Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was 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 of B. 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. Gorging B. divergens infected 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 produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.

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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 g/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 bovis is a wall-less bacterium responsible for severe infections in cattle, including pneumonia, mastitis, arthritis, and otitis (1). In young cattle, it is now recognized as a major contributor to economic losses associated with bovine respiratory diseases (BRD). The infection pressure of BRD usually peaks 2 to 3 weeks after calves are mingled in fattening units following transportation from their respective birth farms (2). Since no efficient vaccines are available and licensed for use outside the United States (3), efforts to control M. bovis infections often rely on antimicrobial treatments, administered either prophylactically or in the early stages of the disease. Antimicrobials used for the treatment or prevention of BRD usually include broad-spectrum cephalosporins (cefquinome and ceftiofur), extended-spectrum fluoroquinolones (enrofloxacin, danofloxacin, and marbofloxacin), florfenicol, and long-lasting macrolides (tulathromycin, gamithromycin, and tildipirosin) (4). This extensive use of antibiotics has predictably resulted in an increase in resistant isolates over time. In France, it was recently shown that contemporary M. bovis strains had become significantly less susceptible than archival strains to 9 of the 12 antimicrobials tested (5). With regard to fluoroquinolones, the decrease in susceptibility was limited (only 1 dilution of the MIC), and no highly resistant isolates were observed in a set of more than 90 strains. This wa...

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 GA and AG in the 23S rRNA alleles for tylosin/tilmicosin and of AT and AT 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.

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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. Ixodes ricinus is a ubiquitous triphasic tick found commonly in Europe. This arthropod feeds on a wide variety of vertebrate hosts, including small rodents and wild and domestic ungulates. It is therefore a potential vector of numerous pathogens, such as bacteria, viruses, and parasites, mainly apicomplexans. Among these pathogens, 2 zoonotic Babesia species have been described in Europe: the well-known cattle parasite Babesia divergens (1) and the more recently reported roe deer parasite Babesia sp. EU1 (2-4). Biological transmission of B. divergens by I. ricinus ticks has been proven by in vivo experimental infections (5); however, quantitative transmission studies that visualize and quantify sporozoites have never been conducted. For Babesia sp. EU1, biological evidence of natural transmission by I. ricinus ticks is still lacking; its presence has been assessed only by DNA amplifi cation from whole ticks (4,(6)(7)(8). Therefore, to analyze transmission of zoonotic Babesia spp. by I. ricinus ticks, we visualized, isolated, and identifi ed infectious sporozoites from dissected tick salivary glands, the transmitting organs. The StudyIn 2008, ticks were collected from animals from 2 different biotopes where each Babesia species had been known to circulate: a farm on which a herd was infected with B. divergens and a reserve on which wild fauna were infected with Babesia sp. EU1. A dairy farm in La Verrie (Vendée, France) was selected as a favorable biotope for B. divergens transmission on the basis not only of the presence of numerous ticks on cows and in pastures in 2007 but also of the parasite circulation in the herd, attested by serologic testing (prevalence of 37.5% by immunofl uorescence antibody test [IFAT]) and confi rmed by its isolation from cattle erythrocytes (prevalence 25% by culture) (9). Of the cows tested by IFAT, 56% had positive results, which indicated that new infections from ticks were occurring within the herd. Because we assumed that sporozoite differentiation is stimulated by blood ingestion and because of experimental proof that female ticks can transmit B. divergens (10), we collected only adult ticks feeding on cows. The 324 collected ticks were morphologically identifi ed as I. ricinus and weighed to estimate their repletion status (range 3-398 mg). Of these, 223 ticks (4.7-339 mg) were dissected under a stereomicroscope to isolate both salivary glands, which were subsequently crushed in 30 μL phosphate-buffered saline in a 1.5-mL microtube with an adapted pestle. A droplet of this suspension was deposited on an 18-well slide, stained with May-Grünwald-Giemsa, and examined under a light microscope. When parasites were seen, and for 41 additional negative sa...

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.

Summary 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.