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Detection and identification of equine Theileria and Babesia species by reverse line blotting: epidemiological survey and phylogenetic analysis Veterinary assistant. Vet Parasitol

Department of Animal Health, Instituto Vasco de Investigación y Desarrollo Agrario (NEIKER) Berreaga 1, Derio, 48160 Bizkaia, Spain.
Veterinary Parasitology (Impact Factor: 2.55). 09/2004; 123(1-2):41-54. DOI: 10.1016/j.vetpar.2004.04.010
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ABSTRACT Specific oligonucleotide probes were designed to develop a new and highly sensitive reverse line blot assay to detect and identify simultaneously different Theileria and Babesia species in horses. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against different generic and species-specific probes. The survey was conducted over 243 samples of equine blood divided into three different groups: group 1, 24 horses presented as possible clinical piroplasmosis; group 2, 181 clinically healthy free-ranging horses exposed to ticks; group 3, 38 riding horses with unrelated pathologies and low or no contact with ticks. The study demonstrated a high piroplasm prevalence in the first two groups of animals. Two Theileria genotypes sharing 96.8% similarity between their 18S rRNA gene sequences and two Babesia genotypes sharing 97.4% similarity, were identified. The biologic meaning of such genotypes is discussed in terms of their phylogenetic relationships and potential pathogenicity.

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Available from: Josune García-Sanmartín, Oct 27, 2014
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    • "In Mediterranean region in which piroplasmosis is an endemic disease, symptomatic illness can occur year round with specific or vague signs (Friedhoff et al., 1990, Zobba et al 2008). Small piriform, circular merozoite in RBCs, which is indicative for T. equi was found via microscopic examination, direct diagnosis includes demonstration of intraerythrocytic merozites in Giemsa stained blood by molecular detection (Nagore et al., 2004; Alhassan et al., 2007). "
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    • ") and similar (99%) to the sequence detected in a horse in Spain (AY534883) (Nagore et al., 2004; Beck et al., 2009; Bhoora et al., 2009). "
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    ABSTRACT: Donkeys, owing to the frequent outdoor activity, are exposed to a high risk of infection with tick-borne pathogens. This work aimed to detect exposure to Theileria equi, Babesia caballi, Anaplasma phagocytophilum and Borrelia burgdorferi s.l. of donkeys reared in Central Italy. For this purpose 122 adult donkeys were selected within 11 herds and submitted to blood collection. IgG antibodies to T. equi, B. caballi, A. phagocytophilum and B. burgdorferi s.l. were detected by IFAT. Conventional PCRs targeting the genes MSP2 and the flagellin were used for the detection of A. phagocytophilum and B. burgdorferi s.l. respectively and a Real Time PCR Sybr Green was used to detect Babesia/Theileria spp…. The species identity was determined by amplicons sequencing. Forty eight (39.3%) and 58 (47.5%) animals tested positive for T. equi and B. caballi antibodies, respectively; nine animals (7.4%) were found positive for antibodies against A. phagocytophilum whereas negative results were obtained for B. burgdorferi s.l. Twenty-six (21.3%) animals showed antibodies for both T. equi and B. caballi. Twenty-three (18.8%) donkeys were positive to Babesia/Theileria spp. PCR assay. Out of 21 sequenced amplicons, 20 were identified as T. equi, belonging to three main groups designated A, B and D and one as B. caballi group A. Neither A. phagocytophilum nor B. burgdorferi PCR results were positive. The study showed a high exposure of donkeys to tick-borne pathogens and provides information on the genetic identity of the T. equi strains circulating in Central Italy.
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    • "HRM analyses are performed in the one run, and the results are available for analysis at the end of the run. Primers for the test were designed from the 18S rRNA, which has been shown to be reliable for the differentiation between T. equi and B. caballi (Nagore et al. 2004; Bhoora et al. 2009). Using this technique, we were able to assess the 20 samples after amplification . "
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    ABSTRACT: The application of high-resolution melting (HRM) analysis in the differentiation between Theileria equi and Babesia caballi was evaluated using control samples from the United States Department of Agriculture and field samples collected from horses in Sudan and China. A region of the 18S rRNA gene, with four known nucleotide differences between the two parasites, was selected for primer design. HRM analysis successfully allowed the detection and differentiation of T. equi and B. caballi without the necessity of performing time-consuming and expensive post-PCR procedures such as sequencing or restriction digestion. Our results suggest that HRM could be an ideal method for rapid genotyping, which is required to determine a drug of choice or to administer an appropriate vaccine during an outbreak.
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