Serological and molecular surveys of Babesia bovis and Babesia bigemina among native cattle and cattle imported from Thailand in Hue, Vietnam.

Serum and DNA from blood samples collected from Vietnamese yellow cattle (n=101) and cattle imported from Thailand (n=54) at a Vietnamese slaughter house were screened for Babesia bovis and Babesia bigemina infections by enzyme-linked immunosorbent assay (ELISA) and PCR. The positive rates determined by ELISA (B. bovis and B. bigemina) or PCR (B. bigemina) in the Vietnamese cattle were significantly higher than those found in Thai cattle. Some PCR-positive Vietnamese animals were ELISA-negative, whereas all PCR-positive Thai cattle were ELISA-positive, suggesting that the animals were infected in Thailand. Importing Babesia-infected cattle may lead to the introduction of new parasite strains, possibly compromising the development of anti-Babesia immune control strategies in Vietnam.

Epidemiology of bovine hemoprotozoa parasites in cattle and water buffalo in Vietnam.

A PCR-based survey of hemoprotozoa parasites detected Babesia bigemina, Theileria orientalis and Trypanosoma theileri among cattle and water buffalo in Vietnam, and a new Babesia sp. closely related to Babesia ovata was detected in cattle only. In addition, Theileria annulata and Trypanosoma evansi were not detected in both cattle and water buffalo. Phylogenetic analysis detected T. orientalis MPSP genotypes 3, 5, 7 and N3 in cattle and 5, 7, N1 and N2 in water buffalo. Additionally, water buffalo-derived T. theileri CATL sequences clustered together with a previously reported cattle-derived sequence from Vietnam. This is the first report of a new Babesia sp. in cattle, and T. orientalis MPSP genotype 7 and T. theileri in water buffalo in Vietnam.

Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam.

Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic diversity of the B. bovis msa-1 gene in Asia.

Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes.

The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam.

Molecular and seroepidemiological survey of Babesia bovis and Babesia bigemina infections in cattle and water buffaloes in the central region of Vietnam.

In the present study, a total of 137 blood samples were collected from cattle and water buffaloes in central region of Vietnam and tested using nested polymerase chain reaction (nPCR), enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) to determine the molecular and serological prevalence of Babesia bovis and Babesia bigemina. In cattle, the prevalence of B. bovis and B. bigemina was 21.3% and 16.0% by nPCR, 73.4% and 42.6% by ELISA and 60.6% and 59.6% by IFAT, respectively, whereas those of water buffalos were 23.3% and 0% by nPCR, 37.2% and 9.3% by ELISA and 27.9% and 18.6% by IFAT, respectively. IFAT and ELISA detected a higher number of infected cattle and water buffaloes than nPCR totally. Statistically significant differences in the prevalence of the two infections were observed on the basis of age. Overall, the current data suggest high incidence of B. bovis and B. bigemina infections in the central region of Vietnam, which is needed to develop comprehensive approach to the modern surveillance, diagnosis and control of bovine babesiosis.

PCR detection and genetic diversity of bovine hemoprotozoan parasites in Vietnam.

Hemoprotozoan infections often cause serious production losses in livestock. In the present study, we conducted a PCR-based survey of Babesia bovis, Babesia bigemina, Theileria annulata, Theileria orientalis, Trypanosoma evansi and Trypanosoma theileri, using 423 DNA samples extracted from blood samples of cattle (n=202), water buffaloes (n=43), sheep (n=51) and goats (n=127) bred in the Hue and Hanoi provinces of Vietnam. With the exception of T. annulata and T. evansi, all other parasite species (B. bovis, B. bigemina, T. orientalis and T. theileri) were detected in the cattle populations with B. bovis being the most common among them. Additionally, four water buffaloes and a single goat were infected with B. bovis and B. bigemina, respectively. The Hue province had more hemoprotozoan-positive animals than those from the Hanoi region. In the phylogenetic analyses, B. bovis-MSA-2b, B. bigemina-AMA-1 and T. theileri-CATL gene sequences were dispersed across four, one and three different clades in the respective phylograms. This is the first study in which the presence of Babesia, Theileria and Trypanosoma parasites was simultaneously investigated by PCR in Vietnam. The findings suggest that hemoprotozoan parasites, some of which are genetically diverse, continue to be a threat to the livestock industry in this country.

Cloning and Expression of Genes Encoding F107-C and K88-1NT Fimbrial Proteins of Enterotoxigenic Escherichia coli from Piglets.

We cloned two genes coding F107-C and K88-1NT fimbrial subunits from strains E. coli C and 1NT isolated from Thua Thien Hue province, Vietnam. The mature peptide of faeG gene from strain E. coli 1NT (called faeG-1NT) is 100 % similarity with faeG gene, while the CDS of fedA gene from strain C (called fedA-C) has a similarity of 97 % with the fedA gene. Expression of the faeG-1NT and fedA-C genes in E. coli BL21 Star™ (DE3) produced proteins of ~31 and 22 kDa, respectively. The effect of IPTG concentration on the K88-1NT and F107-C fimbriae production was investigated. The results showed that 0.5 mM IPTG is suitable for higher expression of K88-1NT subunit, while 0.75 mM IPTG strongly stimulated expression of F107-C subunit. The optimal induction time for expression was also examined. Generally, highest expression of K88-1NT subunit occurred after 6 h of induction, while that of F107-C subunit is after 14 h.

A PCR based survey of Babesia ovata in cattle from various Asian, African and South American countries.

Babesia ovata is a tick-transmitted hemoprotozoan parasite that infects cattle. In our study, bovine blood samples (n=2,034) were collected from 10 different countries (Brazil, China, Ghana, Japan, Mongolia, the Philippines, South Africa, Sri Lanka, Thailand and Vietnam) and DNA extracted. The DNA samples were screened using an established and specific polymerase chain reaction (PCR) assay targeting the Apical membrane antigen 1 (AMA-1) gene. Parasite DNA was detected among samples collected from Japan, Mongolia and Thailand. Sequence analyses confirmed that the PCR assay detected only B. ovata AMA-1, and that amplicons from different geographical locations were conserved. Our findings highlight the importance of designing adequate strategies to control B. ovata infection in Japan, Mongolia, and Thailand.

Molecular epidemiological survey of Theileria orientalis in Thua Thien Hue Province, Vietnam.

Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).