Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka.

Bovine babesiosis is a serious threat to the cattle industry. We prepared blood DNA samples from 13 cattle with clinical babesiosis from the Badulla (n = 8), Jaffna (n = 3), and Kilinochchi (n = 2) districts in Sri Lanka. These DNA samples tested positive in PCR assays specific for Babesiabovis (n = 9), Babesia bigemina (n = 9), and Babesiaovata (n = 1). Twelve cattle were positive for B. bovis and/or B. bigemina One cow was negative for the tested Babesia species but was positive for Babesia on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infected with Babesia sp. Mymensingh, which was recently reported from a healthy cow in Bangladesh. We then developed a novel Babesia sp. Mymensingh-specific PCR assay and obtained positive results for one other sample. Analysis of gene sequences from the cow with positive B. ovata-specific PCR results demonstrated that the animal was infected not with B. ovata but with Babesia sp. Hue-1, which was recently reported from asymptomatic cattle in Vietnam. The virulence of Babesia sp. Hue-1 is unclear, as the cow was coinfected with B. bovis and B. bigemina However, Babesia sp. Mymensingh probably causes severe clinical babesiosis, as it was the sole Babesia species detected in a clinical case. The present study revealed the presence of two bovine Babesia species not previously reported in Sri Lanka, plus the first case of severe bovine babesiosis caused by a Babesia species other than B. bovis, B. bigemina, and Babesiadivergens.

First detection of Theileria equi in free-roaming donkeys (Equus africanus asinus) in Sri Lanka.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses, donkeys, zebras, and mules. It is globally endemic with significant economic impact on the equine industry. Infected animals may serve as carriers, and they may be a source of infection for ticks, thereby posing a great challenge for disease management. Sri Lanka is a tropical country, where infections by various tick-borne parasites are common among livestock animals. However, infections by T. equi and B. caballi remain unstudied in Sri Lanka. Therefore, in the present study, we conducted an epidemiological survey to investigate the presence of T. equi and B. caballi in apparently healthy free-roaming donkeys. Blood samples were randomly taken from 111 donkeys in Mannar (n = 100) and Kilinochchi (n = 11) districts in Sri Lanka. Thin blood smears were prepared from the blood samples and subjected to microscopic examination. Additionally, blood DNA samples were prepared and screened for T. equi and B. caballi infections using species-specific PCR assays. Our results showed that 64 (57.7%) and 95 (85.6%) of the donkeys were positive for T. equi by microscopy and PCR, respectively. However, all samples were negative for B. caballi. Phylogenetic analysis of the T. equi 18S rRNA sequences detected two distinct genotypes, namely C and D. To our knowledge, this is the first report of T. equi in Sri Lanka and of genotype C in donkeys. The present study highlights the importance of monitoring the shrinking donkey population in Sri Lanka owing to EP caused by T. equi.

Phylogenetic analyses of the mitochondrial, plastid, and nuclear genes of Babesia sp. Mymensingh and its naming as Babesia naoakii n. sp.

BACKGROUND: The recently discovered Babesia sp. Mymensingh, which causes clinical bovine babesiosis, has a wide geographical distribution. We investigated the phylogenetic position of Babesia sp. Mymensingh using its mitochondrial, plastid, and nuclear genes. Based on morphological and molecular data, Babesia sp. Mymensingh is a unique species and we named it as Babesia naoakii n. sp. METHODS: A blood DNA sample from a Babesia sp. Mymensingh-infected cow was subjected to genome sequencing to obtain the sequences of mitochondrial, plastid, and nuclear genes. Six phylogenetic trees were then constructed with (1) concatenated amino acid sequences of cytochrome oxidase subunit I, cytochrome oxidase subunit III, and cytochrome b genes of the mitochondrial genome; (2) 16S rRNA of the plastid genome; (3) nucleotide sequences of the elongation factor Tu gene of the plastid genome; (4) ITS1-5.8S rRNA-ITS2; (5) concatenated nucleotide sequences of 89 nuclear genes; and (6) concatenated amino acid sequences translated from the 89 nuclear genes. RESULTS: In all six phylogenetic trees, B. naoakii n. sp. formed a sister clade to the common ancestor of Babesia bigemina and B. ovata. The concatenated nuclear genes of B. naoakii n. sp. and their translated amino acid sequences shared lower identity scores with the sequences from B. bigemina (82.7% and 84.7%, respectively) and B. ovata (83.5% and 85.5%, respectively) compared with the identity scores shared between the B. bigemina and B. ovata sequences (86.3% and 87.9%, respectively). CONCLUSIONS: Our study showed that B. naoakii n. sp. occupies a unique phylogenetic position distinct from existing Babesia species. Our findings, together with morphological differences, identify B. naoakii n. sp. as a distinct parasite species.

