Anaplasma capra in sheep and goats on Corsica Island, France: A European lineage within A. capra clade II?

Anaplasmosis is a tick-transmitted disease due to several species of the genus Anaplasma. In 2019, we demonstrated the presence of Anaplasma capra in two deer species at a zoological park in mainland France. As we suspected its presence in Corsica, we surveyed 11 geographically distant sheep or goat farms. Using molecular tools such as nested PCR targeting 16S ribosomal RNA (rRNA), citrate synthase (gltA) and heat-shock protein (groEL) genes, we detected the presence of A. capra on 5/11 farms, in 26/108 blood samples (24%), in sheep as well as in goats. Genotyping and phylogenetic analysis of A. capra revealed that isolates from Corsica island grouped closely with A. capra isolates reported in red deer and swamp deer from a zoological reserve in mainland France, as well as in roe deer from Spain, in a separate and well supported clade within A. capra clade II. This third report of the tick-borne bacterium A. capra in Europe suggests a potentially larger presence of this pathogen on the European continent, on domestic, native as well as wild ruminants, a broad host range already described in Asian countries for this species.

Human babesiosis in Alsace.

OBJECTIVES: Human babesiosis is a rare parasitic anthropozoonosis transmitted to humans by tick bites. Fifty-six cases of human babesiosis have been recorded in Europe. Two cases of babesiosis were reported in Alsace, France, in 2009. We performed a retrospective observational descriptive study to assess the epidemiology of the disease in Alsace. METHODS: Patients were included if they had a positive serology result for Babesia and/or a positive blood smear and/or a positive PCR result. The tests were performed in the microbiology laboratories of the university hospitals of Strasbourg, the civil hospitals of Colmar, and the hospital of Mulhouse between January 1, 2005 and December 31, 2015. Included patients were divided into three groups: definite case group (positive PCR or positive blood smear or seroconversion), possible case group (positive serology results without seroconversion with a compatible clinical picture and without other confirmed diagnoses), and incompatible case group (positive serology results without seroconversion, without compatible clinical picture and/or with other confirmed diagnoses). The compatible clinical picture was defined by the presence of flu-like symptoms and fever (≥38°C). RESULTS: Fifty-one patients had at least one positive result. Three cases were excluded (missing files). There were six definite cases, 12 possible cases, and 30 incompatible cases. All patients in the definite case group were immunocompetent. No deaths occurred. CONCLUSIONS: Human babesiosis is probably underdiagnosed due to its non-specific symptoms, lack of awareness about the disease, and the difficulty in making a diagnosis.

Low prevalence of zoonotic Babesia in small mammals and Ixodes ricinus in Brittany, France.

In order to evaluate the zoonotic risk due to Babesia spp., especially B. microti, we investigated their presence in 597 individuals of five small mammal species and in 2620 questing nymphs of Ixodes ricinus in rural landscapes of Western France (Brittany). Small mammals (rodents and shrews) are indeed suspected to be reservoir hosts for B. microti, and the tick I. ricinus is the vector of the three main zoonotic species in Europe, i.e. B. divergens, B. venatorum and B. microti. Only one bank vole carried B. microti (genotype "Munich") and only 13 and 2 nymphs of Ixodes ricinus ticks carried B. venatorum and B. capreoli respectively. According to these results, prevalences observed for zoonotic Babesia (0.17% for small mammals and 0.50% for ticks), indicate that exposure of humans to this infectious agent is probably low in western France.

Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique.

Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence of B. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. Gorging B. divergens infected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.

Metalloproteinases and tumor necrosis factor-alpha activities in synovial fluids of horses: correlation with articular cartilage alterations.

Early detection of osteoarthritis in horses represents a challenge for equine practitioners. Several biological markers have been implicated in the pathological processes involved in articular cartilage destruction. To further document cartilage matrix proteases production, synovial fluid was collected from 14 horses (90 joints) before they were subjected to euthanasia. Growth macroscopic examination of the joints gave information on cartilage alterations. Samples were analyzed for matrix metalloproteinase (MMPs) activities by gelatin zymography and tumor necrosis factor alpha (TNF-alpha) cytotoxicity using L929 cells. Significant increase of MMP-9 monomer and dimer were found in synovial fluids of joints with severe cartilage alterations. On the contrary, the activity of TNF-alpha was not correlated to the degree of joint damage. The levels of MMP-9 monomer and dimer in the synovial fluid could reflect cartilage alteration in arthritis in the horse.

Analysis of myosin heavy chains at the protein level in horse skeletal muscle.

