Luc Chabanne

Prevalence of Bartonella clarridgeiae and Bartonella henselae in Domestic Cats from France and Detection of the Organisms in Erythrocytes by Immunofluorescence

ABSTRACT The prevalence of Bartonella infection in a pet cat population from France was found to be 8.1% (8 of 99 cats). The intraerythrocytic location of Bartonella clarridgeiae is shown for the first time, and we show that immunofluorescence detection of the organism in erythrocytes correlates with the number of bacteria in blood.

Diagnosis and incidence risk of clinical canine monocytic ehrlichiosis under field conditions in Southern Europe

BackgroundCanine Monocytic Ehrlichiosis (CME), due to the bacterium Ehrlichia canis and transmitted by the brown dog tick Rhipicephalus sanguineus, is a major tick-borne disease in southern Europe. In this area, infections with other vector-borne pathogens (VBP) are also described and result in similar clinical expression. The aim of the present study was to evaluate the incidence risk of clinical CME in those endemic areas and to assess the potential involvement of other VBP in the occurrence of clinical and/or biological signs evocative of the disease.MethodsThe study was conducted from April to November 2011 in veterinary clinics across Italy, Spain and Portugal. Sick animals were included when fitting at least three clinical and/or biological criteria compatible with ehrlichiosis. Serological tests (SNAP®4Dx, SNAP®Leish tests, Idexx, USA) and diagnostic PCR for E. canis, Anaplasma platys, Anaplasma phagocytophilum, Babesia spp, Hepatozoon canis and Leishmania infantum detection were performed to identify the etiological agents. Ehrlichiosis was considered when three clinical and/or biological suggestive signs were associated with at least one positive paraclinical test (serology or PCR). The annual incidence risk was calculated and data were geo-referenced for map construction. The probabilities of CME and other vector-borne diseases when facing clinical and/or biological signs suggestive of CME were then evaluated.ResultsA total of 366 dogs from 78 veterinary clinics were enrolled in the survey. Among them, 99 (27%) were confirmed CME cases, which allowed an estimation of the average annual incidence risk of CME amongst the investigated dog population to be 0.08%. Maps showed an increasing gradient of CME incidence risk from northern towards southern areas, in particular in Italy. It also suggested the existence of hot-spots of infections by VBP in Portugal. In addition, the detection of other VBP in the samples was common and the study demonstrated that a dog with clinical signs evocative of CME is as likely to be positive to Ehrlichia canis as to another VBP.ConclusionsThe study confirms the endemicity of CME in southern Europe and highlights the difficulties encountered by veterinarians to differentiate CME from other vector-borne diseases under field conditions.

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.

First evidence and molecular characterization of Babesia vogeli in naturally infected dogs and Rhipicephalus sanguineus ticks in southern France.

Babesiosis is an emerging tick-borne disease of animals and humans caused by intraerythrocytic protozoa of the genera Babesia and Theileria. In France canine babesiosis has a high prevalence with Babesia canis thought to be the main aetiological agent of the disease. Babesia vogeli has already been reported to occur in Europe and in other countries around the Mediterranean Sea. The tick Rhipicephalus sanguineus, the main known vector of B. vogeli, occurs in southern France. However, only one case of a B. vogeli infected dog has been reported to date in France. To gain further insight into the prevalence of Babesia and Theileria infections in dogs and ticks of the R. sanguineus complex, a study was conducted in a veterinary practice in the south of France from January to September 2010. Twelve bloods from dogs and 36 R. sanguineus ticks were analyzed using PCR and sequencing. For the analysis of ticks, a new primer was designed to specifically amplify the B. vogeli 18S rRNA gene. Four dogs (33.3%) and 8 ticks (22.2%) were found to be infected with B. vogeli. This approach has thus revealed for the first time a cluster of cases of canine babesiosis caused by B. vogeli in France and highlights the need to systematically screen for pathogens potentially responsible for canine babesiosis at the species level using suitable molecular tools.

