Agnès Leblond

A new multiple-locus variable-number tandem repeat analysis reveals different clusters for Anaplasma phagocytophilum circulating in domestic and wild ruminants

BackgroundAnaplasma phagocytophilum is a tick-borne intragranulocytic alpha-proteobacterium. It is the causative agent of tick-borne fever in ruminants, and of human granulocytic anaplasmosis in humans, two diseases which are becoming increasingly recognized in Europe and the USA. However, while several molecular typing tools have been developed over the last years, few of them are appropriate for in-depth exploration of the epidemiological cycle of this bacterium. Therefore we have developed a Multiple-Locus Variable number tandem repeat (VNTR) Analysis typing technique for A. phagocytophilum.MethodsFive VNTRs were selected based on the HZ human-derived strain genome, and were tested on the Webster human-derived strain and on 123 DNA samples: 67 from cattle, 7 from sheep, 15 from roe deer, 4 from red deer, 1 from a reindeer, 2 from horses, 1 from a dog, and 26 from ticks.ResultsFrom these samples, we obtained 84 different profiles, with a diversity index of 0.96 (0.99 for vertebrate samples, i.e. without tick samples). Our technique confirmed that A. phagocytophilum from roe deer or domestic ruminants belong to two different clusters, while A. phagocytophilum from red deer and domestic ruminants locate within the same cluster, questioning the respective roles of roe vs red deer as reservoir hosts for domestic ruminant strains in Europe. As expected, greater diversity was obtained between rather than within cattle herds.ConclusionsOur technique has great potential to provide detailed information on A. phagocytophilum isolates, improving both epidemiological and phylogenic investigations, thereby helping in the development of relevant prevention and control measures.

Adaptation of a real-time PCR method for the detection and quantification of pathogenic leptospires in environmental water

Leptospirosis is a major zoonotic disease that affects humans and animals in all continents, in both rural and urban areas. In Europe, metropolitan France is the most affected country, with about 300 human cases declared per year. In France, although leptospirosis is now mostly considered as a recreational disease related to freshwater areas, isolation of pathogenic leptospires from environmental water samples still remains difficult. It thus seemed important to set up an efficient method to detect and quantify these bacteria in this environment. We determined a DNA extraction method suitable for freshwater samples and adapted a real-time quantitative PCR based on the detection of the LipL32 gene using the SYBR green chemistry. The method developed is specific for pathogenic Leptospira. It permits the detection of all the pathogenic strains tested and none of the saprophytic strains. Quantification is possible between 10 and 10(7) bacteria/mL, and therefore, the method represents a tool that could be integrated into future public health surveillance programs for recreational freshwater areas.

Can horses be clinically screened for West Nile Fever

In Europe, the frequency of West Nile Fever (WNF) outbreaks in horses and/or human beings is on the increase, especially in mid-eastern and southern Europe (Dauphin and others 2004, Rabel and others 2011). However, in several western European countries no activity of the virus has been detected so far, for example, in The Netherlands (Rockx and others 2006), Belgium and the UK (Morgan 2006)). However, considering the presence of migratory birds and suitable vectors in those countries, and the reports of changing epidemiology of the virus (Petersen and Marfin 2005, Blitvich 2008), the West Nile virus (WNV) is a genuine threat. Horses are considered good sentinels for WNV infection surveillance (Petersen and Marfin 2005) by the use of syndromic surveillance followed by laboratory confirmation. Syndromic surveillance aims at early identification of disease clusters before laboratory confirmation, thereby reducing morbidity and mortality (Leblond and others 2007). Clinical signs of WNF in horses are, however, difficult to distinguish from those of other neurological diseases (Leblond and others 2007, Porter and others 2011). The aim of this study was to identify clinical variables that could be indicators for WNF in horses, which will be attempted …

Antimicrobial resistance trends in Escherichia coli isolated from diseased food‐producing animals in France: A 14‐year period time‐series study

