Molecular detection and genetic diversity of bovine Babesia spp., Theileria orientalis, and Anaplasma marginale in beef cattle in Thailand.

Babesia spp., Theileria orientalis, and Anaplasma marginale are significant tick-borne pathogens that affect the health and productivity of cattle in tropical and subtropical areas. In this study, we used PCR to detect the presence of Babesia bovis, Babesia bigemina, and T. orientalis in 279 beef cattle from Western Thailand and A. marginale in 608 beef cattle from the north, northeastern, and western regions. The PCRs were performed using species-specific primers based on the B. bovis spherical body protein 2 (BboSBP2), B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. orientalis major piroplasm surface protein (ToMPSP), and A. marginale major surface protein 4 (AmMSP4) genes. To determine the genetic diversity of the above parasites, amplicons of B. bovis and B. bigemina ITS1-5.8s rRNA gene-ITS2 regions (B. bovis ITS, B. bigemina ITS), ToMPSP, and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the prevalence of B. bovis, B. bigemina, T. orientalis, and A. marginale in the Western region was 11.1, 12.5, 7.8, and 39.1 %, respectively. Coinfections of two or three parasites were observed in 17.9 % of the animals sampled. The study revealed that the prevalence of A. marginale in the western region was higher than in the north and northeastern regions (7 %). Sequence analysis showed the BboSBP2 gene to be more conserved than B. bovis ITS in the different isolates and, similarly, the BbiRAP-1a was more conserved than B. bigemina ITS. In the phylogenetic analysis, T. orientalis MPSP sequences were classified into types 3, 5, and 7 as previously reported. A. marginale MSP4 gene sequences shared high identity and similarity with each other and clustered with isolates from other countries. This study provides information on the prevalence and genetic diversity of tick-borne pathogens in beef cattle and highlights the need for effective strategies to control these pathogens in Thailand.

Jirapattharasate C ，Adjou Moumouni PF ，Cao S ，Iguchi A ，Liu M ，Wang G ，Zhou M ，Vudriko P ，Efstratiou A ，Changbunjong T ，Sungpradit S ，Ratanakorn P ，Moonarmart W ，Sedwisai P ，Weluwanarak T ，Wongsawang W ，Suzuki H ，Xuan X ... -  《-》

Molecular detection and genetic identification of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in Turkey.

Babesia spp., Theileria spp. and Anaplasma spp. are significant tick-borne pathogens of livestock globally. In this study, we investigated the presence and distribution of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in cattle from 6 provinces of Turkey using species-specific PCR assays. The PCR were conducted using the primers based on the B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. annulata merozoite surface antigen-1 (Tams-1), T. orientalis major piroplasm surface protein (ToMPSP) and A. marginale major surface protein 4 (AmMSP4) genes, respectively. Fragments of B. bigemina internal transcribed spacer (BbiITS), T. annulata internal transcribed spacer (TaITS), ToMPSP and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the overall infections of A. marginale, T. annulata, B. bigemina and T. orientalis were 29.1%, 18.9%, 11.2% and 5.6%, respectively. The co-infection of two or three pathogens was detected in 29/196 (15.1%) of the cattle samples. The results of sequence analysis indicated that BbiRAP-1a, BbiITS, Tams-1, ToMPSP and AmMSP4 were conserved among the Turkish samples, with 99.76%, 99-99.8%, 99.34-99.78%, 96.9-99.61% and 99.42-99.71% sequence identity values, respectively. In contrast, the Turkish TaITS gene sequences were relatively diverse with 92.3-96.63% identity values. B. bigemina isolates from Turkey were found in the same clade as the isolates from other countries in phylogenetic analysis. On the other hand, phylogenetic analysis based on T. annulata ITS sequences revealed significant differences in the genotypes of T. annulata isolates from Turkey. Additionally, the T. orientalis isolates from Turkish samples were classified as MPSP type 3 genotype. This is the first report of type 3 MPSP in Turkey. Moreover, AmMSP4 isolates from Turkey were found in the same clade as the isolates from other countries. This study provides important data for understanding the epidemiology of tick-borne diseases and it is expected to improve approach for diagnosis and control of tick-borne diseases in Turkey.

Zhou M ，Cao S ，Sevinc F ，Sevinc M ，Ceylan O ，Moumouni PFA ，Jirapattharasate C ，Liu M ，Wang G ，Iguchi A ，Vudriko P ，Suzuki H ，Xuan X ... -  《-》

Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya.

Infections with Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya. Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes. B. bovis, B. bigemina, T. parva, T. velifera, T. taurotragi, T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp. (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo-derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents. The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya.