Multilocus sequence analysis of Anaplasma phagocytophilum reveals three distinct lineages with different host ranges in clinically ill French cattle

Molecular epidemiology represents a powerful approach to elucidate the complex epidemiological cycles of multi-host pathogens, such as Anaplasma phagocytophilum. A. phagocytophilum is a tick-borne bacterium that affects a wide range of wild and domesticated animals. Here, we characterized its genetic diversity in populations of French cattle; we then compared the observed genotypes with those found in horses, dogs, and roe deer to determine whether genotypes of A. phagocytophilum are shared among different hosts. We sampled 120 domesticated animals (104 cattle, 13 horses, and 3 dogs) and 40 wild animals (roe deer) and used multilocus sequence analysis on nine loci (ankA, msp4, groESL, typA, pled, gyrA, recG, polA, and an intergenic region) to characterize the genotypes of A. phagocytophilum present. Phylogenic analysis revealed three genetic clusters of bacterial variants in domesticated animals. The two principal clusters included 98% of the bacterial genotypes found in cattle, which were only distantly related to those in roe deer. One cluster comprised only cattle genotypes, while the second contained genotypes from cattle, horses, and dogs. The third contained all roe deer genotypes and three cattle genotypes. Geographical factors could not explain this clustering pattern. These results suggest that roe deer do not contribute to the spread of A. phagocytophilum in cattle in France. Further studies should explore if these different clusters are associated with differing disease severity in domesticated hosts. Additionally, it remains to be seen if the three clusters of A. phagocytophilum genotypes in cattle correspond to distinct epidemiological cycles, potentially involving different reservoir hosts.

Clinical signs, seasonal occurrence and causative agents of canine babesiosis in France: Results of a multiregional study

Canine babesiosis (or piroplasmosis) is an emerging tick-borne disease caused by intraerythrocytic protozoa of the genera Babesia and Theileria. In dogs, infection by these parasites usually induces a haemolytic syndrome that can be fatal when complicated. Canine babesiosis prevalence is high in France, with Babesia canis thought to be the main etiological agent of the disease. This article presents the results of a multiregional prospective longitudinal survey on canine babesiosis conducted in France from October 2006 to December 2007. A total of 836 cases were reported by veterinarians using a multiple choice questionnaire and blood samples from 70 dogs were analyzed using PCR-RFLP to identify species responsible for canine babesiosis cases across the country. The main clinical signs reported were lethargy (98%), anorexia (98%) and hyperthermia ≥ 39 °C (80%) followed by pale mucous membranes (54%), modification of urine aspect (45%) and splenomegaly (33%). The dog population at risk was mainly represented by young dogs living in rural areas. Twenty-five out of the 70 blood samples (36%) tested by diagnostic PCR were found to contain Babesia/Theileria genus-specific DNA and all had profiles similar to that of Babesia canis genomic DNA after restriction fragment length polymorphism analyses. The survey results provide a reference for further molecular studies to assess the species and vectors involved in the transmission of the disease in France and across the Mediterranean basin.

Clinical and laboratory features of canine Anaplasma platys infection in 32 naturally infected dogs in the Mediterranean basin

Since the first description of Anaplasma platys Infection (ApI), the disease has been sporadically reported worldwide. Whereas it is considered a subclinical disease in the United States or in Australia, severe cases are reported in Europe. Thus far, little information is available regarding the clinical and laboratory findings associated with the disease and the implication of co-infections with other vector-borne pathogens (VBPs) in Southern Europe. The purpose of the study was to describe clinical and laboratory findings in PCR-confirmed naturally infected dogs in the Mediterranean Basin, and to assess the potential impact of co-infections with other VBPs. This is a retrospective analysis of medical records from 32 client-owned dogs diagnosed with ApI using PCR-based assays. Anorexia (62.5%) and weight loss (43.8%) were the major changes, whereas lethargy was less frequent (34.4%). Lymphadenomegaly (43.8%), hyperthermia (40.6%) and hemorrhage (37.5%) were frequent clinical abnormalities, whereas cutaneous signs (31.3%), musculoskeletal disorders (21.9%), splenomegaly (15.6%), dehydration and ocular inflammation (12.5%) were less common. Hematological abnormalities included thrombocytopenia (81.0%), anemia (81.0%), leukocytosis (33.3%) and leucopenia (23.8%). Seven dogs (33.3%) were severely thrombocytopenic. Among the 28 dogs with complete testing, 15 and 13 were mono- and co-infected, respectively. Co-infections included Ehrlichia canis (3 dogs), Leishmania infantum (4), Babesia vogeli (2) and Hepatozoon canis (5). One dog was infected concurrently with Anaplasma platys, Ehrlichia canis and Babesia vogeli. The 1-month mortality rate was 23.9% and only 38.1% improved. In the univariate analysis the 15 mono- and the 13 co-infected dogs did not differ regarding the relative frequencies of clinical and laboratory findings. Sequencing and phylogenetic analyses suggested the existence of 2 different groups of strains: one of them might have higher pathogenicity. In all, ApI was associated with a wide variety of non-specific clinical findings. The most common laboratory findings were thrombocytopenia and anemia. Co-infections were frequent but appeared of limited clinical impact. The absence of improvement despite appropriate treatment, high frequency of hemorrhagic disorders, and case fatalities, suggested the existence of pathogenic European strains supported by subsequent molecular analyses.

