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Bovine respiratory diseases (BRD) are widespread in veal calf feedlots. Several pathogens are implicated, both viruses and bacteria, one of which, Mycoplasma bovis, is under-researched. This worldwide-distributed bacterium has been shown to be highly resistant in vitro to the main antimicrobials used to treat BRD. Our objective was to monitor the relative prevalence of M. bovis during BRD episodes, its diversity, and its resistance phenotype in relation to antimicrobial use. For this purpose, a two-year longitudinal follow-up of 25 feedlots was organized and 537 nasal swabs were collected on 358 veal calves at their arrival in the lot, at the BRD peak and 4 weeks after collective antimicrobial treatments. The presence of M. bovis was assessed by real-time PCR and culture. The clones isolated were then subtyped (polC subtyping and PFGE analysis), and their susceptibility to five antimicrobials was determined. The course of the disease and the antimicrobials used had no influence on the genetic diversity of the M. bovis strains: The subtype distribution was the same throughout the BRD episode and similar to that already described in France, with a major narrowly-variable subtype circulating, st2. The same conclusion holds for antimicrobial resistance (AMR) phenotypes: All the clones were already multiresistant to the main antimicrobials used (except for fluoroquinolones) prior to any treatments. By contrast, changes of AMR phenotypes could be suspected for Pasteurellaceae in two cases in relation to the treatments registered.

Q fever is a zoonotic abortive disease of ruminants mostly transmitted by inhalation of aerosols contaminated by Coxiella burnetii. Clusters of cases or even epidemics regularly occur in humans but, to date, there is no consensus about the best way to carry out outbreak investigations in order to identify potential farms at risk. Although environmental samples might be useful during such investigations, there are few baseline data on the presence of C. burnetii in the environment of ruminant farms. We thus investigated dust samples from cattle, sheep and goat farm buildings in order to (a) estimate C. burnetii detection frequency and bacterial loads in the environment, and (b) determine whether this environmental contamination is associated with series of abortions attributed to Q fever. We considered 113 herds with a recent abortive episode potentially related (n = 60) or not (n = 53) to C. burnetii. Dust was sampled using a swab cloth and tested by a quantitative PCR method targeting the IS1111 gene. Coxiella burnetii DNA was detected on 9 of 50 cattle farms, 13 of 19 goat farms and 30 of 40 sheep farms. On 16 cloths, bacterial loads were higher than 108 genome equivalents, levels as high as in infectious materials such as placentas and aborted foetuses. Overall, the probability of detecting C. burnetii DNA was higher on small ruminant farms than cattle farms, in herds suspected of Q fever and in large herds. We conclude that swab cloths are a putative indicator of contamination of ruminant farms by C. burnetii.

Mycoplasma bovis is considered a major contributor to respiratory diseases in young cattle. Resistant M. bovis isolates have increasingly been reported worldwide due to extensive use of antimicrobials to treat bovine pneumonia. The frequency of isolates resistant to fluoroquinolones varies considerably from one country to another. The MICs of isolates collected in France have only increased from “very low” to “low.” The present study was conducted to investigate whether alterations in the quinolone resistance-determining regions (QRDRs) could account for this slight modification in susceptibility. No correlation between QRDR alterations and increased MICs was evidenced in clinical isolates. In addition, all clinical isolates were subtyped, and the tendencies of the different sequence types to develop resistance through mutations in QRDRs under selective pressure in vitro were examined. In vitro , 3 hot spots for mutations in QRDRs (position 83 in GyrA and positions 80 and 84 in ParC) were associated with a high level of resistance when cumulated. We showed that the point mutations in the QRDRs observed in vitro were different (in location and selection rapidity) between the different subtypes. Our in vitro observations were corroborated by the recent detection of a clinical isolate highly resistant to fluoroquinolones (MIC ≥ 16 μg/ml) and belonging to the subtype which easily accumulates QRDR alterations in vitro . The current increased prevalence of this subtype in clinical isolates highlights the urgent need to control fluoroquinolone usage in veterinary medicine.

Mycoplasma (M.) bovis has recently emerged as a major, worldwide etiological agent of bovine respiratory diseases leading to huge economic losses mainly due to high morbidity and mortality as well as poor growth rates. The spread of M. bovis infections between different animals, herds, regions or countries has been often reported to be connected to the movement of animals. However, despite recent considerable efforts, no universal subtyping method is yet available to trace M. bovis isolates circulation at an international scale. Moreover in France, the overall population diversity of M. bovis isolates has not been assessed since the early 1990s. This study was conducted to fill in these gaps. The genotypic diversity between sixty isolates collected in France over the last 35 years was assessed using two molecular subtyping methods that addressed either the long-term epidemiological relationships (Multi Locus Sequence Typing, MLST) or the genetic microvariations (Multiple Locus VNTR Analysis, MLVA) between isolates. Phenotypic diversity was also analyzed by using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) to compare the main protein patterns of isolates. All proposed subtyping approaches were optimized and led to the same pattern in the French M. bovis population that consisted of two clusters, the first one comprising isolates collected before 2000 and the second, those collected after 2000. Recent strains were further shown to be more homogeneous than older ones, which is consistent with the spread of a single clone throughout the country. Because this spread was concomitant with the emergence of multiresistant M. bovis isolates, several hypotheses are discussed to explain the homogeneity of M. bovis isolates in France, even though the M. bovis species is fully equipped to generate diversity.

