Background and Objectives: Neospora caninum is one of main etiologic factors of abortion in in cattle. The mass production of protozoa in laboratory conditions is performed based on growing in different cell lines. Since using suspension cell lines are very cost effective for mass production of biological products, this study aimed to evaluate the suspension cell culturing for production of this protozoan.Materials and Methods: This study was experimentally performed bases on growth of N. caninum tachyzoites on Ka6 cell line (a cell line obtained from cattle infected with Theileria LESTOQUARDI, Razi Institute, Shiraz, Iran. Next, based on MTT assay, ability of this cell culture for production of N. caninum was compared with Vero cell as the best current cell line for this purpose.Results: The results showed that N. caninum tachyzoites could enter into K6a cell lines and after replication released from the cells successfully. Replication of the tachyzoites was significant in both Vero and K6a cell lines in compare to the control.Conclusion: To our knowledge, this is the first and successful report of suspension culture of N. caninum tachyzoites. Although, the rate of N. caninum proliferation on Ka6 cell line did not show any significant difference in compare to Vero cell line, since Ka6 cell line is a transformed lymphocytic cell and it is possible to grow massively this cell line as suspension bioreactors, it is preferred to Vero cell line.

Introduction and Objectives: Malignant sheep and goats theileriosis is one of the most important protozoan diseases of sheeps in Iran. Theileria LESTOQUARDI is the only intracellular eukaryotic pathogen that is able to transform its host cells reversibly. Therefore, design of a cell line of ovine lymphocyte that is transformed by the T. LESTOQUARDI is very valuable.Materials and Methods: The breeding ticks was infected by feeding on the sheeps that were carrier of Theileria LESTOQUARDI sporozoite in order to obtain homogeneous infected ticks. Then, ovine lymphocytes were infected with Theileria LESTOQUARDI sporozoite stabilate to produce lymphocytic cell line containing T. LESTOQUARDI.Result: This research resulted in the development of a new ovine lymphocytic cell line called as Booket. Molecular study confirmed that the cell lines were transformed by T. LESTOQUARDI. The gene sequence of this isolate has been submitted to GenBank with accession no. GU233776.Conclusion: In this study the second lymphocytic cell line containing T. LESTOQUARDI was established in Iran.

The aim of this study was to detect and differentiate Theileria annulata and T. LESTOQUARDI (hirci) by PCR. Members of the genus Theileria are tick-borne hemoprotozoan parasites; those cause fatal and enervating diseases of cattle and sheep in Iran. In order to develop a specific method for detecting and identification of Theileria species, specific primers from the surface protein (SP) sequence were designed that allowed the specific diagnosis of T. annulata and T. LESTOQUARDI infection simultaneously. Theileria surface protein genes have consensus and variable sequences regions that allowed us to design the common primers for both species, which amplified two different PCR products. The results of this study demonstrated that a novel, simple, and high specific PCR for detecting and identifying T. annulata and T. LESTOQUARDI infection.

BACKGROUND: Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions which causes high economic loss in the livestock industry. OBJECTIVES: The aim of this study was the differential detection of Theileria species in sheep using PCR method. METHODS: Two hundred blood samples of sheep were investigated in order to differentially diagnose Theileria species. DNA was extracted from blood samples and DNA samples were amplified using specific primers designed for 18S rRNA, TamS1 and TaSp genes. RESULTS: In this study, from 200 examined samples, 42 samples (21%) were infected by Theileria spp. and none of them were infected by Babesia spp. Moreover, from these 42 positive samples, 24 samples (57. 1%) were only infected by T. ovis. 12 samples (28. 5%) were only infected by T. LESTOQUARDI, 2 samples (4. 7%) were only infected by T. annulata and 4 samples (9. 5%) were simultaneously infected by T. LESTOQUARDI and T. ovis. The results of nucleotide sequencing showed that PCR product of 18S rRNA from T. LESTOQUARDI has 99 and 95% similarity with T. annulata and T. ovis respectively. T. LESTOQUARDI and T. annulata showed 86% similarity. Also TaSp gene of T. ovis in comparison with T. annulata and T. LESTOQUARDI showed 96 and 86% similarity, respectively. CONCLUSIONS: In the present study could be shown that the two genes (TamS1 and TaSp) from examined three genes could be used for Theileria species specific diagnosis by PCR.