Study of ehrlichiosis in kennel dogs under treatment and prevention during seven months in Dakar (Senegal).

In Dakar kennels where morbidity and mortality attributed to diseases transmitted by ticks were high, we conducted a field study to assess the prevalence of Ehrlichia canis, Anaplasma platys and Babesia spp. infections in two kennels (n = 34 dogs) and to study the impact of tick protection. The first day of the study, the E. canis PCR were positive in 18 dogs (53%). A. platys was found in one dog and all dogs were negative for Babesia spp. After one month of doxycycline treatment, the number of PCR positive dogs decreased significantly to 2 (5.9%). During seven months, all dogs were treated monthly topically with a novel combination (Certifect(®), Merial) delivering at least 6.7 mg fipronil/kg body weight, 8.0mg amitraz/kg and 6 mg (S)-methoprene/kg. The number of PCR positive dogs remained stable all over the seven months, with 4 dogs being positive at Day 90 and 2 at Day 210. The combination of treatment and monthly prevention had a significant effect in the two kennels. All dogs remained healthy, which was not the case in previous years.

Bacterial microbiota associated with Rhipicephalus sanguineus ( s.l. ) ticks from France, Senegal and Arizona

BackgroundTicks of the group Rhipicephalus sanguineus (sensu lato) are distributed worldwide and are major pathogen vectors of both dogs and humans. Previous phylogenetic reconstructions have suggested the existence of two main lineages within this group, “Tropical” and “Temperate”. Symbiotic interactions contribute to vector development, survival, reproduction and competence. The diversity of microbial communities associated with different populations of R. sanguineus (s.l.) remains poorly characterized, however, this knowledge will aid in future studies of hosts-microbiota-pathogen interactions. To gain insight into the bacterial communities associated with R. sanguineus (s.l.) ticks, 40 specimens from France, Senegal and Arizona were analyzed by high-throughput 16S amplicon sequencing. All tick specimens were taxonomically classified using the mitochondrial 12S rDNA gene, which provides sufficient phylogenetic resolution to discriminate different lineages of R. sanguineus.ResultsRhipicephalus sanguineus (s.l.) samples from Senegal belonged to the “Tropical” lineage, samples from France belonged to the “Temperate” lineage, whereas both lineages were identified in samples from Arizona. Regardless of origin, each bacterial microbiota was dominated by three genera: Coxiella, Rickettsia and Bacillus. Rickettsia and Coxiella were the two main genera found in females whereas males had a higher proportion of Bacillus. Significant differences of relative abundances were evidenced between specimens from different geographical origins.ConclusionsThis study highlights differences in the microbiota composition within R. sanguineus (s.l.) specimens from different genotypes, genders and geographical origins. This knowledge will help in future studies of the symbiotic interactions, biology and vector competence of the R. sanguineus (s.l.) complex.

Diversité génétique d’Anaplasma Phagocytophilum, agent de l’anaplasmose granulocytaire, conséquences épidemiologiques et implications pour le contrôle en France

Anaplasma phagocytophilum is a tick-borne bacterium and the etiologic agent of granulocytic anaplasmosis, an emerging disease that affects a wide range of mammals. In this paper, we present the recent knowledge gained from studies on the genetic diversity of this pathogen in France. Multilocus sequence analysis (MLSA) was used to characterize the genetic diversity of A. phagocytophilum in populations of French cattle, horses, dogs, and roe deer. MLSA was based on nine loci (ankA, msp4, groESL, typA, pled, gyrA, recG, polA, and an intergenic region). Phylogenic analysis revealed three genetic clusters of bacterial variants in domesticated animals. The two principal clusters included 98% of the bacterial genotypes found in cattle, which were only distantly related to those in roe deer. One cluster comprised only cattle genotypes, while the second contained genotypes from cattle, horses, and dogs. The third contained all roe deer genotypes and three cattle genotypes. These results suggest that roe deer do not contribute to the spread of A. phagocytophilum in cattle in France. A Multiple-Locus Variable number tandem repeat (VNTR) Analysis typing technique was developed for A. phagocytophilum. Five 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. This study 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. The molecular techniques recently developed have 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.