International audience; Mycoplasma bovis is considered an emerging threat to bovine production in industrialized countries. Its control depends on good husbandry and efficient chemotherapy practices. In France, clinical isolates collected after 2009 showed a drastic loss of susceptibility to most antimicrobials when compared with isolates collected in 1978-1979. The aim of the present study was to analyze the molecular mechanisms underlying the shift toward resistance to macrolides and tetracyclines and to assess whether the clinical origin of the isolates or their molecular subtypes could have influenced their pattern of evolution. We demonstrated that all M. bovis isolates collected as early as 2000 should already be considered resistant to tylosin, tilmicosin, and oxytetracycline, whatever the associated clinical signs. The shift toward resistance happened earlier for oxytetracycline and more progressively for tylosin/tilmicosin. Isolates belonging to the major subtype ST2 (n = 40) showed a homogeneous genotype for resistance, with combined alterations of G 748 A and A 2058 G in the 23S rRNA alleles for tylosin/tilmicosin and of A 965 T and A 967 T in the 16S rRNA alleles for oxytetracycline. The genotypes of ST3 or ST1 isolates (n = 9 and 25, respectively) in the process of becoming resistant were more varied. In recent years, the convergence of both ST2 and ST3 isolates toward the same resistance genotype suggests that the corresponding mutations have been selected for providing an appropriate balance between fitness cost and resistance.

Contagious agalactia is an important disease worldwide that affects small ruminants. Clinical manifestations vary from mastitis, pneumonia, arthritis and keratoconjunctivitis to septicemia. Four mycoplasmal etiological agents have been identified: Mycoplasma (M.) agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. The current procedure for direct diagnosis, recommended by the World Organization for Animal Health, involves the isolation of one or several causative agents from clinical specimens and further time-consuming identification steps. The present study reports the development of a new multiplex real-time PCR (including an internal positive control) that detects all four pathogens simultaneously and distinguishes M. agalactiae from the others. First, intra- and inter-species polymorphisms of the two target house-keeping genes, namely polC and fusA, were analyzed to design primers and probes adapted to the diversity of currently circulating strains. The specificity and the sensitivity of the assay were then challenged and the limit of detection was found to be as low as 6 to 12 copies of the target genes. The assay requires further assessment on clinical specimens but its performances (notably low intra- and inter-assay variability) are already very promising for use in large-scale diagnosis and prophylactic surveys of contagious agalactia.

AlthoughBabesia divergensis the the principal confirmed zoonoticBabesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tickIxodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, anin vitroanimal skin feeding technique on blood containingin vitrocultivatedB. divergenswas developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence ofB. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. GorgingB. divergensinfected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be producedin vitro. To our knowledge, this is the first report of artificial feeding ofI. ricinusvia membrane as well asin vitrotransmission ofB. divergensto its vector. The opportunities offered by the use of such a transmission model of a pathogen byI. ricinusare discussed.

To determine characteristics of natural transmission of Babesia sp. EU1 and B. divergens by adult Ixodes ricinus ticks, we examined tick salivary gland contents. We found that I. ricinus is a competent vector for EU1 and that their sporozoites directly invade erythrocytes. We conclude that EU1 is naturally transmitted by I. ricinus.

International audience; Babesiosis is a tick-transmitted disease of mammalian hosts, caused by the intraerythrocytic protozoan parasites of the genus Babesia. Transmission of Babesia parasites from the vertebrate host to the tick is mediated by sexual stages, the gametocytes which are the only intraerythrocytic stages that survive and develop inside the vector. Very few data are available concerning these parasite stages and some markers are needed in order to refine our knowledge of Babesia life cycle inside the tick and to permit the monitoring of parasite transmission from vertebrate to vector. We previously identified some potential markers of the Babesia divergens gametocytes using an in silico post-genomic approach based on sequence identity between the available genomes of Plasmodium and Babesia spp. Here, one of the identified proteins, BdCCp2, was validated as a marker of sexual stages of B. divergens, in infected ticks challenged with antisera directed against recombinant BdCCp2 protein. The BdCCp2 protein was detected by Western blot in some infected ticks, as a discrete band of approximately 171 kDa, while no signal was detected in the laboratory-reared non-infected tick. BdCCp2 was also detected, by immunohistochemical analyses, in piriform or ovoid bodies, measuring 2.5-4.5 μm in diameter, in the gut of partially engorged ticks that were experimentally infected. This molecular marker can then be used in the future to characterize and analyze the biology of B. divergens gametocytes.

International audience; Babesia divergens, a tick-borne protozoan parasite of red blood cells, is the main agent of bovine and human babesiosis in Europe. Very few data are available concerning its life cycle and sexual reproduction inside the tick vector, Ixodes ricinus. The aim of this study was to define some markers of the B. divergens sexual stage. An in silico post-genomic approach was used to analyze genomic, transcriptomic and proteomic data and to select specific sexual stage proteins of the related apicomplexan genus Plasmodium. Three proteins, based on sequence identity between the available genomes of Plasmodium and Babesia spp., were chosen, as members of a highly conserved and apicomplexan sexual stages specific protein family (CCp) potentially involved in adhesive functions. Degenerate primers were used to amplify and clone three B. divergens orthologs (bdccp1, bdccp2, and bdccp3) corresponding to newly identified genes in this parasite. The opportunities offered by such markers to study parasite development in its vector are discussed. (C) 2010 Elsevier B.V. All rights reserved.