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.

Prevalence of Mycoplasma bovis udder infection in dairy cattle: preliminary field investigation in southeast France.

AIMS: To update epidemiological data on Mycoplasma bovis infection in dairy herds from six departments in the southeast of France, in order to obtain a first estimate of the prevalence of M. bovis infection through bacteriological investigations on bulk tank milk, and estimate the prevalence of M. bovis in clinical mastitis in this population of cattle. METHODS: To estimate a prevalence of M. bovis of 2%, with 95% confidence, a sample of >270 herds was required. Bulk tank milk samples were collected from herds between January and February 2005 and milk samples from cows with clinical mastitis were collected between January 2007 and March 2008. Bulk tank milk and composite mastitic samples were analysed for M. bovis using culture and/or PCR. RESULTS: Mycoplasma bovis was not detected by either technique in any of the 345 bulk tank milk samples. The prevalence of M. bovis infection in this population of dairy herds was <1%, with 95% confidence. Mycoplasma bovis was not isolated from any of the 166 composite samples obtained from 828 samples of mastitic milk. The prevalence of M. bovis in clinical mastitis was <0.44%, with 95% confidence. CONCLUSIONS: These preliminary results suggest that the prevalence of udder infections with M. bovis is very low in dairy herds in the southeast of France. These two studies provide preliminary data, that can be used to derive working hypotheses for future statistically representative investigations at the national level.

Comparison of isolates of Mycoplasma mycoides subspecies capri from asymptomatic and septicaemic goats.

Strains of Mycoplasma mycoides subspecies capri (Mmc) are frequently isolated from goats with contagious agalactia, but they can also be recovered from herds that have shown no clinical signs of mycoplasmosis for several years. The present study was conducted in order to explore the potential genetic and antigenic differences existing between an Mmc strain isolated from an outbreak (septicaemic) and a strain isolated from the ear canal of a goat belonging to a herd with no recent episode of mycoplasmosis (carriage strain). The genomes of the two strains, compared by suppression subtractive hybridization, were shown to be poorly divergent. The two strains were inoculated into goats to produce specific antisera, but both induced fatal mycoplasmosis. These results indicate that septicaemic and carriage strains cannot be distinguished by their genetic background or by their pathogenic capacity under experimental conditions.

Expression of Mycoplasma bovis variable surface membrane proteins in the respiratory tract of calves after experimental infection with a clonal variant of Mycoplasma bovis type strain PG45.

The pathomorphological findings and the expression and distribution of variable surface protein antigens (Vsp) of Mycoplasma (M.) bovis were characterised immunohistochemically in lungs of eight calves following inoculation with a Vsp A-expressing clonal variant of M. bovis type strain PG45. Within 48 h post inoculation (p.i.) an innate immune response dominated by macrophages and neutrophils develops. The monoclonal antibodies (mAbs) 1A1 and 1E5 detected M. bovis Vsp antigens in paraffin tissue sections of seven calves. Vsp antigens were widely distributed and were already present at day two p.i. within macrophages and other lung compartments. Taken together, the results demonstrate that the bovine is unable to eliminate M. bovis during the time period examined. Based on the different immunohistochemical labelling patterns obtained with the mAbs, the results also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease.

Epidemiological surveillance of mycoplasmas belonging to the 'Mycoplasma mycoides' cluster: is DGGE fingerprinting of 16S rRNA genes suitable?

AIMS: The analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rRNA gene was previously shown to detect and differentiate a large number of human and animal mycoplasmas. In this study, we further assessed the suitability of the technique for epidemiological surveillance of mycoplasmas belonging to the 'Mycoplasma mycoides' cluster, a phylogenetic group that includes major ruminant pathogens. METHODS AND RESULTS: The V3 region of 16S rRNA genes from approx. 50 field strains was amplified and analysed by DGGE. Detection and identification results were compared with the ones obtained by antigenic testing and sequence analysis. CONCLUSIONS: The DGGE technique is robust and valuable as a first-line test, but the patterns obtained for strains belonging to the 'M. mycoides' cluster were too variable within a taxon and in contrast too conserved between taxa to allow an unequivocal identification of isolates without further analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Issues raised by the quest for a single universal test able to detect and identify any mycoplasma in one clinical sample are thoroughly documented.

A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: application to the diagnosis of contagious agalactia.

