Hemoprotozoa of domestic animals in France: Prevalence and molecular characterization

Very limited information is available on epizootiology of haematozoan infections in French domestic animals. In an attempt to address this issue, prevalence of piroplasmida was studied in carnivores and ruminants, whereas prevalence of Hepatozoon spp. was only investigated in carnivores. In total, 383 animals were included in the survey (namely 116 cats, 108 dogs, 91 sheep and 68 cows). Parasite diagnosis was carried out using molecular methods such as PCR and sequencing of the 18S rRNA gene. In addition, ruminant samples were analyzed with the reverse line blotting technique (RLB). Results of RLB and PCR plus sequencing were in total agreement. In carnivores, haematozoan prevalence was close to 1%. Two cats were infected by H. canis (1.7% prevalence) and one of them was co-infected by Cytauxzoon sp. (0.8%). This represents the first finding of both pathogens in French cats. One dog was infected by H. canis (0.9%) and another by Babesia canis vogeli (0.9%). In ruminants, haematozoan prevalence (piroplasmida) was significantly higher than in carnivores (4.8% in sheep and 8.8% in cow). Theileria ovis was found in 1 sheep, Theileria sp. in 2 sheep, Theileria buffeli in 5 cows and B. major in 1 cow. Evidence presented in this contribution indicates that haematic protozoa are not widely distributed in domestic mammal populations of France

Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids.

This paper reports two new quantitative PCR (qPCR) assays, developed in an attempt to improve the detection of bovine piroplasmids. The first of these techniques is a duplex TaqMan assay for the simultaneous diagnosis of Babesia bovis and B. bigemina. This technique is ideal for use in South America where bovids harbour no theilerids. The second technique, which is suitable for the diagnosis of both babesiosis and theileriosis worldwide, involves fluorescence resonance energy transfer (FRET) probes. In FRET assays, Babesia bovis, B. divergens, Babesia sp. (B. major or B. bigemina), Theileria annae and Theileria sp. were all identifiable based on the melting temperatures of their amplified fragments. Both techniques provided linear calibration curves over the 0.1fg/microl to 0.01ng/microl DNA range. The assays showed good sensitivity and specificity. To assess their performance, both procedures were compared in two separate studies: the first was intended to monitor the experimental infection of calves with B. bovis and the second was a survey where 200 bovid/equine DNA samples from different countries were screened for piroplasmids. Comparative studies showed that duplex TaqMan qPCR was more sensitive than FRET qPCR in the detection of babesids

Evaluating amplified rDNA restriction analysis assay for identification of bacterial communities

Amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism were originally used for strain typing and for screening clone libraries to identify phylogenetic clusters within a microbial community. Here we used ARDRA as a model to examine the capacity of restriction-based techniques for clone identification, and the possibility of deriving phylogenetic information from ARDRA-based dendrograms. ARDRA was performed in silico on 48,759 sequences from the Ribosomal Database Project, and it was found that the fragmentation profiles were not necessarily unique for each sequence in the database, resulting in different species sharing fragmentation profiles. Although ARDRA-based clusters separated clones into different genera, these phylogenetic clusters did not overlap with trees constructed according to sequence alignment, calling into question the intra-genus ARDRA-based phylogeny. It is thus suggested that the prediction power of ARDRA clusters in identifying clone phylogeny be regarded with caution

Emergence of new types of Theileria orientalis in Australian cattle and possible cause of theileriosis outbreaks

Theileria parasites cause a benign infection of cattle in parts of Australia where they are endemic, but have, in recent years, been suspected of being responsible for a number of outbreaks of disease in cattle near the coast of New South Wales. The objective of this study was to identify and characterize the species of Theileria in cattle on six farms in New South Wales where disease outbreaks have occurred, and compare with Theileria from three disease-free farms in Queensland that is endemic for Theileria. Special reference was made to sub-typing of T. orientalis by type-specific PCR and sequencing of the small subunit (SSU) rRNA gene, and sequence analysis of the gene encoding a polymorphic merozoite/piroplasm surface protein (MPSP) that may be under immune selection. Nucleotide sequencing of SSU rRNA and MPSP genes revealed the presence of four Theileria genotypes: T. orientalis (buffeli), T. orientalis (ikeda), T. orientalis (chitose) and T. orientalis type 4 (MPSP) or type C (SSU rRNA). The majority of animals showed mixed infections while a few showed single infection. When MPSP nucleotide sequences were translated into amino acids, base transition did not change amino acid composition of the protein product, suggesting possible silent polymorphism. The occurrence of ikeda and type 4 (type C) previously not reported to occur and silent mutation is thought to have enhanced parasite evasion of the host immune response causing the outbreak

A survey of canine tick-borne diseases in India

Background: There are few published reports on canine Babesia, Ehrlichia, Anaplasma, Hepatozoon and haemotropic Mycoplasma infections in India and most describe clinical disease in individual dogs, diagnosed by morphological observation of the microorganisms in stained blood smears. This study investigated the occurrence and distribution of canine tick-borne disease (TBD) pathogens using a combination of conventional and molecular diagnostic techniques in four cities in India. Results: On microscopy examination, only Hepatozoon gamonts were observed in twelve out of 525 (2.3%; 95% CI: 1.2, 4) blood smears. Using polymerase chain reaction (PCR), a total of 261 from 525 dogs (49.7%; 95% CI: 45.4, 54.1) in this study were infected with one or more canine tick-borne pathogen. Hepatozoon canis (30%; 95% CI: 26.0, 34.0) was the most common TBD pathogen found infecting dogs in India followed by Ehrlichia canis (20.6%; 95% CI: 17.2, 24.3), Mycoplasma haemocanis (12.2%; 95% CI: 9.5, 15.3), Anaplasma platys (6.5%; 95% CI: 4.5, 8.9), Babesia vogeli (5.5%, 95% CI: 3.7, 7.8) and Babesia gibsoni (0.2%, 95% CI: 0.01, 1.06). Concurrent infection with more than one TBD pathogen occurred in 39% of cases. Potential tick vectors, Rhipicephalus (most commonly) and/or Haemaphysalis ticks were found on 278 (53%) of dogs examined. Conclusions: At least 6 species of canine tick-borne pathogens are present in India. Hepatozoon canis was the most common pathogen and ticks belonging to the genus Rhipicephalus were encountered most frequently. Polymerase chain reaction was more sensitive in detecting circulating pathogens compared with peripheral blood smear examination. As co-infections with canine TBD pathogens were common, Indian veterinary practitioners should be cognisant that the discovery of one such pathogen raises the potential for multiple infections which may warrant different clinical management strategies