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Surveillance of bovine tuberculosis (bTB) is partly based on the sanitary inspection of carcasses at the abattoir to detect bTB-like lesions which, in compliance with EU recommendations, are analysed by bacteriology and histopathology to disclose Mycobacterium bovis (or M. caprae) infection. Moreover, since 2012, a PCR method with similar sensitivity and specificity values of histopathology and bacteriology respectively is additionally employed in France, partially compensating for the weaknesses of classical diagnostic methods. We analysed a collection of bTB-like lesions from cattle presenting positive histological results albeit with negative PCR results. We present here the results of these samples, recovered from 292 animals culled between 2013 and 2016, analysed with a second line molecular diagnosis approach that consists in a combination of PCRs targeting the M. tuberculosis-M. avium complexes as well as the Mycobacterium genus and sequencing of hsp65 gene. These molecular analyses disclosed to identify the presence of non-tuberculous bacteria which could be responsible for most of these non-specific TB lesions: non tuberculous mycobacteria (24%) or Actinomycetales (56%) such as Rhodococcus equi (53%); 24% of the samples were negative. M. bovis -or any other MTBC members- was neither detected by molecular methods nor isolated in any of them at the end of the 3 months of culture. In conclusion, these results highlight the lack of specificity of histopathology and the usefulness of a first line PCR with a second line molecular diagnostic test to circumvent it. This diagnostic strategy makes it possible to reduce the number of suspect bTB cases raised at the abattoir or shortening their lock-up periods. By simplifying diagnostic schemes, the use of this tool could improve bTB surveillance and make eradication programs more efficient in the future.

INTRODUCTION:Ticks are the most common arthropod vectors of both human and animal diseases in Europe, and the Ixodes ricinus tick species is able to transmit a large number of bacteria, viruses and parasites. Ticks may also be co-infected with several pathogens, with a subsequent high likelihood of co-transmission to humans or animals. However few data exist regarding co-infection prevalences, and these studies only focus on certain well-known pathogens. In addition to pathogens, ticks also carry symbionts that may play important roles in tick biology, and could interfere with pathogen maintenance and transmission. In this study we evaluated the prevalence of 38 pathogens and four symbionts and their co-infection levels as well as possible interactions between pathogens, or between pathogens and symbionts. METHODOLOGY/PRINCIPAL FINDINGS:A total of 267 Ixodes ricinus female specimens were collected in the French Ardennes and analyzed by high-throughput real-time PCR for the presence of 37 pathogens (bacteria and parasites), by rRT-PCR to detect the presence of Tick-Borne encephalitis virus (TBEV) and by nested PCR to detect four symbionts. Possible multipartite interactions between pathogens, or between pathogens and symbionts were statistically evaluated. Among the infected ticks, 45% were co-infected, and carried up to five different pathogens. When adding symbiont prevalences, all ticks were infected by at least one microorganism, and up to eight microorganisms were identified in the same tick. When considering possible interactions between pathogens, the results suggested a strong association between Borrelia garinii and B. afzelii, whereas there were no significant interactions between symbionts and pathogens. CONCLUSION/SIGNIFICANCE:Our study reveals high pathogen co-infection rates in ticks, raising questions about possible co-transmission of these agents to humans or animals, and their consequences to human and animal health. We also demonstrated high prevalence rates of symbionts co-existing with pathogens, opening new avenues of enquiry regarding their effects on pathogen transmission and vector competence.

Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.

BACKGROUND AND AIMS: Ticks are highly susceptible to global environmental and socio-economical changes. Several tick-borne pathogens have been reported in new geographical regions while new species, strains or genetic variants of tick-borne microorganisms are continually being detected. However, tick-borne pathogens are still poorly understood, and it is estimated that half of all human tick-borne disease has an unknown origin. Therefore in order to prevent these diseases, more effort is required to identify unknown or unexpected tick-borne pathogens. Ixodes ricinus is the vector for a broad range of bacterial pathogens and the most prevalent tick in Europe. The aim of the present study was to evaluate the capability of Next Generation Sequencing (NGS) to extend the inventory of pathogenic bacteria carried by this species of tick in France. METHODS: RNA and DNA were extracted from 1450 I. ricinus questing nymphs collected by flagging in Alsace, France. RNA was pooled and used for NGS. Following de novo assembly, bacterial contigs were assigned to the closest known taxonomy. DNA was used for real time PCR to confirm taxonomic species assignment of NGS-derived contigs for the doubtful cases, and for determination of prevalence. RESULTS: We have generated a global in-depth picture of tick-borne bacteria. We identified RNA from the main pathogenic bacterial species known to be transmitted by I. ricinus. In addition we also identified unanticipated bacterial species for which we have estimated the prevalence within those ticks inhabiting the studied areas. CONCLUSIONS: The data obtained from this study has proven that NGS has an enormous potential to detect the unexpected and provides the means to monitor pathogen occurrence.