae possesses a capacity for phenotypic diversification of its surface antigenicity. The emphasis was on dot immunobinding screening of the field isolates with MAbs recognizing permanently expressed epitopes. Eight different profiles could be defined. Great differences in epitope conservation were demonstrated with some area-specific strains completely lacking certain specific determinants. These results indicate that the antigenic variability of M. agalactiae relies not only upon surface switching mechanisms but also upon true epitope differences, partially related to the geographic origin of the isolates.