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

BACKGROUND: Ticks 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. RESULTS: Rhipicephalus 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. CONCLUSIONS: This 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.

First clinical case report of Cytauxzoon sp. infection in a domestic cat in France.

BACKGROUND: Feline cytauxzoonosis is an emerging infection caused by tick-transmitted apicomplexan parasites of the genus Cytauxzoon. The association of clinical disease with Cytauxzoon infection appears to be limited to C. felis infections in the Americas. Sporadic infections of wild and domestic felids with Cytauxzoon sp. were recently described in European countries but clinical reports of the infection are rare and incomplete. This case report brings new interesting information on cytauxzoonosis expression in Europe. CASE PRESENTATION: A 9-years-old castrated European shorthair cat living in rural area of north-eastern France (Saint Sauveur, Bourgogne-Franche-Comté region), without any travel history was presented for consultation due to hyperthermia, anorexia, depression and prolonged fever that didn't respond to antibiotic therapy. The cat had outdoor access with a history of vagrancy and was adequately vaccinated (core vaccines and FeLV vaccine). During biological investigations, intraerythrocytic inclusions were observed on blood smear and were further investigated by PCR analysis and sequencing. Molecular analyses confirmed Cytauxzoon sp. infection. The cat was treated with a subcutaneous injection of imidocarb dipropionate (3.5 mg/kg). One week after treatment, the cat improved clinically, although parasitic inclusions within erythrocytes persisted, and only a mild lymphocytosis was found. Two weeks after treatment, the cat appeared in excellent health, appetite was normal and parasitemia was negative. However, one month after treatment the cat relapsed with hyperthermia, anorexia, and depression. Blood smears and PCR were once again positive. Subsequently, the cat received an additional dose of imidocarb dipropionate (3.5 mg/kg SC) and recovered rapidly without other clinical signs. Two weeks after the second imidocarb injection, the cat was hit by a car and died. CONCLUSION: This case provides the first clinical description of infection by Cytauxzoon sp. in a domestic cat in France. These findings support the fact that cytauxzoonosis should be considered in the differential diagnosis of acute febrile illness which does not respond to antibiotic in cats with outdoor access especially in areas where populations of wild felids are present.

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.

Update on epidemiology of canine babesiosis in Southern France.

BACKGROUND: Canine babesiosis is an emerging or re-emerging disease caused by Babesia and Theileria protozoans, also called piroplasms, transmitted by Ixodid ticks. In Europe, four etiological agents have been identified to date, namely Babesia canis, B. vogeli, B. gibsoni and Theileria annae. France has a high prevalence of canine babesiosis and two tick species, Dermacentor reticulatus and Rhipicephalus sanguineus, are supposed to transmit B. canis and B. vogeli respectively. In southern France, where dog infections with B. vogeli were recently confirmed, no comprehensive study was performed to date on piroplasm species infecting dogs. Thus, a large scale survey involving veterinary clinics, kennels and tick collection from the environment was conducted from 2010 to 2012 in this area. RESULTS: From 2010 to 2012, 140 dog blood samples and 667 ticks were collected. All blood and a subset of ticks were screened for the presence of piroplasms by PCR amplification of 18S rDNA. B. vogeli, B. canis and T. annae were detected in 13.6, 12.9 and 0.7 % dogs respectively. B. vogeli and B. canis were detected in 10.5 % and in 1.6 % R. sanguineus ticks including 1.3 % co-infections. B. canis was the only species detected in D. reticulatus ticks (9.7 %). B. canis infections were only recorded in the southwest of France whereas B. vogeli was mainly found in the southeast. Finally, a significantly higher prevalence of B. vogeli infection was found in Gard compared to Corsica and Drôme regions, both in dogs (p < 0.002) and R. sanguineus ticks (p < 0.02) although R. sanguineus was the main ticks species removed from dogs in those three areas. CONCLUSIONS: The survey confirmed the circulation of both B. canis and B. vogeli in dogs in southern France with differences in distribution probably linked to the distribution of their respective vectors. It also showed differences in prevalence of B. vogeli infection in areas similar in terms of risk of dogs infestation with R. sanguineus. Further studies focusing on genetic and microbiota of R. sanguineus ticks should be conducted to explore other biological interactions that may explain the differences observed.

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

BACKGROUND: Canine 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. METHODS: The 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. RESULTS: A 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. CONCLUSIONS: The 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.

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.

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

BACKGROUND: Anaplasma 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. METHODS: 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: 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. RESULTS: From 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. CONCLUSIONS: Our 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.

Comparative microbiological features of Bartonella henselae infection in a dog with fever of unknown origin and granulomatous lymphadenitis.