Theileriosis is caused by an intracellular protozoan parasite ofTheileria species, which is transmitted by Ixodidae ticks. Recognition of the vector ticks is essential in each area for establishing some integrated preventive measures against the disease. Therefore, 219 ticks were collected from the body surface of 150 sheep suffering from fever and anemia, in different parts of the province, during April - August 2012. Also, thin blood films were prepared from the peripheral blood of these animals. DNA of the tick salivary glands including 152Rhipicephalus sanguineus, 13 R. bursa and 54 Hyalomma anatolicum anatolicumwere extracted. Then PCR, using a pair of 520 bp specific primer of SSurRNA gene of Theileria ovis, as well as a pair of specific primer for amplification of 785bp of T. LESTOQUARDImerozoite surface antigens, was performed. The PCR revealed that 37 out of 152 R. sanguineus(24.34%) were positive for Theileria ovis, whereas none of R. bursa ticks were positive. Also, 5 out of 54H. a anatolicum (9.25%) were positive for T. LESTOQUARDI. The microscopic examinations of blood smears showed that 19 out of 150 blood smears (12.66%) contained the piroplasmic forms of Theileria species. Regarding the vast distribution of R. sanguineusin the area, it seems that this tick may be the main vector of Theileria ovis in Lorestan province, Iran.

Theileria species are obligatory intracellular hemoprotozoan parasites transmitted by various species of hard ticks and cause Theleriosis. Geographically, the disease has a global distribution and is often occurred in the tropical and subtropical areas of the world. In Iran, the small ruminant Theleriosis is caused by Theileria ovis and T. LESTOQUARDI (Hirci). This study was aimed to identify Theileria ovis and T. LESTOQUARDI among the small ruminants of East Azerbaijan province based on molecular method. For this purpose, a total of 166 whole blood samples (including 125 sheep and 41 goats) were collected from different regions of East Azerbaijan province, and then, all samples were examined under routine microscopic method and molecular assay. According to the molecular findings, 30(18%) of 166 specimens were only infected with T. ovis, whereas no infection was found on microscopic examination. The infection was only related to sheep samples. There was no significant difference between infection and age and sex, statistically. Sequencing showed that the amplified fragments in the present study was 98-100% identical to the corresponding sequences on GeneBank. Phylogenetic analysis indicated that the sequence obtained in this study with many sequences from different parts of the world in different years in a set and is closely related to these sequences. As a result, it can be noted that Theileriosis has a clinical and subclinical feature among the small ruminants of the East Azarbaijan province and T. ovis is the main cause of Theileriosis.

Background: This study was carried out to identify Theileria spp. infections in goats and ticksin Kermanshah Province, western Iran from May– Sep 2015. Methods: For differentiation of different Theileria spp. both blood and tick samples were examined by nested PCRRFLP. Results: Light microscopy of blood smears revealed Theileria spp. infection in 22 (5. 5%), while 68 (17%) of blood samples were positive using nested PCR. Out of 68 positive samples, 85. 3% (58/68) and 11. 7% (8/68) were respectively positive for Theileria ovis and T. LESTOQUARDI. Mixed infection was detected in 3% (2/68) cases. Overall, 420 ixodid ticks belong to seven different hard ticks species were collected from goats. Rhipicephalus turanicus 112 (26. 7%), R. sanguineus 95 (22. 6%), R. bursa, 91(21. 7%), Hyalomma anatolicum, 55(13. 1%), H. excavatum 27(6. 4%), H. marginatum, 22(5. 3%) and Dermacentor marginatus, 18(4. 2%) were the main tick species infesting goats. The PCR products obtained from ticks were subjected to the differentiation of Theileria species. Respectively, 2 and 8 pools of H. marginatum and R. turanicus salivary glands were infected with T. ovis and T. LESTOQUARDI. In addition, T. annulata and T. LESTOQUARDI infection weredetected in three pools of H. anatolicum. Conclusion: This is the first report of goats and collected ticks to Theileria spp infection in Iran. The results suggest that T. ovis has a higher prevalence than T. LESTOQUARDI. It is also postulated H. marginatum, R. turanicus and H. anatolicum might play an important role in the field as a vector of Theileria spp in this area.