Antimicrobial resistance (AMR) among bacteria isolated from food‐producing animals is a growing concern with implications for public health. AMR surveillance is essential to identify resistance trends and help in the design of effective and efficient control strategies. The aim of the study was to describe the antimicrobial susceptibility of pathogenic Escherichia coli isolated from three livestock productions in France (cattle, swine and poultry). The trend in resistance to the most commonly prescribed antibiotics in animal health was analysed as follows: amoxicillin (penicillin), spectinomycin or streptomycin (aminoglycoside), tetracycline and trimethoprim‐sulfamethoxazole/Enrofloxacin and ceftiofur were also taken into account as members of critically important antimicrobial families in human and veterinary medicine, that is fluoroquinolones and third‐generation cephalosporins, respectively. Data collected between 2002 and 2015 by the French national surveillance network of AMR referred to as RESAPATH were analysed. Resistance trends were investigated using non‐linear analysis (generalized additive models) applied to time‐series stratified by livestock production and antibiotic. Irrespective of the species and the antibiotic considered, resistance signals over time showed no significant annual cycle. Resistance to third‐generation cephalosporins emerged during the period of the study, with a peak at 22% [20.5; 24.0] in poultry in 2010, decreasing afterwards, while it remained consistently below 10% for the other species. The proportion of resistance to fluoroquinolones was broadly similar between species and remained under 30%, with a slight decreasing trend after 2009. Resistances to tetracycline and amoxicillin remained high, between 90% and 40% over time in cattle and swine. After 2010, there was a decrease in resistance to these antibiotics for all species, especially to tetracycline for poultry with a drop from 84% in 2009 to 43% in 2015. These results contribute to risk assessment and constitute objective evidence on which to evaluate the efficacy of control measures implemented to limit AMR occurrence.

An Integrative Eco-Epidemiological Analysis of West Nile Virus Transmission

West Nile disease, caused by the West Nile virus (WNV), is a mosquito-borne zoonotic disease affecting humans and horses that involves wild birds as amplifying hosts. The mechanisms of WNV transmission remain unclear in Europe where the occurrence of outbreaks has dramatically increased in recent years. We used a dataset on the competence, distribution, abundance, diversity and dispersal of wild bird hosts and mosquito vectors to test alternative hypotheses concerning the transmission of WNV in Southern France. We modelled the successive processes of introduction, amplification, dispersal and spillover of WNV to incidental hosts based on host–vector contact rates on various land cover types and over four seasons. We evaluated the relative importance of the mechanisms tested using two independent serological datasets of WNV antibodies collected in wild birds and horses. We found that the same transmission processes (seasonal virus introduction by migratory birds, Culex modestus mosquitoes as amplifying vectors, heterogeneity in avian host competence, absence of ‘dilution effect’) best explain the spatial variations in WNV seroprevalence in the two serological datasets. Our results provide new insights on the pathways of WNV introduction, amplification and spillover and the contribution of bird and mosquito species to WNV transmission in Southern France.

A retrospective study on equine herpesvirus type-1 associated myeloencephalopathy in France (2008-2011).

Diagnosis of equine herpesvirus-1 associated myeloencephalopathy (EHM) can be troublesome, but early recognition and knowledge of risk factors are essential for prevention and control. The objectives for this study are to (1) describe EHM in France, (2) improve clinical recognition, (3) identify risk factors. Through epidemiosurveillance of acute neurological cases (all considered to be potentially infectious cases) in France (2008-2011), 26 EHM cases were identified and 29 EHM negative control cases. EHM cases were described and compared to controls with univariate, multivariate and classification and regression tree analysis. EHM cases had a 46% fatality rate and were frequently isolated cases. Most showed ataxia, paresis and a cauda equina syndrome, yet presence of other neurological signs was variable. Statistical analysis identified the following variables to be significantly associated to EHM compared to controls: introduction of a new horse to the herd, cauda equina syndrome, larger herd size, saddle horses and month of occurrence. The presence of many isolated cases, and less typical and variable clinical presentations emphasize the difficulty in diagnosing EHM. Nevertheless, history and clinical examination of acute neurological cases can be valuable in recognizing EHM early as well in order to select those cases that need further laboratory testing and infection control measures. Moreover, with a different study format and geographic location, risk factors were found to be similar to previous studies, therefore strengthening their significance to the spread of EHM.