We report the first documented case of Bartonella henselae infection in a dog from France and the first isolation of B. henselae from a dog with fever of unknown origin. This observation contributes to the "One Health" concept focusing on zoonotic pathogens emerging from companion animals. A 1-year-old female German shepherd dog was referred for evaluation of fever of unknown origin of 1 month duration. Diagnostic investigations confirmed diffuse pyogranulomatous lymphadenitis. The dog became afebrile, and lymph node size normalized in response to a 6-week course of doxycycline. Retrospectively, Bartonella DNA was amplified from an EDTA-anticoagulated blood sample obtained before antimicrobial therapy, with the gtlA fragment sharing 99 % identity with the 350-bp gtlA fragment of the B. henselae Houston-1 strain. The same strain was isolated in the blood of three healthy cats from the household. Two months after discontinuation of doxycycline, the dog experienced a febrile relapse. Bartonella DNA was again amplified from blood prior to and immediately after administration of a 6-week course azithromycin therapy. However, without administration of additional medications, PCR was negative 9 months after azithromycin therapy and the dog remains clinically healthy 12 months following the second course of antibiotics. The medical management of this case raises several clinically relevant comparative infectious disease issues, including the extent to which Bartonella spp. contribute to fever of unknown origin and pyogranulomatous inflammatory diseases in dogs and humans, and the potential of doxycycline and azithromycin treatment failures. The possibility that dogs could constitute an underestimated reservoir for B. henselae transmission to people is also discussed.

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.

Causes, diagnostic signs, and the utility of investigations of fever in dogs: 50 cases.

This study aimed to determine the distribution of diseases causing fever in dogs in France. Dogs with fever were reviewed and 50 dogs were retrospectively assigned to disease groups. Fever profile and intensity, the time taken to reach a diagnosis, and inflammatory status were compared among groups. Almost half the dogs (48%) were diagnosed with non-infectious inflammatory diseases. No final diagnosis was reached in 14 dogs, 13 of which belonged to owners who did not wish to pursue the investigations. No association was found between disease group and the intensity of fever, fever profile, or serum C-reactive protein concentration. Cytological examinations were most frequently found to be the most important determinant for diagnosis (55.7%). This study confirms the predominance of non-infectious inflammatory diseases as causes of fever. Neither clinical nor biological factors were found to be predictive of disease group.

Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendritic cells (cMo-DC).

Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs.

CD86 molecule is a specific marker for canine monocyte-derived dendritic cells.

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.

Evaluation of elutriation and magnetic microbead purification of canine monocytes.

An elutriation technique was developed to obtain large quantities of pure canine monocytes. Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient. Then, the PBMC were separated by an elutriation procedure. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64% +/- 7.9 when labelled with anti-CD14 antibody. This purity increased to 83% +/- 2.2 after separation by magnetic anti-CD14 microbeads. The cell viability was more than 95% and apoptotic cells were less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR). A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9% +/- 10.7 of CD5+, 7.9% +/- 3.5 of CD21+ and 1.78% +/- 2.53 of CD14+. Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.

Immunopathology of vesicular cutaneous lupus erythematosus in the rough collie and Shetland sheepdog: a canine homologue of subacute cutaneous lupus erythematosus in humans.

Clinical and histological features of an erosive disease in the rough collie and Shetland sheepdog are most consistent with a vesicular variant of cutaneous lupus erythematosus (VCLE). This paper reports the immunopathological findings of canine VCLE using samples from 17 affected dogs. Lesional skin sections were stained with monoclonal antibodies specific for CD3 (11 dogs) or a panel of monoclonal antibodies specific for leukocyte antigens (two dogs). Apoptotic cells were detected using the TUNEL method in 12 cases. Direct (14 dogs) and indirect immunofluorescence tests (five dogs) were also performed. Circulating antibodies to extractable nuclear antigens (ENA) were surveyed in 11 dogs by immunoblotting and ELISA. The predominant cells at the dermal-epidermal interface were identified as CD3(+) T lymphocytes expressing CD4 or CD8 and CD1(+) dendritic antigen presenting cells. In 7/12 dogs (58%), apoptosis of basal keratinocyte nuclei was present. Up-regulation of MHCII and ICAM-1 was observed on basal keratinocytes from the two dogs examined. Direct immunofluorescence revealed deposition of immunoglobulins bound to the cytoplasm of keratinocytes (6/14 dogs; 43%), to the dermal-epidermal junction (7/14 dogs; 50%), or to superficial dermal venules (13/14 dogs; 93%). Circulating IgG auto-antibodies targeting one or more ENA were detected in nine (82%) and eight (73%) of 11 dogs by immunoblotting and ELISA, respectively. These auto-antibodies recognized Ro/SSA and/or La/SSB in four (36%) and six (55%) of 11 dogs respectively by these two methods. Altogether, results of these studies provide evidence supporting the hypothesis that canine VCLE is an immunological homologue of subacute cutaneous lupus erythematosus in humans.

Prevalence of Bartonella clarridgeiae and Bartonella henselae in domestic cats from France and detection of the organisms in erythrocytes by immunofluorescence.

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.