Contagious agalactia monitoring in caprine herds through regular bulk tank milk sampling.

Surveillance and control of Mycoplasma spp. responsible for contagious agalactia (CA) in caprine herds are important challenges in countries with a large small-ruminant dairy industry. In the absence of any clinical signs, being able to determine the potential circulation of mycoplasmas within a herd could help to prevent biosecurity issues during animal exchanges between farms and improve health management practices. The objective of this study was to determine whether regular sampling of bulk tank milk was suitable for such surveillance. Twenty farms were sampled once a month for 2 yr and CA-responsible mycoplasmas were detected by real-time PCR on DNA extracted from milk, using 3 different DNA extraction methods. The pattern of mycoplasma excretion in bulk tank milk was assessed over time and several herd characteristics were recorded together with any event occurring within the herds. In general, the results obtained with the different detection methods were comparable and mainly agreed with the culture results. Several patterns of excretion were observed but were not related to herd characteristics (size, breed, and so on). Recurrence of the same (sub)species and same pulsed-field gel electrophoresis subtype during the 2-yr period is indicative of the considerable persistence of mycoplasmas. This persistence was associated with intermittent excretion. In conclusion, bulk tank milk sampling could be valuable for controlling CA in caprine herds provided it is repeated several times, yet to be defined, per year and analyzed using an appropriate methodology and the right cut-off for interpretation.

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.

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).

[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.

The discovery of orally available thrombin inhibitors: optimisation of the P1 pharmacophore.

Thrombin inhibitors have been designed with the replacement of the strongly basic guanidine P1 pharmocophore with a group that exploits the lipophilicity of the S1 pocket. The approach has lead to the discovery of potent thrombin inhibitors demonstrating good intra-duodenal absorption.

Studies towards the identification of potent, selective and bioavailable thrombin inhibitors.

The application of selection criteria, based on potency and physicochemical parameters, to a candidate library of thrombin inhibitors is described. The utility of the approach is exemplified by the discovery of a potent, selective and bioavailable thrombin inhibitor 62.

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.

The discovery of orally available thrombin inhibitors: studies towards the optimisation of CGH1668.

The chemical optimisation of CGH1668 1 is described employing an in vivo model of absorption to determine the influence on bioavailability of single point modifications to five key molecular templates. The discovery of an orally bioavailable and selective thrombin inhibitor, 24, highlights the utility of this approach.

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.

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.

[Genital Ureaplasma diversum infection: investigations in cattle in France]. / Infection génitale à Ureaplasma diversum: enquête chez les bovins en France.

A first bacteriological study of infection by Ureaplasma diversum in cattle was performed in France on 50 bulls and 565 dairy cows. U diversum was isolated in 74% (37/50) of the bull semen and 40% (227/565) of the cows. No significant relationship was found in cows between infection and lesions of granular vulvitis, nor between infection and breeding performances. Serological studies of isolates by membrane filtration dot immunobinding showed a predominance of the serogroups B and C in males, and serogroup B in females.