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1.  Staphylococcus aureus from 152 cases of bovine, ovine and caprine mastitis investigated by Multiple-locus variable number of tandem repeat analysis (MLVA) 
Veterinary Research  2014;45(1):97.
Staphylococcus aureus is one of the main etiological agents of mastitis in ruminants. In the present retrospective study, we evaluated the potential interest of a previously described automated multiple loci Variable Number of Tandem Repeats (VNTR) Assay (MLVA) comprising 16 loci as a first line tool to investigate the population structure of S. aureus from mastitis. We determined the genetic diversity of S. aureus strains from cases of clinical and subclinical mastitis in dairy cattle (n = 118, of which 16 were methicillin-resistant), sheep (n = 18) and goats (n = 16). The 152 strains could be subdivided into 115 MLVA genotypes (including 14 genotypes for the ovine strains and 15 genotypes for the caprine strains). This corresponds to a discriminatory index (D) value of 0.9936. Comparison with published MLVA data obtained using the same protocol applied to strains from diverse human and animal origins revealed a low number (8.5%) of human-related MLVA genotypes among the present collection. Eighteen percent of the S. aureus mastitis collection belonged to clonal complexes apparently not associated with other pathological conditions. Some of them displayed a relatively low level of diversity in agreement with a restricted ecological niche. These findings provide arguments suggesting that specific S. aureus lineages particularly adapted to ruminant mammary glands have emerged and that MLVA is a convenient tool to provide a broad overview of the population, owing to the availability via internet of databases compiling published MLVA genotypes.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-014-0097-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4195859  PMID: 25315988
2.  Experimental Infections with Mycoplasma agalactiae Identify Key Factors Involved in Host-Colonization 
PLoS ONE  2014;9(4):e93970.
Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.
PMCID: PMC3974822  PMID: 24699671
3.  Comparative assessment of two commonly used commercial ELISA tests for the serological diagnosis of contagious agalactia of small ruminants caused by Mycoplasma agalactiae 
Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated.
Two commercial ELISA kits are widely used, one involving a fusion protein as target antigen and the other a total antigen. The objectives were to compare these tests by evaluating:
i. Their diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the two tests, and, the correlation between serology data and the milk shedding of M. agalatiae;
ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections.
A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test.
The average diagnostic sensitivity was 56% [51.8–59.8] for the fusion protein ELISA and 84% [81.3–87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9–100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4–99.9] and 95.7% [93.8–97.2] respectively.
Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples.
Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor.
These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use.
PMCID: PMC3439703  PMID: 22776779
4.  High Throughput Multiple Locus Variable Number of Tandem Repeat Analysis (MLVA) of Staphylococcus aureus from Human, Animal and Food Sources 
PLoS ONE  2012;7(5):e33967.
Staphylococcus aureus is a major human pathogen, a relevant pathogen in veterinary medicine, and a major cause of food poisoning. Epidemiological investigation tools are needed to establish surveillance of S. aureus strains in humans, animals and food. In this study, we investigated 145 S. aureus isolates recovered from various animal species, disease conditions, food products and food poisoning events. Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis (MLVA), known to be highly efficient for the genotyping of human S. aureus isolates, was used and shown to be equally well suited for the typing of animal S. aureus isolates. MLVA was improved by using sixteen VNTR loci amplified in two multiplex PCRs and analyzed by capillary electrophoresis ensuring a high throughput and high discriminatory power. The isolates were assigned to twelve known clonal complexes (CCs) and –a few singletons. Half of the test collection belonged to four CCs (CC9, CC97, CC133, CC398) previously described as mostly associated with animals. The remaining eight CCs (CC1, CC5, CC8, CC15, CC25, CC30, CC45, CC51), representing 46% of the animal isolates, are common in humans. Interestingly, isolates responsible for food poisoning show a CC distribution signature typical of human isolates and strikingly different from animal isolates, suggesting a predominantly human origin.
PMCID: PMC3342327  PMID: 22567085
5.  Transcriptomic analysis of milk somatic cells in mastitis resistant and susceptible sheep upon challenge with Staphylococcus epidermidis and Staphylococcus aureus 
BMC Genomics  2011;12:208.
The existence of a genetic basis for host responses to bacterial intramammary infections has been widely documented, but the underlying mechanisms and the genes are still largely unknown. Previously, two divergent lines of sheep selected for high/low milk somatic cell scores have been shown to be respectively susceptible and resistant to intramammary infections by Staphylococcus spp. Transcriptional profiling with an 15K ovine-specific microarray of the milk somatic cells of susceptible and resistant sheep infected successively by S. epidermidis and S. aureus was performed in order to enhance our understanding of the molecular and cellular events associated with mastitis resistance.
The bacteriological titre was lower in the resistant than in the susceptible animals in the 48 hours following inoculation, although milk somatic cell concentration was similar. Gene expression was analysed in milk somatic cells, mainly represented by neutrophils, collected 12 hours post-challenge. A high number of differentially expressed genes between the two challenges indicated that more T cells are recruited upon inoculation by S. aureus than S. epidermidis. A total of 52 genes were significantly differentially expressed between the resistant and susceptible animals. Further Gene Ontology analysis indicated that differentially expressed genes were associated with immune and inflammatory responses, leukocyte adhesion, cell migration, and signal transduction. Close biological relationships could be established between most genes using gene network analysis. Furthermore, gene expression suggests that the cell turn-over, as a consequence of apoptosis/granulopoiesis, may be enhanced in the resistant line when compared to the susceptible line.
Gene profiling in resistant and susceptible lines has provided good candidates for mapping the biological pathways and genes underlying genetically determined resistance and susceptibility towards Staphylococcus infections, and opens new fields for further investigation.
PMCID: PMC3096985  PMID: 21527017
6.  Prions in Milk from Ewes Incubating Natural Scrapie 
PLoS Pathogens  2008;4(12):e1000238.
Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.
Author Summary
A decade ago, a new variant form of Creutzfeldt-Jakob disease was identified. The emergence of this prion disease in humans was the consequence of the zoonotic transmission of bovine spongiform encephalopathy through dietary exposure. Since then, the control of human exposure to prions has become a priority, and a policy based on the exclusion of known infectious materials from the food chain has been implemented. Because all investigations carried out failed to reveal evidence of infectivity in milk from affected ruminants, this product has continuously been considered as safe. In this study, we demonstrate the presence of prions in colostrum and milk from sheep incubating natural scrapie and displaying apparently healthy mammary glands. This finding indicates that milk from small ruminants could contribute to the transmission of prion disease between animals. It also raises some concern with regard to the risk to humans associated with milk products from ovine and other dairy species.
PMCID: PMC2587713  PMID: 19079578
7.  Characterization of P40, a Cytadhesin of Mycoplasma agalactiae  
Infection and Immunity  2002;70(10):5612-5621.
An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGATrp codon to the universal TGGTrp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.
PMCID: PMC128363  PMID: 12228289
8.  Characterization and Analysis of a Stable Serotype-Associated Membrane Protein (P30) of Mycoplasma agalactiae 
Journal of Clinical Microbiology  2001;39(8):2814-2822.
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.
PMCID: PMC88244  PMID: 11473997

Results 1-8 (8)