Adjou Moumouni PF ，Aboge GO ，Terkawi MA ，Masatani T ，Cao S ，Kamyingkird K ，Jirapattharasate C ，Zhou M ，Wang G ，Liu M ，Iguchi A ，Vudriko P ，Ybanez AP ，Inokuma H ，Shirafuji-Umemiya R ，Suzuki H ，Xuan X ... -  《Parasites & Vectors》

Molecular epidemiology of bovine Babesia spp. and Theileria orientalis parasites in beef cattle from northern and northeastern Thailand.

Beef cattle production represents the largest cattle population in Thailand. Their productivity is constrained by tick-borne diseases such as babesiosis and theileriosis. In this study, we determined the prevalence of Babesia bigemina, Babesia bovis and Theileria orientalis using polymerase chain reaction (PCR). The genetic markers that were used for detection of the above parasites were sequenced to determine identities and similarity for Babesia spp. and genetic diversity of T. orientalis. Furthermore the risk factors for the occurrence of the above protozoan parasites in beef cattle from northern and northeastern parts of Thailand were assessed. A total of 329 blood samples were collected from beef cattle in 6 provinces. The study revealed that T. orientalis was the most prevalent (30.1%) parasite in beef cattle followed by B. bigemina (13.1%) and B. bovis (5.5%). Overall, 78.7% of the cattle screened were infected with at least one of the above parasites. Co-infection with Babesia spp. and T. orientalis was 30.1%. B. bigemina and T. orientalis were the most prevalent (15.1%) co-infection although triple infection with the three parasites was observed in 3.0% of the samples. Sequencing analysis revealed that B. bigemina RAP1 gene and B. bovis SBP2 gene were conserved among the parasites from different cattle samples. Phylogenetic analysis showed that the T. orientalis MPSP gene from parasites isolated from cattle in north and northeast Thailand was classified into types 5 and 7 as reported previously. Lack of tick control program was the universal risk factor of the occurrence of Babesia spp. and T. orientalis infection in beef cattle in northern and northeastern Thailand. We therefore recommend training of farmers on appropriate tick control strategies and further research on potential vectors for T. orientalis and elucidate the effect of co-infection with Babesia spp. on the pathogenicity of T. orientalis infection on beef in northern and northeastern Thailand.

Jirapattharasate C ，Adjou Moumouni PF ，Cao S ，Iguchi A ，Liu M ，Wang G ，Zhou M ，Vudriko P ，Changbunjong T ，Sungpradit S ，Ratanakorn P ，Moonarmart W ，Sedwisai P ，Weluwanarak T ，Wongsawang W ，Suzuki H ，Xuan X ... -  《-》

Molecular characterization of South Indian field isolates of bovine Babesia spp. and Anaplasma spp.

Ticks and tick-borne diseases (TTBDs) are considered major causes of economic loss in the livestock sector which incur an annual control cost estimated at US$ 498.7 million in India. Among these diseases, babesiosis, theileriosis and anaplasmosis are listed among the top ten livestock diseases in India and cause significant mortality and morbidity among cattle. However, molecular characterization of bovine Babesia and Anaplasma species are scant; thus, the aim of this study is to perform molecular characterization of field isolates of Babesia spp. and Anaplasma spp. infecting bovines in Kerala, South India. Blood smears and whole blood samples were collected from a total of 199 apparently healthy adult female cattle in Kerala. Based on microscopy, Babesia spp., Theileria orientalis and Anaplasma spp. organisms were detected in 9 (4.5%), 40 (20%) and 6 (3%) samples, respectively. Genus-specific polymerase chain reactions for amplification of 18S rRNA of Babesia spp. and 16S rRNA of Anaplasma spp. revealed positive results with 18 (9%) and 14 (7%) samples. The phylogenetic analysis of 18S rRNA gene sequences of Babesia spp. confirmed the existence of two different populations of Babesia spp. circulating in the blood of infected cattle viz., Babesia bigemina and a Babesia sp. genetically related to Babesia ovata. Further phylogenetic analysis using rap-1a sequences of isolates of B. bigemina revealed higher levels of genetic heterogeneity. However, the field isolates of B. bigemina displayed only slight heterogeneity when the rap-1c gene was examined. Polymerase chain reaction followed by sequencing and phylogenetic analysis of 16S rRNA gene of Anaplasma spp. revealed the existence of Anaplasma marginale, Anaplasma bovis and Anaplasma platys in bovines in South India. Based on msp4 gene sequences, all the field isolates of A. marginale from Kerala were clustered in a single clade with others isolated from around the world. To our knowledge, this study forms the first report on occurrence of B. ovata-like parasites and A. platys in cattle from India.

Pradeep RK ，Nimisha M ，Sruthi MK ，Vidya P ，Amrutha BM ，Kurbet PS ，Kumar KGA ，Varghese A ，Deepa CK ，Dinesh CN ，Chandrasekhar L ，Juliet S ，Pradeepkumar PR ，Ravishankar C ，Ghosh S ，Ravindran R ... -  《-》