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Neurocysticercosis (NC), caused by the larval stage of Taenia solium , is one of the most common parasitic diseases of the central nervous system. The diagnosis of NC is mostly based on costly brain neuroimaging (computed tomography and/or nuclear magnetic resonance), which is rarely accessible in most affected areas. The most sensitive and specific tools for NC diagnosis are imagery techniques. The identification of specific antibodies and antigens is currently used only to support NC diagnosis due to their limited specificity and sensitivity. This study was performed to compare immunodiagnostic assays (antibody detection by enzyme-linked immunosorbent assay [ELISA] and enzyme-linked immunoelectrotransfer blotting [EITB] and HP10 antigen detection by ELISA) with the detection of parasite DNA by PCR amplification of a repetitive element of the parasite genome in the cerebrospinal fluid (CSF) of 121 radiologically and clinically characterized NC patients. Patients were divided into six groups according to the stage of the parasites and their localization. The CSF cellularity of each patient was also recorded. When all patients were considered, PCR exhibited the highest sensitivity (95.9%) and variable specificity (80% or 100%) depending on the controls used. The sensitivities of antibody detection by ELISA and EITB were not significantly different, and ELISA identified HP10 antigen mostly when vesicular cysticerci were located in the subarachnoideal basal cisterns. These results can help in the selection of different individual assays or combinations of assays to be used in NC diagnosis according to different requirements.

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex (MTBC), a group of major human and animal pathogens such as Mycobacterium tuberculosis and Mycobacterium bovis . M microti was originally described as the cause of tuberculosis (TB) in wild rodents, but it can also infect other animal species and has even been described as a potential zoonotic agent (Cavanagh and others 2002, Panteix and others 2010). A recent retrospective study reported 35 animal cases in France between 2002 and 2014 (Michelet …

International audience; An unusual outbreak of teat/udder skin lesions occurred in a dairy goat flock in France. Lesions first appeared as circular, indurated, erythematous areas of skin and progressed to form dark raised haemorrhagic crusts and ulcerative plaques. Histopathological examination revealed marked granulomatous dermatitis with multifocal ulceration. The granulomatous inflammation, with frequent Langhans type multinucleated cells and central caseous necrosis, was indicative of mycobacterial infection. The presence of non-cultivable mycobacteria was confirmed by sequencing PCR products from DNA extracted directly from the lesions and sequences matched a novel mycobacterial pathogen closely related to M. leprae and M. lepromatosis and previously identified in cattle thelitis. The association of nodular gross lesions and tuberculoid granulomas on the teat and lower udder, and the presence of mycobacteria DNA support a diagnosis of tuberculoid nodular thelitis in goats due to mycobacterial infection