Epidemiological survey of hemoprotozoan parasites in cattle from low-country wet zone in Sri Lanka.

The diseases caused by hemoprotozoan parasites in cattle often result in economic losses. In Sri Lanka, previous studies found that the up-country wet zone, which is located in central Sri Lanka, was characterized by a high rate of Theileria orientalis and a low rate of Theileria annulata compared with the dry zone. In this study, DNA samples were prepared from the blood of 121 cattle in Galle, a coastal district located in low-country wet zone in Sri Lanka, and were PCR-screened for Babesia bovis, Babesia bigemina, T. annulata, T. orientalis, and Trypanosoma theileri. All the parasite species, except B. bovis, were detected among the surveyed cattle. The animals had a high rate of T. orientalis (100%) and a low rate of T. annulata (1.6%), as in the up-country wet zone. Babesia bigemina and Tr. theileri were detected in 19.0% and 20.6% of the animals, respectively, and their infection rates were higher in the animals reared in extensive management systems (32.8% and 27.9%, respectively) than in those managed in intensive/semi-intensive systems (5.0% and 13.3%, respectively). Genotypic analyses found that the T. orientalis mpsp type 5 was predominant similar to up-country wet zone, and that Tr. theileri consisted of seven catl genotypes, including two new genotypes (IL and IM) and four previously detected genotypes (IA, IB, II, and IK). These findings suggest that the hemoprotozoan infection profiles are largely conserved within the wet zone, despite differences in the geography, cattle breeds, and management practices between the up-country and low-country wet zones.

Genetic characterization of Babesia and Theileria parasites in water buffaloes in Sri Lanka.

Water buffaloes are thought to be the reservoir hosts for several hemoprotozoan parasites that infect cattle. In the present study, we surveyed Sri Lankan bred water buffaloes for infections with Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis using parasite-specific PCR assays. When 320 blood-derived DNA samples from water buffaloes reared in three different districts (Polonnaruwa, Mannar, and Mullaitivu) of Sri Lanka were PCR screened, B. bovis, B. bigemina, and T. orientalis were detected. While T. orientalis was the predominant parasite (82.5%), low PCR-positive rates were observed for B. bovis (1.9%) and B. bigemina (1.6%). Amplicons of the gene sequences of the Rhoptry Associated Protein-1 (RAP-1) of B. bovis, the Apical Membrane Antigen-1 (AMA-1) of B. bigemina, and the Major Piroplasm Surface Protein (MPSP) of T. orientalis were compared with those characterized previously in Sri Lankan cattle. While the B. bigemina AMA-1 sequences from water buffaloes shared high identity values with those from cattle, B. bovis RAP-1 sequences from water buffaloes diverged genetically from those of cattle. For T. orientalis, none of the MPSP sequence types reported previously in Sri Lankan cattle (types 1, 3, 5, and 7) were detected in the water buffaloes, and the MPSP sequences analyzed in the present study belonged to types N1 or N2. In summary, in addition to reporting the first PCR-based survey of Babesia and Theileria parasites in water buffaloes in Sri Lanka, the present study found that the predominant variants of water buffalo-derived B. bovis RAP-1 and T. orientalis MPSP sequences were different from those previously described from cattle in this country.

Genetic diversity within Theileria orientalis parasites detected in Sri Lankan cattle.

