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      A comparison of two different techniques for the detection of blood parasite, Theileria annulata, in cattle from two districts in Khyber Pukhtoon Khwa Province (Pakistan) Translated title: Comparaison de deux techniques de détection de Theileria annulata chez des bovins de deux districts de la province de Khyber Pukhtoon Khwa (Pakistan)

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          The present study was carried out to determine the prevalence of Theileria annulata in large ruminants from two districts, Peshawar and Kohat, in Khyber Pukhtoon Khwa (Pakistan). Blood samples were collected from 95 cattle. Data on the characteristics of animals and herds were collected through questionnaires. No significant risk factors were found associated with the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa stained slides, were compared and it was found that PCR amplification is a more sensitive tool (33.7% parasite detection), as compared to smear scanning (5.2% parasite detection) for the detection of Theileria annulata. 32 out of 95 animals, from both districts, produced the 721-bp fragment specific for Theileria annulata.

          Translated abstract

          Une étude a été menée afin de déterminer la prévalence de Theileria annulata chez 95 bovidés de deux districts (Peshawar et Kohat) de la province de Khyber Pukhtoon Khwa au Pakistan. L’âge des bovins, la présence de tiques chez ceux-ci, ainsi que la présence de tiques chez les chiens du troupeau ne sont pas des facteurs de risque impliqués dans la diffusion de la theilériose dans la zone étudiée. La comparaison de deux techniques de détection du parasite (PCR et frottis sanguin coloré au Giemsa) a montré que la PCR était plus sensible (33,7 %) que le frottis (5,2 %).

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          Most cited references 8

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          Detection of Theileria annulata in blood samples of carrier cattle by PCR.

          We report the detection of Theileria annulata, the causative agent of tropical theileriosis, by PCR in blood samples obtained from carrier cattle. The assay employs primers specific for the gene encoding the 30-kDa major merozoite surface antigen of T. annulata. A 721-bp fragment was amplified from blood samples taken monthly from calves experimentally infected with one of four different stocks of T. annulata originating in either Mauritania, Portugal, Spain, or Turkey. At the end of the experiment, five animals carried the infection for 12 months and two animals remained infected for 15 months. DNAs from six other Theileria species, T. parva, T. mutans, T. sergenti, T. buffeli, T. velifera, and T. taurotragi, were not amplified. Moreover, DNAs from four other hemoparasites (Anaplasma centrale, Anaplasma marginale, Babesia bovis, and Babesia bigemina) were also not amplified. As a control, primers derived from the small subunit rRNA gene of Theileria spp. amplified a 1.1-kb DNA fragment from all Theileria species examined but not from the other four hemoparasites. As few as two to three parasites per microliter of infected blood in a 50-microliters sample volume were detected by Southern or microplate hybridization with a T. annulata-specific cDNA probe. In addition, 92 field samples obtained from cattle in Spain were tested; 22% were positive in blood smears, 40% were positive by immunofluorescent antibody test, and 75% were positive for T. annulata by PCR. The method provides a useful diagnostic tool for detecting T. annulata carrier cattle.
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            Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa.

            A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; approximately 1600bp) was amplified and sequenced from 33 of these 488 samples. Sequences were compared with published sequences from the public sequence databases. Twelve distinct T. equi and six B. caballi 18S rRNA sequences were identified. Alignments demonstrated extensive sequence variation in the V4 hypervariable region of the 18S rRNA gene within T. equi. Sequence variation was also found in B. caballi 18S rRNA genes, although there was less variation than observed for T. equi. Phylogenetic analysis based on 18S rRNA gene sequences revealed three T. equi clades and two B. caballi clades in South Africa. The extent of sequence heterogeneity detected within T. equi and B. caballi 18S rRNA genes was unexpected since concerted evolution is thought to maintain homogeneity within repeated gene families, including rRNA genes, in eukaryotes. The findings reported here show that careful examination of variants of the 18S rRNA gene of T. equi and B. caballi is required prior to the development of molecular diagnostic tests to detect these parasites in horses. Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.
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              Molecular detection of Theileria and Babesia infections in cattle.

              This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.

                Author and article information

                Parasite : journal de la Société Française de Parasitologie
                EDP Sciences
                February 2012
                15 February 2012
                : 19
                : 1 ( publisher-idID: parasite/2012/01 )
                : 91-95
                [1 ] Department of Zoology, Kohat University of Science and Technology Kohat Pakistan
                [2 ] Institute of Pure and Applied Biology, Zoology Division, Bahauddin Zakariya University Multan Pakistan
                [3 ] Institute of Biotechnology, Bahauddin Zakariya University Multan Pakistan
                Author notes
                [* ]Correspondence: Furhan Iqbal, Department of Zoology, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan 60800, Pakistan. Tel.: 92 61 9210053 – Fax: 92 61 9210098. E-mail: furhan.iqbal@ 123456bzu.edu.pk
                parasite2012191p91 10.1051/parasite/2012191091
                © PRINCEPS Editions, Paris, 2012

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 0, Tables: 2, Equations: 0, References: 21, Pages: 5
                Research Note


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