To the Editor: Mycobacterium microti is a member of the Mycobacterium tuberculosis complex (MTBC). This complex also includes M. tuberculosis, which causes human tuberculosis, and M. bovis and M. caprae, which cause bovine tuberculosis. M. microti was initially described as a pathogen of small rodents and also frequently affects domestic animals, especially cats, and has also been described in wildlife, especially wild boars and badgers (1). This mycobacterium has also been involved in human pulmonary tuberculosis cases, which highlights its potential zoonotic risk (2). We report a case of M. microti infection in a dairy goat herd, which underlines the risk for confounding bovine tuberculosis diagnosis and potential consequences for livestock management. France has been considered officially free of bovine tuberculosis by the European Union since 2001. Surveillance of this disease is based on antemortem testing with tuberculin skin tests and on systematic postmortem inspection at abattoirs through sanitary inspection of carcasses to detect bovine tuberculosis–like lesions. However, because bovine tuberculosis evolves in an insidious manner and antemortem or postmortem diagnostic tests are not efficient for detecting latently infected animals, an infected herd may remain unidentified for long periods and can be responsible for contamination of other herds by animal movement or contact with animals of neighboring herds. For this reason, to detect other potentially associated cases, investigations in herds epidemiologically linked to the index outbreak are also performed, either through skin testing or through diagnostic culling of those animals introduced from the infected herd or any other animal with a skin test–positive result. This case of bovine tuberculosis in a goat was reported in a region of the Alps Mountains in France. The herd was composed of 140 dairy goats, which were used for raw milk cheese production. Goats were semi-extensively bred and kept in pastures >6 months per year. Investigations conducted after identification of a case of bovine tuberculosis in a neighboring cattle herd infected with M. bovis, in which the index case was identified at an abattoir, highlighted the epidemiologic link with the goat herd because animals in both populations shared the same pastures. Thus, the goat herd was subjected to single intradermal tuberculin tests. Three adult goats (goats A, B, and C) showed positive results and were culled for direct diagnosis. Goats A and B showed no lesion at abattoir inspections, but goat C had bovine tuberculosis–like lesions on the retropharyngeal and mediastinal lymph nodes. Retropharyngeal, tracheobronchial, and mediastinal lymph nodes were sampled from the 3 goats. Retromammary lymph nodes were also sampled from goats B and C (both females). Lesions from goat C were examined by histopathologic analysis and showed a profile suggestive of bovine tuberculosis with necrosis, Langhans giant cells, and few acid–alcohol-resistant bacilli by Ziehl-Neelsen staining (Figure). All samples were subjected to bacterial culture and molecular diagnosis (3). Figure Goat lymph node granuloma with numerous Langhans-type multinucleated giant cells from a goat in France infected with Mycobacterium microti (hematoxylin and eosin stain). Scale bar indicates 50 μm. Although after 3 months culture results were negative for all samples, DNA extracted from the retropharyngeal lymph node of goat C showed a positive PCR result for MTBC DNA by the LSI VetMAX Mycobacterium tuberculosis Complex Real-Time PCR Kit (Thermo Fisher Scientific, Villebon sur Yvette, France). Further characterization of this DNA was performed by using molecular analysis specific for the regions of difference, which enables differentiation of MTBC members (4), and spoligotyping (5). The infectious agent from goat C was identified as M. microti spoligotype SB0118. Moreover, a bovine tuberculosis investigation in wildlife (6) identified M. microti spoligotype SB0118 infection case in a dead badger found 8 km from the goat farm. Thus, the long time during which goats remain in pastures might have favored environmental contamination by interaction with wildlife. Furthermore, an additional case of M. microti infection in a cat reported in 2011 in the same region also had the SB0118 spoligotype (7), which demonstrated that this bacillus is actively circulating in animals from this area. M. microti was previously isolated on the basis of a skin test–positive result for cattle in the United Kingdom (8), which demonstrated the risk for infection in livestock. These findings raise concern on reliability of diagnostic tests used for bovine tuberculosis surveillance. M. microti, which is phylogenetically similar to M. bovis or M. caprae and widely disseminated in the environment, could be responsible for misleading diagnostic results, as demonstrated in this study. Highly specific tests are needed to accurately identify M. bovis (or M. caprae) infection at antemortem examination through use of specific antigens, such as ESAT 6 and CFP10, which are absent in M. microti and are currently used in the interferon-γ test in France (9). In addition, at postmortem diagnosis, use of specific molecular tools capable of rapidly distinguishing members of the MTBC should be considered. Histopathologic analysis lacks specificity, and obtaining results for bacterial culture takes too much time for these particularly slow-growing and fastidious mycobacteria. M. microti has already been reported to cause tuberculosis in immunocompromised and immunocompetent patients in France (10). Thus, potential risk for infection of humans by consumption of raw goat milk cheese cannot be ruled out.

We describe here 35 animal cases of tuberculosis due to Mycobacterium microti in France (2002–2014). Recently, molecular tools that overcome the difficulty of confirming infection by this potentially zoonotic agent have revealed an increasing number of cases, suggesting that its prevalence may have been underestimated.