In spite of presence of the vector ticks and susceptible hosts of Ovine Malignant Theileriosis in all parts of Iran, the endemic areas of the disease are restricted to certain foci in the South and center of the country. Finding the reason of this point, this study was conducted in seven experiments to transmit Theileria LESTOQUARDI from carrier sheep to susceptible host by Hyalomma anatolicum anatolicum. The carrier sheep were collected from three different areas including Fars, Ilam and Tehran provinces and the vector ticks were collected from Fars, Ilam and Urmia. The results showed that the ticks from non-endemic areas could potentially transmit the parasite. Therefore the assumption that the restricted foci of the disease are due to adaptation of the parasite in the endemic areas to the local vector tick is not much valid. It may be concluded that in some areas (non-endemic) the limiting factor is low temperature and in the others, the extreme high temperature that reduce tick ratio per animal. Ovine and Caprine Malignant Theileriosis (OCMT) occurs in certain foci of Iran with a mean annual temperature between 20 - 25° C. The clinical signs and the variation of parasitemia were recorded in the experimentally infected animals.

English: 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. (French: La pré, sente é, tude a examiné,la relation phylogé, né, tique basé, e sur les sé, quences du gè, ne du cytochrome b parmi les espè, ces pathogè, nes de Theileria (spp. ) en Iran, y compris Theileria annulata et Theileria LESTOQUARDI, ainsi que d'autres donné, es disponibles dans la GenBank. Les é, chantillons de sang de 136 bovins et 80 moutons suspecté, s d'infection par le piroplasme ont é, té,pré, levé, s dans six provinces diffé, rentes d'Iran. Des mé, thodes microscopiques et molé, culaires utilisant des amorces spé, cifiques à,l'espè, ce ont é, té,utilisé, es pour cribler des é, chantillons positifs pour T. annulata et T. LESTOQUARDI. Enfin, le gè, ne partiel du cytochrome b de 30 T. annulata et 5 T. LESTOQUARDI ont é, té,amplifié, s, sé, quencé, s et dé, posé, s dans la GenBank. Les ré, sultats ont indiqué,qu'il y avait 12 gé, notypes diffé, rents parmi les isolats de T. annulata, alors qu'un seul gé, notype a é, té,observé,parmi les isolats de T. LESTOQUARDI. Une infection à, T. LESTOQUARDI chez les bovins a é, té,dé, tecté, e dans un é, chantillon, et aucune co-infection à,T. annulata et T. LESTOQUARDI n'a é, té,dé, tecté, e chez les moutons et les bovins. Dans l'arbre phylogé, né, tique, diffé, rentes Theileria spp. Ont é, té,placé, es dans des clades sé, paré, s, et la fiabilité,de l'arbre repré, senté,et de la monophylie des endogroupes de T. annulata et de T. LESTOQUARDI a é, té,soutenue par la valeur de bootstrap de 94% qui a indiqué,de maniè, re significative que ces deux espè, ces ont é, volué,d'un ancê, tre commun. L'arbre a é, galement montré,que ces deux spp. pathogè, nes partageaient un ancê, tre commun plus ré, cent, comparé,à,une autre espè, ce de parasites Theileria. À,notre connaissance, cette é, tude est la premiè, re analyse phylogé, nique de Theileria spp. pathogè, ne en Iran basé, e sur les sé, quences du gè, ne du cytochrome b. De plus, le premier gè, ne du cytochrome b de T. LESTOQUARDI a é, té,sé, quencé,et dé, posé,dans la GenBank. )