Contagious agalactia is a mycoplasmal infection caused by Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens. Identification of the causative organisms is usually performed by isolation and classical biochemical and serological tests, though this is a lengthy and cumbersome process for mycoplasmas. Specific PCR assays have been developed for the identification of Mycoplasma agalactiae and M. putrefaciens. For members of the M. mycoides cluster existing PCR tests are based on the amplification of highly conserved genes coding for ribosomal proteins, hence a possibility of cross-reactions. The gene glk, coding for a glucokinase, that is found in this cluster is very distantly related to any other bacterial glucokinase described so far. It was therefore chosen as target to design a new PCR test. The validation was performed independently in three laboratories in France and India using over 100 mycoplasma strains of various geographical origins. All strains belonging to the M. mycoides cluster were detected by amplification of the expected PCR product (428 bp) while no amplification was obtained from M. agalactiae strains. Our results demonstrate the universality of this PCR in spite of the great heterogeneity found within this cluster. This new tool may be of great help for the implementation of control measures directed towards contagious agalactia.

Mycoplasma mycoides subsp. mycoides biotype large colony isolates from healthy and diseased goats: prevalence and typing.

Most severe goat mycoplasmosis outbreaks in France are caused by Mycoplasma mycoides subsp. mycoides biotype LC (MmmLC). However, MmmLC can also be recovered from ear canals of healthy goats or from bulk milk collected in herds showing no clinical signs of mycoplasmosis. To improve our understanding of how MmmLC strains are balanced between pathogenic ones and asymptomatically carried ones, descriptive epidemiological data were analysed, together with the genomic fingerprints of isolates generated using pulsed-field gel electrophoresis (PFGE). PGFE analyses were performed with isolates collected from the ear canals of goats or bulk milk in healthy herds, from individual clinical cases in different diseased herds at different times, and within a single herd during a severe outbreak, from various body sites including the ear canals at autopsy. Results showed that each isolate collected in healthy herds yielded a unique and characteristic PFGE profile. Isolates from diseased herds had profiles that were distinct for each outbreak and the group of 41 isolates from a single severe outbreak had 2 predominant PFGE profiles that persisted throughout the outbreak. These data suggest that while several distinct isolates are carried by healthy animals, only a few are responsible for the clinical signs observed within one herd during an outbreak. Whether this reflects differences in virulence between different field strains of MmmLC remains to be demonstrated.

Serological prevalence of Mycoplasma bovis infection in suckling beef cattle in France.

The prevalence of Mycoplasma bovis infection in France was assessed by means of a serological survey of suckling beef cattle, using an ELISA. The survey included 824 randomly selected herds in eight French counties and a total of 32,197 animals more than one year old. In each county, the number of herds tested was determined statistically on the basis of the hypothesis that about 40 per cent of herds are infected. The proportion of herds containing at least one infected animal ranged from 28 to 90 per cent depending on the county. Among the 32,197 sera tested, the animal infection rate ranged between 2 per cent and 13 per cent. In infected herds, the average number of positive animals per herd was between 10 and 20 per cent, and the infection was unevenly distributed among the areas tested.

Efficacy of spectinomycin against Mycoplasma bovis induced pneumonia in conventionally reared calves.

Sixteen 3-week-old calves were intratracheally inoculated with Mycoplasma bovis. Follow-up consisted of regular bronchoalveolar lavages (BALs) and clinical examinations. Animals were slaughtered from 4 to 21 days after inoculation. Counts were made of the mycoplasmas and other bacteria systematically isolated from the BAL liquids and lung lobes after slaughter. On the 6th day, spectinomycin 20mg/kg was given intramuscularly in three repeated doses at 24h intervals to six randomly chosen calves. All animals had developed a persistent M. bovis infection with a maximum BAL count on the 6th day (start of treatment). Co-occuring Pasteurella multocida infection was found in most animals with a maximum rate on the 14th day. The extent of lung surface lesions varied widely (0-64%) but was greater in the later slaughtered calves. Average counts of M. bovis and P. multocida in the BAL liquids were lower in treated calves than in untreated ones but the difference was not statistically significant. However, M. bovis and P. multocida counts in the lungs of the treated group were significantly lower than in the untreated group (p=0.003 and 0.009, respectively).

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.

[The seroprevalence of Mycoplasma bovis in lactating cows in Switzerland, particularly in the republic and canton of Jura]. / Etude sur la séroprévalence de Mycoplasma bovis chez la vache laitière en Suisse, en particulier dans la République et Canton du Jura.

Sporadic cases of infection with Mycoplasma bovis have been observed in Switzerland since 1983. However, five severe outbreaks of endemic mastitis in a geographically distinct, small region (Canton du Jura) during the period 1995 to 1997 prompted the present investigation on the seroprevalence of infection with M. bovis among milking cows in Switzerland. A commercially prepared indirect enzyme immunoassay was used. Among a stratified random sample of 118 herds of milking cows in Switzerland, at least one positive animal was detected in 56 (47%) of the herds, whereas 6.1% of the 1816 individual animals tested positive. An epidemiological study was performed in the Canton du Jura region among 51 herds in order to assess the importance of management factors in the spread of M. bovis infection. The herd-level prevalence was 78%, and the seroprevalence at the level of the 1354 individual animals tested was 13.4%. A multivariate analysis of possible risk factors showed purchase of animals to be the only variable significantly associated with serological status of the herd with an "odds ratio" of 10.8.

