Prevalence of Mycoplasma bovis in Algeria and Characterisation of the Isolated Clones.

Bovine respiratory disease (BRD) is common in calves in Algeria, but to date, Mycoplasma bovis has never been monitored as a potential etiological agent. Here, to assess the presence (direct detection) and circulation (indirect detection) of M. bovis, broncho-alveolar lavage fluids (BALF) and serum samples were collected from 60 veal calf farms in Algeria. A commercial ELISA kit (ID Screen® ELISA) was used to screen for the presence of specific antibodies against M. bovis in 351 blood sera collected from both diseased and healthy calves, and 69% (241 sera) tested positive. BALFs from the 176 diseased calves were used to screen for M. bovis by real-time-PCR (rt-PCR), and 102 (58%) tested positive. A non-exhaustive set of 53 clones were isolated from 44 calves and further subtyped using polC gene sequencing. No predominant subtype was found, and two clones exhibited a new subtype. Fourteen clones were further characterized by multilocus sequence typing, and results showed a high degree of genetic diversity, with some clones having new alleles and subtypes. The minimum inhibitory concentrations (MICs) of 5 antimicrobials regularly used to treat BRD was determined on 45 clones. Susceptibility profiles showed very broad diversity, confirming the variety of clones actively circulating. We detected clones with high MICs, including increased MICs of enrofloxacin (n = 5). This is the first study to report the presence of M. bovis in Algeria in calves with BRD. This research also finds broad genetic and phenotypic diversity in the actively circulating isolates.

Monitoring Mycoplasma bovis Diversity and Antimicrobial Susceptibility in Calf Feedlots Undergoing a Respiratory Disease Outbreak.

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.

Swab cloths as a tool for revealing environmental contamination by Q fever in ruminant farms.

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.

Monitoring the Decrease in Susceptibility to Ribosomal RNAs Targeting Antimicrobials and Its Molecular Basis in Clinical Mycoplasma bovis Isolates over Time.

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 G748A and A2058G in the 23S rRNA alleles for tylosin/tilmicosin and of A965T and A967T 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.

Alterations in the Quinolone Resistance-Determining Regions and Fluoroquinolone Resistance in Clinical Isolates and Laboratory-Derived Mutants of Mycoplasma bovis: Not All Genotypes May Be Equal.

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.

Loss of diversity within Mycoplasma bovis isolates collected in France from bovines with respiratory diseases over the last 35 years.

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.

Validation of BdCCp2 as a marker for Babesia divergens sexual stages in ticks.

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.

Development of a multiplex real-time PCR for contagious agalactia diagnosis in small ruminants.

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.

A Vegetation Index qualifying pasture edges is related to Ixodes ricinus density and to Babesia divergens seroprevalence in dairy cattle herds.

Babesia divergens, transmitted by the tick Ixodes ricinus, is the main agent of bovine piroplasmosis in France. This Apicomplexa often is present in asymptomatic carriers; however, clinical cases are rare. While numerous factors are known to influence tick density, no risk factor of contact with B. divergens has been identified for cattle. Our study aimed to explore whether a Vegetation Index could serve as an indirect indicator of within-herd B. divergens seroprevalence. In February 2007, blood samples were taken from all of the cows in 19 dairy cattle herds in Western France and IFAT serology was performed individually to measure B. divergens seroprevalence. The following spring, I. ricinus nymphs were collected by drag sampling along transects on the vegetation of each farm's pasture perimeters. Tick density was related significantly to a Vegetation Index (V.I., ranging from 1 to 5) that took into account the abundance of trees and bushes on the edge of pastures: most ticks (57%) were found in transects with the highest V.I. (covering 15% of the explored surface in the study area). At the farm level, the proportion of transects presenting I. ricinus nymphs was significantly related to B. divergens seroprevalence: the farms with more than 15% of transects with I. ricinus had a significantly higher risk of high seroprevalence. The proportion of pasture perimeters where the V.I.=5 also was significantly related to B. divergens seroprevalence: the farms where more than 20% of transects had a V.I.=5 had a significantly higher risk of high seroprevalence. Given that the Vegetation Index is a steady indicator of the potential I. ricinus density in the biotope, we recommend that the risk of high B. divergens seroprevalence in cows be evaluated using this tool rather than drag samplings.

Natural transmission of Zoonotic Babesia spp. by Ixodes ricinus ticks.

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.