Detection of Hepatocystis sp. Infection in Vervet Monkeys (Chlorocebus Pygrythrus) Imported from Tanzania Using Molecular and Microscopic Methods.

The present study examined and reported Hepatocystis sp. infection in wild-caught vervet monkeys imported from Tanzania into the Razi vaccine and serum research institute (RVSRI). Polymerase chain reaction (PCR) targeting of 18S rRNA gene, followed by sequencing, Basic Local Alignment Search Tool (BLAST), and phylogenetic studies revealed that 82.8% of the imported monkeys were infected with Hepatocystis. Nevertheless, as illustrated by a routine parasitological examination of blood smears and histopathological examination of liver collected samples, the rates of Hepatocystis infection were obtained at 33.9% and 38.1%, respectively. Two isolated 18S rRNA gene sequences of Hepatocystis sp. from Tanzanian vervet monkeys were registered under the accession numbers OM281567 and OM281564 in GenBank. Although Hepatocystis infections do not cause clinical disease, they may interfere with the research data. The results of the current study pointed out that after proper nutrition and implementation of good physical environmental conditions for 3-4 months, the imported monkeys obviously gained weight and most of their hematological parameters, even in the presence of the parasite, returned to the normal levels and the experimental monkeys would be ready for use in studies.

Molecular Identification and Genotyping of Theileria Orientalis Type 3 (Buffeli) Isolated from Cattle Using Nested-PCR Assay in Guilan Province, Iran.

Protozoan parasites of the genus Theileria are tick-borne parasites that have been found in many species of mammals. More than a dozen species of Theileria have been found in cattle, water buffalo, sheep, and goats. Theileria orientalis is a non-pathogenic blood protozoan parasite that was detected and identified during a regular investigation of piroplasmida infection in indigenous cattle in the spring of 2019 in Northern Provinces of Iran. In total, 92 blood samples were collected from different areas of Guilan and Mazandaran Provinces, Iran during the spring. The Giemsa stained blood smears did not show any parasitic infection; however, T. orientalis was identified by 18S rRNA gene polymerase chain reaction (PCR) and DNA sequencing. The specific sequenced DNA for T. orientalis was registered in GenBank under the accession number MN453385. The partial 18S rRNA gene sequence of the obtained DNA showed 100% nucleotide identity with reference sequences for the T. orientalis that have been registered from Europe, Africa, and Asia. Additionally, molecular phylogenetic studies have shown that T. orientalis Iran GC98-01 isolate belongs to nonpathogenic T. orientalis type 3 (buffeli). In this study, the indigenous Bos indicus cattle were detected as asymptomatic carriers of Theileria spp. infection. Here, we identified and genotyped T. orientalis for the first time as T. orientalis type 3 (buffeli) in Iran using molecular phylogenetic analysis and registered the 18S rRNA gene sequence of the T. orientalis GC98-01 isolate in GenBank. Moreover, rare T. annulata infection was detected in cattle using semi-nested PCR in Mazandaran (Miankaleh peninsula). The T. orientalis can be differentiated from other Theileria and Babesia haemoprotozoan parasites by specific molecular assays.

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.

Molecular detection of Theileria spp. and Babesia ovis Infection in Sheep in Baneh, Iran.

The purpose of the present study was to investigate the Theileria and Babesia infection in sheep using polymerase chain reaction (PCR) assay in Baneh, Iran. Theileria and Babesia are apicomplexan parasites that have both vertebral and invertebrate hosts. These protozoa, which are transmitted by tick vectors, are considered to be the most important causes of parasitic diseases in Iran.The detection methods of Babesia and Theileria spp. are morphological examination, serology tests, and more recently, molecular assays, such as PCR. In this study, a total of 66 blood samples were collected and analyzed using specific primers for Theileria annulata, T. ovis, T. lestoquardi, and Babesia ovis. Two PCR methods were used, namely semi-nested PCR and competitive PCR. Based on the results of the PCR assay of 66 sheep blood samples, B. ovis, T. ovis, T. lestoquardi, and T. annulata were detected in 57 (86.4%), 28 (42.4%), 0, and 16 (24%) cases, respectively. Detection of low levels of protozoan infection with high morbidity in the tested animals shows their status as a carrier that keeps the infection in the region and extends the protozoan life cycle. Another important factor is the geographical situation of Baneh as a border city since the hemoprotozoan infection is present in this region. Moreover, piroplasmida infection was found in Iraq and other neighboring provinces. Therefore, animal husbandry in Baneh is at the risk of infection with Babesia and Theileria. The collected data in this study are useful for reaching a better understanding of the epizootiology of theileriosis and babesiosis, in order to control and prevent the diseases in this region.

Phylogenetic Analysis of Theileria annulata Infected Cell Line S15 Iran Vaccine Strain.

BACKGROUND: Bovine theileriosis results from infection with obligate intracellular protozoa of the genus Theileria. The phylogenetic relationships between two isolates of Theileria annulata, and 36 Theileria spp., as well as 6 outgroup including Babesia spp. and coccidian protozoa were analyzed using the 18S rRNA gene sequence. METHODS: The target DNA segment was amplified by PCR. The PCR product was used for direct sequencing. The length of the 18S rRNA gene of all Theileria spp. involved in this study was around 1,400 bp. RESULTS: A phylogenetic tree was inferred based on the 18S rRNA gene sequence of the Iran and Iraq isolates, and other species of Theileria available in GenBank. In the constructed tree, Theileria annulata (Iran vaccine strain) was closely related to other T. annulata from Europe, Asia, as well as T. lestoquardi, T. parva and T. taurotragi all in one clade. CONCLUSION: Phylogenetic analyses based on small subunit ribosomal RNA gene suggested that the percent identity of the sequence of Iran vaccine strain was completely the same as Iraq sequence (100% identical), but the similarity of Iran vaccine strain with other T. annulata reported from China, Spain and Italy determined the 97.9 to 99.9% identity.