International audience; Due to increased travel, climatic, and environmental changes, the incidence of tick-borne disease in both humans and animals is increasing throughout Europe. Therefore, extended surveillance tools are desirable. To accurately screen tick-borne pathogens (TBPs), a large scale epidemiological study was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae) pathogens in the three European countries. Moreover we detected Borrelia spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the first time in Danish ticks. This surveillance method represents a major improvement in epidemiological studies, able to facilitate comprehensive testing of TBPs, and which can also be customized to monitor emerging diseases.

Background: In France as elsewhere in Europe the most prevalent TBD in humans is Lyme borreliosis, caused by different bacterial species belonging to Borrelia burgdorferi sensu lato complex and transmitted by the most important tick species in France, Ixodes ricinus. However, the diagnosis of Lyme disease is not always confirmed and unexplained syndromes occurring after tick bites have become an important issue. Recently, B. miyamotoi belonging to the relapsing fever group and transmitted by the same Ixodes species has been involved in human disease in Russia, the USA and the Netherlands. In the present study, we investigate the presence of B. miyamotoi along with other Lyme Borreliosis spirochetes, in ticks and possible animal reservoirs collected in France. Methods: We analyzed 268 ticks (Ixodes ricinus) and 72 bank voles (Myodes glareolus) collected and trapped in France for the presence of DNA from B. miyamotoi as well as from Lyme spirochetes using q-PCR and specific primers and probes. We then compared the French genotypes with those found in other European countries. Results: We found that 3% of ticks and 5.55% of bank voles were found infected by the same B. miyamotoi genotype, while co-infection with other Lyme spirochetes (B. garinii) was identified in 12% of B. miyamotoi infected ticks. Sequencing showed that ticks and rodents carried the same genotype as those recently characterized in a sick person in the Netherlands. Conclusions: The genotype of B. miyamotoi circulating in ticks and bank voles in France is identical to those already described in ticks from Western Europe and to the genotype isolated from a sick person in The Netherlands. This results suggests that even though no human cases have been reported in France, surveillance has to be improved. Moreover, we showed that ticks could simultaneously carry B. miyamotoi and Lyme disease spirochetes, increasing the problem of co-infection in humans.

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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., Author Summary Diseases transmitted by ticks have diverse etiology (viral, bacterial, parasitic) and are responsible for high morbidity and mortality rates around the world, both in humans and animals. The emergence or re-emergence of tick-borne diseases is increasingly becoming a problem as the geographical distribution of several tick species is expanding, as well as the numbers of potential or known tick-borne pathogens are constantly evolving. It is thus necessary to know which microorganisms circulate within communities of this major vector to ensure adequate epidemiological surveillance. In this study, we evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce, without a priori, an inventory of both predicted and non-expected parasites carried by Ixodes ricinus, the most prevalent human biting tick in France. Our findings suggest that NGS strategies could be used to produce an inventory of live parasites residing in ticks from a selected area, thereby expanding our knowledge base of tick-associated parasites.

Ticks can transmit a large spectrum of pathogens including bacteria, viruses and parasites with a significant number of these pathogens being agents of emerging infectious diseases. In Europe, the most prevalent tick-borne disease is Lyme Borreliosis, caused by the bacteria Borrelia burgdorferi s.l. In most cases, Lyme Borreliosis is well diagnosed. However, beside these typical cases, patients bitten by ticks can be infected by many other pathogens (bacteria: Anaplasma spp., Bartonella spp., Rickettsia spp.; parasites: Babesia spp., Theileria spp.; and arboviruses: TBEV) that are more difficult to diagnose. Moreover, co-infections between several of these pathogens might also occur. Clinical surveys show that patients coinfected by several tick-borne pathogens present more severe symptoms and a longer duration of illness than those infected by a single pathogen. The overall objective of our study was to evaluate tick-borne pathogen coinfection in ticks and the consequence of those coinfections for human health. Using the high throughput real-time PCR chip, we detected the DNA of the 37 major tick-borne pathogens in a cohort of questing adult of Ixodes ricinus ticks collected in Ardennes (France). We identified that 60% of ticks were infected by at least one pathogen and half of the infected ticks were coinfected. We then evaluated the risk for simultaneous infection of those pathogens to humans by detecting the DNA of the most prevalent tick-borne pathogens in the blood of patients bitten by ticks. Our data illustrated the importance of coinfection, and highlighted the necessity to evaluate coinfection in the context of tick-borne diseases.

