There is need to confirm the presence of Theileria orientalis among the cattle population in Peninsular Malaysia and to evaluate the risk factors associated with the infection. To this effect, blood samples were collected from 1045 cattle from 43 farms throughout the entire States of Peninsular Malaysia. The collected blood samples were subjected to DNA extraction and subsequent PCR amplification of the major piroplasm surface protein (MPSP) gene of the haemoprotozoan. Representative positive amplicons were purified, sequenced and compared with other sequences of the MPSP gene of T. orientalis curated from the GenBank. A well-structured questionnaire was used to get information about each cattle, it's demography, the bio-security, environmental and management factors. Univariate and multivariate analysis were used for the statistical evaluation, with significance set at p < 0.05. A total prevalence of 49.76% (520/1045; 95% CI: 46.73 - 52.79) was obtained. Types of breeds, age, production type, herd size, level of farm biosecurity, farm size, presence of other animal species in the farm, management systems and prophylaxis were significantly (p < 0.05) associated with the prevalence of T. orientalis. This study confirmed the presence of T. orientalis and establish that the haemoprotozoan is endemic among cattle in Peninsular Malaysia.
Classification of Theileria parasites of south-east Asian countries is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, we included 6 known species and 14 unclassified Theileria parasite isolates: Theileria annulata, Theileria parva, Theileria taurotragi, Theileria sergenti, Theileria buffeli, Theileria types Sable, Theileria types A, B, B1, B2, C, D, E, F, G, G1, Theileria type Medan (Indonesia), Theileria type Ipoh (Malaysia) and Theileria type Thong Song (Thailand). Small subunit ribosomal RNA (srRNA) nucleotide sequence data were collected by PCR, cloning and dideoxy sequencing. The srRNA nucleotide sequences were aligned and analyzed by distance methods, maximum parsimony algorithms and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. The data indicated that all of the tree-building methods gave very similar results. This study identified two groups of Theileria, the pathogenic and benign groups, which are strongly supported by bootstrap analysis. The analysis also indicated that three subgroups (A, B and C) were generated within the benign Theileria group whereas the classification of Theileria type D and Thong Song is questionable. However, more basic information such as life cycle differences, vectors, modes of transmission, virulent and genetic/sexual compatability is essential for clearer taxonomic definition of the benign Theileria parasites.
Genetic characterization of Theileria species infecting bovines in India was attempted targeting the 18S ribosomal RNA region of the parasite. Blood samples of bovines (n = 452), suspected for haemoprotozoan infections, from 9 different states of the country were microscopically examined for Theileria species infection. Four Theileria spp. positive blood samples from each state were randomly utilized for PCR amplification of the 18S rRNA gene (approx. 1529 bp) followed by cloning and sequencing. The sequence data analysis of all the 36 isolates revealed that 33 isolates had high sequence similarity with published sequences of T. annulata, whereas 3 isolates (MF287917, MF287924 and MF287928) showed close similarity with published sequences of T. orientalis. Sequence homology within the isolates ranged between 95.8 and 100% and variation in the length of targeted region was also noticed in different isolates (1527-1538 nt). Phylogenetic tree created for T. annulata sequences revealed that a total of 24 Indian isolates formed a major clade and grouped together with isolates originating from countries like China, Spain, Turkey and USA. Remaining 09 isolates clustered in a separate group and were closely related to the TA5 isolate of T. annulata (a new genotype) originating from India and also with the isolates from East Asian countries like Japan and Malaysia. All the three T. orientalis isolates had minimal intraspecific variation (99-100% homology) amongst themselves. Further, in the phylogenetic analysis T. orientalis Indian isolates were found to cluster away from other 14 isolates of T. buffeli/sergenti/orientalis originating from different countries (Australia, China, Indonesia and Spain). However, these 3 isolates clustered together with the T. buffeli Indian isolate (EF126184). Present study confirmed the circulation of different genotypes of T. annulata in India, along with T. orientalis isolates.