Development of a recombinant antigen for antibody-based diagnosis of Mycoplasma bovis infection in cattle.

Mycoplasma bovis induces various clinical manifestations in cattle, such as mastitis, arthritis, and pneumonia. We have evaluated the immunoreactivity of three variable surface proteins (Vsps) of M. bovis, namely VspA, VspB, and VspC, with sera collected from herds with mycoplasmosis or from cattle experimentally infected with M. bovis. Western blot analysis revealed that the Vsps are the predominant antigens recognized by the host humoral response during M. bovis infection. The immunoreactivity of VspA, VspB, and VspC with host antibodies was independent of the clinical manifestations, the geographical origin of the M. bovis isolates, the mode of infection, and the animal's history. Moreover, the results showed that Vsp-specific host antibodies can be detected about 10 days after experimental infection and for up to several months. The full-length or truncated versions of the VspA product were overexpressed in Escherichia coli as fusion proteins (FP-VspA). Recombinant products showed strong immunoreactivity with the Vsp-specific monoclonal antibodies 1A1 and 1E5, with the corresponding epitopes localized at the VspA N-terminal and C-terminal ends, respectively. Anti-M. bovis sera of cattle naturally or experimentally infected also strongly recognized the full-length FP-VspA. The seroreactivity of sera collected from cattle between 6 and 10 days after experimental infection was weaker with truncated versions of VspA lacking the 1E5 epitope than with the full-length VspA or the truncated versions lacking the 1A1 epitope. Overall, the results indicate that the Vsps, despite their inter- and intraclonal variability, may be applied as target antigens in serodiagnostic assays for epidemiological studies.

Vsp antigens and vsp-related DNA sequences in field isolates of Mycoplasma bovis.

The expression of the 1E5 epitope which is common to the three characterized variable lipoproteins VspA, VspB and VspC of Mycoplasma bovis type strain PG45 and the presence of vsp gene DNA sequences were assessed in field isolates randomly collected from cattle showing clinical manifestations due to M. bovis infection. Among 250 isolates tested, only four failed to react with mAb 1E5. Southern blot analysis of these four isolates and of 20 isolates expressing the 1E5 epitope were performed using synthetic oligonucleotide probes corresponding to a sequence located in the Vsp signal peptide coding region common to all known Vsp products or to selected regions of previously characterized vsp genes, vspA, vspE and vspF. The results demonstrate the presence of multiple vsp-related DNA sequences in all M. bovis field isolates tested and indicate that the vsp repertoire varies in size and composition among isolates.

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.

Adaptive surface antigen variation in Mycoplasma bovis to the host immune response.

The variability of predominant Mycoplasma bovis surface antigens in the presence of specific immune pressure was analyzed in an in vitro assay to determine if M. bovis could escape immune destruction. We have shown that serum antibodies from immunized or experimentally infected calves and monoclonal antibodies which specifically react with previously characterized or as yet undefined major M. bovis membrane surface proteins cause repression of expression or shortening of the target protein, or induce switching to expression of an antigenically distinct variant protein. We have further demonstrated that removal of the inducing antibody results in reversion to the original phenotype. These results suggest that the level of expression and the length of M. bovis surface antigens in the host is modulated by cognate antibodies. According to the surface antigenic variation systems, random selection of preexisting variants resistant to antibody-mediated inhibition or direct regulation of gene expression may be means by which this organism evades host immune defences.

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.

A newly identified immunodominant membrane protein (pMB67) involved in Mycoplasma bovis surface antigenic variation.

Mycoplasma bovis is a bovine pathogen able to cause systemic disease. It possesses a series of prominent, structurally related yet clearly distinguishable membrane lipoproteins on the cell surface. These variable surface proteins (Vsps) undergo highly dynamic and spontaneous changes in size and expression and are key immunogenic components. They may play a critical role as mediators of adherence to host cells and in escaping immune destruction. In this report, we define a novel, Vsp-unrelated membrane protein also associated with M. bovis surface antigenic variation. This protein has an apparent molecular mass of 67,000 Da in the type strain PG45 and was designated pMB67. Immunological and biochemical characterization of pMB67 demonstrated that it: (i) contains a specific epitope, (ii) is not modified by lipid but does contain cysteine, (iii) does not contain a Vsp-like repetitive periodic protein structure, (iv) is a predominant antigen recognized during M. bovis infections, (v) undergoes a high rate of phase variation in vitro and (vi) is size-variable. These results showed that M. bovis employs two types of specialized membrane proteins for surface diversification. The pMB67 protein may be useful in diagnostic assays and as a vaccine component.