International audience; Francisella tularensis, the causative agent of tularemia, is commonly transmitted by ticks. To ensure accurate F. tularensis reporting rates in epidemiological surveys, specific discrimination between F. tularensis and Francisella-like tick endosymbionts (FLEs) is absolutely critical. Four molecular available techniques capable of distinguishing Francisella spp. were compared here for the first time in French Dermacentor reticulatus ticks in order to estimate their specificity as well as their ease and speed of use. Results showed that tul4 and fopA real-time PCR assays can easily and effectively discriminate between F. tularensis and FLEs in D. reticulatus. In addition, a high prevalence of FLEs in D. reticulatus collected in France was reported by the use of fopA real-time PCR assay (79%). Finally, phylogenetic analysis showed that FLEs isolated from D. reticulatus correspond to a well-defined group compared to FLEs originating from various tick species. (C) 2013 Elsevier B.V. All rights reserved.

The taeniasis/cysticercosis complex is included in the list of neglected zoonotic diseases by the World Health Organization due to its significant impact on public health in tropical areas. Cysticercosis is still endemic in many regions of Asia, Africa and Latin America. Long absent in Europe and in other developed countries, cysticercosis has recently re-emerged in the United States and Canada, due to immigration, travel and local transmission. This has encouraged the use of molecular data to understand better the influence of animal and human hosts on the emergence and spread of Taenia species. The increasing number of mitochondrial sequences now available from human tapeworms and recent advances in computational tools has enabled reconstruction of the biogeography and evolutionary history of these organisms. New molecular data have provided insights into the biogeography of T. solium, T. asiatica and T. saginata. A Bayesian statistical framework using variable evolutionary rates from lineage to lineage has allowed an improved timescale analysis of human tapeworms. The dates of divergence obtained were compared to the timing of evolutionary events in the history of their hosts, based on the hypothesis that Taenia spp. and their hosts share a common history. Herein, we review changes in the definitive and secondary hosts and human interactions that underlie the differentiation and evolution of tapeworms. Species diversification of Taenia seems to be closely linked with the evolution of intermediate hosts in response to climatic events during the Pleistocene. Different genotypes of T. solium emerged when European and Asian wild boar Sus spp. populations diverged. Taenia saginata emerged when wild cattle Bos primigenius evolved and when zebu Bos indicus and taurine Bos taurus ancestors separated. Humans through migrations and later with the development of farming and animal husbandry may have had a significant impact on the spread and diversification of tapeworms. Migrations of Homo erectus from Africa to Asia and later of Homo sapiens facilitated the diversification and dispersal of T. solium and T. saginata populations. The development of animal husbandry, making Sus scrofa and Bos taurus preferential intermediate hosts, led to the worldwide distribution of parasites. New molecular data combined with an innovative dating method allow us to explain the ways in which ancient human migrations promoted the emergence and spread of taeniasis and cysticercosis around the world. Another intriguing phenomenon explained better by our approach is the influence of human settlement on the spread of these parasites in recently inhabited areas. The diverse nature of T. solium currently observed in Madagascar may correspond to multiple imports of the parasite during Austronesian migrations, while in Mexico a recent influence of humans during the colonial period is more likely. Human activities, especially food preparation and husbandry methods, remain responsible for the transmission and persistence of these parasites.

International audience; Cysticercosis is a serious public health problem in Madagascar. The prevalence rate of active cysticercosis reached 21% in regions with a high level of livestock farming. Taenia solium of African-American and Asian genotypes are both present on the island. The times of divergence of the 13 specimens studied suggests a very ancient diversification of T. solium. These events are widely thought to be prior to the domestication of pigs, and seem to follow the expansion of Homo in Asia. Multiple human migrations and the diversity of potential intermediate hosts may have led to a complex epidemiological situation on the island.