In the present study, we investigated the genetic diversity of Theileria orientalis parasites circulating among Sri Lankan cattle. Nucleotide sequence analysis of the major piroplasm surface protein (MPSP) gene fragments amplified from T. orientalis-positive DNA samples (from bovine blood) revealed the presence of 4 parasite genotypes. The genotypes consisted of types 1, 3, 5, and 7. Phylogenetic analysis indicated that the Sri Lankan MPSP sequences were closely related to those reported from Vietnam (types 3 and 5), Mongolia (types 1 and 5), Thailand (types 1, 5, and 7), and Japan (type 7). Subsequently, genotype-specific PCR assays determined that the most common genotype was type 7, followed by types 5, 3, and 1. Genotype 7 has been reported to be involved in disease outbreaks in India. Therefore, preventive and control measures are essential to avoid potential economic losses due to T. orientalis infection in Sri Lanka. This is the first report that describes the genetic diversity of T. orientalis circulating among Sri Lankan cattle.

Genetic diversity of merozoite surface antigens in Babesia bovis detected from Sri Lankan cattle.

Babesia bovis, the causative agent of severe bovine babesiosis, is endemic in Sri Lanka. The live attenuated vaccine (K-strain), which was introduced in the early 1990s, has been used to immunize cattle populations in endemic areas of the country. The present study was undertaken to determine the genetic diversity of merozoite surface antigens (MSAs) in B. bovis isolates from Sri Lankan cattle, and to compare the gene sequences obtained from such isolates against those of the K-strain. Forty-four bovine blood samples isolated from different geographical regions of Sri Lanka and judged to be B. bovis-positive by PCR screening were used to amplify MSAs (MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b), AMA-1, and 12D3 genes from parasite DNA. Although the AMA-1 and 12D3 gene sequences were highly conserved among the Sri Lankan isolates, the MSA gene sequences from the same isolates were highly diverse. Sri Lankan MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b sequences clustered within 5, 2, 4, 1, and 9 different clades in the gene phylograms, respectively, while the minimum similarity values among the deduced amino acid sequences of these genes were 36.8%, 68.7%, 80.3%, 100%, and 68.3%, respectively. In the phylograms, none of the Sri Lankan sequences fell within clades containing the respective K-strain sequences. Additionally, the similarity values for MSA-1 and MSA-2c were 40-61.8% and 90.9-93.2% between the Sri Lankan isolates and the K-strain, respectively, while the K-strain MSA-2a/b sequence shared 64.5-69.8%, 69.3%, and 70.5-80.3% similarities with the Sri Lankan MSA-2a1, MSA-2a2, and MSA-2b sequences, respectively. The present study has shown that genetic diversity among MSAs of Sri Lankan B. bovis isolates is very high, and that the sequences of field isolates diverged genetically from the K-strain.

A PCR-based survey of selected Babesia and Theileria parasites in cattle in Sri Lanka.

Hemoprotozoan parasites are responsible for significant economic losses in cattle. We screened Sri Lankan cattle populations for the presence of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using species-specific PCR assays. Out of 316 samples collected from animals in four different districts of Sri Lanka (Nuwara Eliya, Polonnaruwa, Ampara, and Jaffna), 231 (73.1%) were positive for at least one parasite species. All four parasite species were detected among the study groups from all of the districts surveyed. The first and second commonest hemoprotozoan parasites identified were T. orientalis (53.5%) and B. bigemina (30.1%), respectively. We found that the dry zones (Polonnaruwa, Ampara, and Jaffna) had more Babesia-positive animals than the hill country wet zone (Nuwara Eliya). In contrast, T. orientalis was the predominant species detected in Nuwara Eliya, while infection with T. annulata was more common in the dry zones. In addition, 81 (35.1%) of the 231 positive samples were infected with more than one parasite species. The presence of multiple parasite species among the different cattle populations is of clinical and economic significance. Therefore, island-wide control and prevention programs against bovine babesiosis and theileriosis are needed to minimize the financial burden caused by these parasites.