Combined methodologies of enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting, traditional myofibrillar ATPase (mATPase) histochemistry and immunocytochemistry of whole biopsied samples were used to study myosin heavy chain (MHC) isoforms in the equine gluteus medius muscle. The ELISA technique allowed the quantification of the three MHC isoforms known to be present in different horse muscles: slow (MHC-I) and two fast (termed MHC-IIA and MCH-IIX). The SDS-PAGE method resolved MHCs in three bands: MHC-I, MHC-IIX and MHC-IIA from the fastest to the slowest migrating band and a quantification by densitometry for each MHC isoform was also possible. The identity of these three MHCs was confirmed by immunoblots with specific monoclonal antibodies. Five fibre types were defined immunohistochemically according to their MHC content: I, I + IIA, IIA, the hybrid IIAX and IIX. When quantitative data obtained with the four different methodologies were combined and compared, they were consistent and, when considered together, showed significant correlation. Nevertheless, the percentage of MHC-IIA histochemically derived was underestimated, while that of MHC-IIX was overestimated in comparison with the immunocytochemical determination of these MHC isoforms. The percentage of MHC-I obtained by ELISA technique was underestimated. In short, these integrated methods for the analysis of MHCs at the protein level demonstrate that equine skeletal muscle does not express the MHC-IIB, so type II fibres have been misclassified in numerous previous studies based upon the vary traditional mATPase histochemistry. They also offer new prospects for muscle fibre typing in equine experimental studies and veterinary medicine.

Heritability of percentage of fast myosin heavy chains in skeletal muscles and relationship with performance.

The purpose of this study was to determine the percentage of fast myosin heavy chains (fast MHCs = MHC 2A + 2B) in 2 propelling muscles to estimate the heritability and to identify any relationship with performance. The gluteus medius and the biceps femoris muscles were biopsied in 100 related French Anglo-Arabian horses. The percentages of slow and fast myosin heavy chains were measured using an ELISA technique. The heritability (s.e.) of the fast MHCs percentage was estimated at 13% (0.1) using a restricted maximum likelihood resolution of a mixed animal model. There were significant (P < 0.05) correlations between the performance level and the fast MHCs percentage of the gluteus medius and biceps femoris muscles: 0.47 and 0.34 respectively. An analysis of variance revealed a significant (P < 0.05) effect of performance level in gallop racing and show jumping on the fast MHCs percentage of the gluteus medius muscle. The good performers in gallop racing (75.5 > 69.6% fast MHCs) and show jumping (74.1 > 67.8% fast MHCs) had a higher percentage of fast MHCs in the gluteus medius than poor performers (P < 0.05). This muscular analysis could be one of the interesting physiological traits to measure for early selection of gallop racing and show jumping horses.

Standardisation of muscular biopsy of gluteus medius in French trotters.

Morphometric measurements were taken from 41 French trotters of various ages and both sexes. Biopsy location was determined for the dorsal compartment as being one-third of the distance from the tuber sacrale to the tuber coxae and for the ventral compartment as being one-third of the distance from the tuber coxae to the caudal Cd1-Cd2 intervertebral joint. Ten horses were biopsied at these 2 sites at a sampling depth equal to half the total depth of the compartment as measured by ultrasonography. The percentage of slow and fast myosin heavy chain fibres was measured by using an immunoenzymatic method. The depth of the dorsal and ventral compartments of the gluteus medius was significantly greater in males than in females. The depth of the ventral compartment was greater in the case of a straight hip than of a wide hip and was greater in young horses than in older horses. The ventral and the dorsal compartments were composed in the mid-portion of 81.3 and 75.6% of fast myosin heavy chains, respectively. It was concluded that the locations and sampling depths in the gluteus medius could be standardised in French trotters by taking into account anatomical references, sex, age and width of the hip.

Enzyme-linked immunosorbent assay for myosin heavy chains in the horse.

The content in slow and fast myosin heavy chains (MHC 1 and MHC 2) of 5 equine muscles was determined using an enzyme-linked immunosorbent assay. The results obtained with this immunoenzymatic method were compared with complementary techniques: electrophoresis and immunohistochemistry. Slices of masseter, diaphragm, tensor faciae latae, semitendinosus and cutaneus trunci were obtained from a 12-year-old saddle horse after slaughter. Muscular proteins were specifically extracted to be analysed by ELISA. The technique used 2 complimentary monoclonal antibodies (MAb). MAb 1 was prepared from a human atrium specimen that reacted specifically against MHC 1. Mab 2 was prepared from myosin of rabbit psoas muscle and reacted against MHC 2. The masseter muscle contained solely MHC 1 (100%) and this was confirmed by electrophoresis and immunohistochemistry. By contrast, the cutaneus trunci was very poor in MHC 1 (1.3%) and was entirely composed of MHC 2 (98.7%) which was confirmed by the other techniques. The diaphragm, tensor fasciae latae and semitendinosus contained 89, 40 and 2% of MHC 1, respectively. It was concluded that this ELISA method made it possible to measure a wide range of MHC contents in equine muscles with a good reproducibility. The results were consistent with those of the other fibre typing techniques. Moreover, this immunoenzymatic method is less time consuming than histological techniques and therefore offers new perspectives for muscle fibre typing in the horse.