Comparative epidemiology of E. coli resistance to third-generation cephalosporins in diseased food-producing animals

Categorized by WHO as critically important antibiotics, third-generation cephalosporins (3GCs) are one of the latest therapeutic alternatives to fight severe infectious diseases in humans. Some antibiotics belonging to this class are prescribed to treat food-producing animals in specific pathological contexts. Preserving the effectiveness of 3GCs requires characterization and careful monitoring of 3GCs resistance and the identification and implementation of measures that can limit this antimicrobial resistance (AMR). Here, we characterized the 3GCs resistance in Escherichia coli isolated from diseased animals. Using data collected from broilers, hens, calves, piglets, sows, turkeys and ducks between 2006 and 2016 by the French surveillance network of AMR in pathogenic bacteria of animal origin (called RESAPATH), we investigated the dynamics of resistance to 3GCs. Our non-linear analysis applied to time series showed that the evolution of E. coli resistance to 3GCs is specific to each animal category. From 2006 to 2010, resistance to 3GCs increased for most animal categories. We observed peaks of high-level of resistance for hens (21.5% in 2010) and broilers (26.7% in 2011), whereas trends stayed below 10% for the other animal categories throughout the study period. Resistance later decreased and, since 2014, 3GCs resistance has dropped below 10% for all animal categories. The parallel between trends and measures to limit AMR over the period shed lights on the impact of practices changes, public policies (EcoAntibio Plan) and sector-led initiatives (moratorium in swine sector). Finally, they highlight the usefulness and importance of AMR surveillance networks in animal health, such as RESAPATH.

Antimicrobial resistance in bacteria isolated from mastitis in dairy cattle in France, 2006–2016

In dairy cattle, mastitis is the most frequent bacterial disease, and the routine use of antibiotics for treatment and prevention can drive antimicrobial resistance (AMR). The aim of our study was to estimate the levels of AMR of the 3 main bacteria isolated from dairy cattle with mastitis in France (Streptococcus uberis, Escherichia coli, and coagulase-positive staphylococci) and to investigate their changes over time. Data collected between 2006 and 2016 by the French surveillance network for AMR in pathogenic bacteria of animal origin (called RESAPATH) were analyzed. The proportions of mono- and multidrug resistance were calculated and the trends were investigated using nonlinear analyses applied to time series. Over the whole period, the lowest proportions of resistance in S. uberis isolates were observed for oxacillin (2.2%) and gentamicin (2.4%) and most resistance levels were below 20%. The trends in resistance showed some significant variation, mainly for S. uberis, but without a common pattern across the various antibiotics examined. For only 2 combinations of bacteria-antibiotic the trend in resistance showed a continuous increase from 2006 to 2016: tetracycline resistance in S. uberis isolates and third-generation cephalosporin resistance in E. coli isolates. In E. coli, the highest proportions of resistance were observed for amoxicillin (28.1%) and tetracycline (23.1%). Resistance to third-generation cephalosporins in E. coli from dairy cattle was almost nil in 2006, but reached 2.4% in December 2016. This increase is particularly concerning because these antibiotics constitute one of the latest therapeutic alternatives to fight severe infectious diseases in humans. Except for penicillin (33.9%), the proportions of resistance in coagulase-positive staphylococci were below 11% during the whole study period. Multidrug resistance (isolates with acquired resistance to at least one antibiotic in 3 or more antibiotic classes) ranged from 2.4% for coagulase-positive staphylococci to 9.9% for S. uberis. These findings can serve as guidelines for practitioners in the choice of the most appropriate antibiotic according to the prevailing epidemiological context. Ultimately, our results contribute to risk assessment of AMR and provide a baseline for setting up and evaluating control measures and designing strategies to limit AMR.

Seroprevalence of horses to Coxiella burnetii in an Q fever endemic area

Coxiella burnetii can infect many animal species, but its circulation dynamics in and through horses is still unclear. This study evaluated horse exposure in an area known to be endemic for ruminants and humans. We assessed antibody prevalence in horse serum by ELISA, and screened by qPCR horse blood, ticks found on horses and dust from stables. Horse seroprevalence was 4% (n = 335, 37 stables) in 2015 and 12% (n = 294, 39 stables) in 2016. Of 199 horses sampled both years, 13 seroconverted, eight remained seropositive, and one seroreverted. Seropositive horses were located close to reported human cases, yet none displayed Q fever-compatible syndromes. Coxiella DNA was detected in almost 40% of collected ticks (n = 59/148 in 2015; n = 103/305 in 2016), occasionally in dust (n = 3/46 in 2015; n = 1/14 in 2016) but never in horse blood. Further studies should be implemented to evaluate if horses may be relevant indicators of zoonotic risk in urban and suburban endemic areas.