Molecular Characterization and Phylogenetic Analysis of Pathogenic Theileria spp. Isolated from Cattle and Sheep based on Cytochrome b Gene in Iran.

The present study investigated the phylogenetic relationship based on cytochrome b gene sequences among pathogenic Theileria species (spp.) in Iran, including Theileria annulata and Theileria lestoquardi, along with other data available in GenBank. A total of 136 (cattle) and 80 (sheep) blood samples suspected of piroplasm infection were obtained from six different provinces of Iran. Both microscopic and molecular methods using species-specific primers were used for screening T. annulata and T. lestoquardi positive samples. Finally, the partial cytochrome b gene of 30 T. annulata and 5 T .lestoquardi were amplified, sequenced, and deposited in GenBank. The results indicated that there were 12 different genotypes among T. annulata isolates, while only one genotype was observed among T. lestoquardi isolates. T. lestoquardi infection in cattle was detected in one sample, and no T. annulata and T. lestoquardi coinfection were detected in sheep and cattle. In the phylogenetic tree, different Theileria spp. were placed in separate clades, and the reliability of depicted tree and monophyly of T. annulata and T. lestoquardi ingroups were supported by the bootstrap value of 94% which significantly indicated that these two species evolved from a common ancestor. The tree also showed that these two pathogenic spp. shared a more recent common ancestor, compared to another species of Theileria parasites. To the best of our knowledge, this study is the first phylogenic analysis of pathogenic Theileria spp. in Iran based on the cytochrome b gene sequences. In addition, the first T. lestoquardi cytochrome b gene was sequenced and deposited in GenBank.

Determination of CD Markers Profile of the Cell Line Infected by S15 Vaccine Strain of Theileria annulata Schizont Using RT-PCR Analysis.

The aim of this study was to identify the cell surface cluster of differentiation (CD) markers of the cell lines infected by Theileria annulata schizont. The CD molecules are very useful for the characterization of cells and different subpopulations of leukocytes. They are usually recognized by specific antibodies using flow cytometry and immunohistochemistry. In the current study, we applied reverse transcriptase-polymerase chain reaction (RT-PCR) to define the profile of cell surface markers in a cell line infected by an attenuated S15 vaccine strain of T. annulata schizont and a new laboratory-established cell line infected by a non-attenuated form. In order to determine the specific markers that can be used for excluding the non-attenuated cell lines, the characterization of the surface proteins profile of the S15 vaccine cell line is important. The RT-PCR was carried out by specifically designed primers using a panel of seven bovine CD markers, as well as beta-actin as an internal control house-keeping gene. We showed that both of the examined cell lines had a consistent expression of CD4, CD5, CD11a, CD14, CD43, and CD45 markers. However, the specific finding in this study was the expression of B-cell markers CD79a and CD5 by the newly-transformed cell line. On the other hand, CD5 as a marker for B-cell subset was expressed by S15 vaccine strain. In conclusion, we consider CD79a surface protein as a new marker for the cell lines infected by non-attenuated T. annulata schizont, while the cell lines infected by the vaccine strain do not express this marker. In addition, the identification of CD marker expression based on the RT-PCR assay might be a suitable and appropriate alternative technique for flow cytometry.

Laboratory Study on Biological Control of Ticks (Acari: Ixodidae) by Entomopathogenic Indigenous Fungi (Beauveria bassiana).

BACKGROUND: Chemical control method using different acaricides as spray, dipping solution or pour-on is routinely used for controlling ticks. Biological control agents are favorable due to their safety for animals and environment. Entomopathogenic fungi such as Beauveria bassiana are well known for controlling ticks. In this study, two Iranian indigenous strains of B. bassiana (B. bassiana 5197 and B. bassiana Evin) were selected and grown on specific media. The pathogenic effects of these strains were evaluated on adult stages of two Iranian Ixodidae members (H. anatolicum anatolicum Koch 1844, and H. marginatum Koch 1844) by dipping method. METHODS: Two Iranian strains of Beauveria bassiana (Beauveria bassiana 5197 and Beauveria bassiana Evin) were selected and were grown successfully on specific media. The pathogenic effects of these strains were evaluated on adult stages of Iranian Ixodidae members such as, Hyalomma anatolicum anatolicum and H. marginatum by dipping method (these ticks were grown up at laboratory conditions during 2002 up to 2003 and still it is continued) . RESULTS: There was no effect of strain 5197 on mortality or fecundity rates for ticks. There was acute phase sign of paralysis in test group after dipping ticks in suspension made from Evin strain of B. bassiana. In addition, the test groups were totally died after four months, but the control groups survived for six months. CONCLUSION: High concentration of fungal spores is needed for inducing fungal infection. Additional study using different strains and fungi on Iranian ticks is proposed.

Comparative evaluation of antibody positive titer by ELISA and IFA in Theileria annulata vaccinated cattle in Iran.

An enzyme linked immunosorbent assay (ELISA) was used to evaluate antibody positive titer in vaccinated and non-vaccinated cattle using schizont infected myeloid cells as an antigen. The result was compared with indirect fluorescent antibody level in the same animals. For this study 116 milking cows, 95 vaccinated and 21 non-vaccinated, were bleeded in order to prepare sera. They were tested with both ELISA and IFA tests. 94 sera had positive antibody titer and 22 sera were negative through ELISA test but, with IFA test, only 89 sera showed positive antibody titer and 27 were negative. Thereby, it was concluded that the sensitivity and specificity of ELISA test in comparison with IFA test was 95.5% and 66.6% respectively. This study generally indicated that ELISA could be an effective test for sero-epidemiological investigations of bovine tropical theileriosis, and it is considered to be valid as an additional test to distinguish the vaccinated from the non vaccinated cattle in order to schedule vaccination programs.