Use of molecular markers can help to understand the genetic diversity of Babesia bovis.

Cattle babesiosis is a tick-borne disease responsible for significant losses for the livestock industries in tropical areas of the world. These piroplasms are under constant control of the host immune system, which lead to a strong selective pressure for arising more virulent or attenuated phenotypes. Aiming to better understand the most critical genetic modifications in Babesia bovis genome, related to virulence, an in silico analysis was performed using DNA sequences from GenBank. Fourteen genes (sbp-2, sbp-4, trap, msa-1, msa-2b, msa-2c, Bv80 (or Bb-1), 18S rRNA, acs-1, ama-1, ß-tub, cp-2, p0, rap-1a) related to parasite infection and immunogenicity and ITS region were selected for alignment and comparison of several isolates of Babesia bovis from different geographic regions around the world. Among the 15 genes selected for the study of diversity, only 7 genes (sbp-2, sbp-4, trap, msa-1, msa-2b, msa-2c, Bv80) and the ITS region presented sufficient genetic variation for the studies of phylogeny. Despite this genetic diversity observed into groups, there was not sufficient information available to associate molecular markers with virulence of isolates. However, some genetic groups no were correlated with geographic region what could indicate some typical evolutionary characteristics in the relation between parasite-host. Further studies using these genes in herds presenting diverse clinical conditions are required. The better understanding of evolutionary mechanisms of the parasite may contribute to improve prophylactic and therapeutic measures. In this way, we suggest that genes used in our study are potential markers of virulence and attenuation and have to be analyzed with the use of sequences from animals that present clinical signs of babesiosis and asymptomatic carriers.

Babesiose cerebral em bezerros / Cerebral babesiosis in calves

Babesiose cerebral é uma enfermidade causada pelo protozoário Babesia bovis. O agente faz parte do complexo Tristeza Parasitária Bovina, uma das mais importantes doenças parasitárias em bovinos. O presente estudo relata um surto causado por B. bovis em vinte bezerros de aproximadamente 7 a 25 dias de idade. O surto ocorreu entre março e junho de 2015, na região sul do Brasil, área de instabilidade enzoótica para a Tristeza Parasitária Bovina. O diagnóstico foi realizado pela epidemiologia, lesões macroscópicas e pela presença de numerosas formas parasitárias de Babesia bovis em capilares encefálicos, observados em imprints corados por Giemsa. Surtos de babesiose por B. bovis cerebral nos primeiros dias de vida de bezerros é incomum, porém não pode ser desconsiderada em surtos com alta letalidade em áreas de instabilidade enzoótica.(AU)

Cerebral babesiosis is a protozoan disease caused by Babesia bovis. This parasite belongs to the bovine parasitic complex of tick-borne diseases that affect livestock worldwide. The present study reports an outbreak caused by B. bovis affected twenty 7-25 day-old calves. Outbreak occurred from May to July 2015 in the south of Brazil, where there is an area of enzootic instability for cattle tick fever. The macroscopic lesions were anemia, hemoglobinuria, splenomegaly, hepatomegaly, yellow liver and cherry-pink discoloration of cerebral and cerebellar cortex. The diagnosis was based on epidemiology, necropsy and microscopic findings in the brain that showed B. bovis in the capillary vessels of the brain in imprints stained by Giemsa. Cases of cerebral babesiosis by Babesia bovis in such young calves are uncommon but should be considered as a diagnosis possibility when there is high mortality rate in areas ofen zootic instability.(AU)

Low levels of genetic diversity associated with evidence of negative selection on the Babesia bovis apical membrane antigen 1 from parasite populations in Thailand.

Babesia bovis, a parasite infecting cattle and buffalo, continues to spread throughout the developing world. The babesial vaccine was developed to be a sustainable alternative treatment to control the parasite. However, genetic diversity is a major obstacle for designing and developing a safe and effective vaccine. The apical membrane antigen 1 (AMA-1) is considered to be a potential vaccine candidate antigen among immunogenic genes of B. bovis. To gain a more comprehensive understanding of B. bovis AMA-1 (BbAMA-1), three B. bovis DNA samples were randomly selected to characterize in order to explore genetic diversity and natural selection and to predict the antigen epitopes. The sequence analysis revealed that BbAMA-1 has a low level of polymorphism and is highly conserved (95.46-99.94%) among Thai and global isolates. The majority of the polymorphic sites were observed in domains I and III. Conversely, domain II contained no polymorphic sites. We report the first evidence of strong negative or purifying selection across the full length of the gene, especially in domain I, by demonstrating a significant excess of the average number of synonymous (dS) over the non-synonymous (dN) substitutions. Finally, we also predict the linear and conformational B-cell epitope. The predicted B-cell epitopes appeared to be involved with the amino acid changes. Collectively, the results suggest that the conserved BbAMA-1 may be used to detect regional differences in the B. bovis parasite. Importantly, the limitation of BbAMA-1 diversity under strong negative selection indicates strong functional constraints on this gene. Thus, the gene could be a valuable target vaccine candidate antigen.

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand.

Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.

Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies are required to confirm this.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Genetic characterization and molecular survey of Babesia bovis, Babesia bigemina and Babesia ovata in cattle, dairy cattle and yaks in China.

BACKGROUND: Babesiosis is an important haemoparasitic disease, caused by the infection and subsequent intra-erythrocytic multiplication of protozoa of the genus Babesia that impacts the livestock industry and animal health. The distribution, epidemiology and genetic characterization of B. bigemina, B. bovis, and B. ovata in cattle in China as well as the prevalence of these protozoan agents were assessed. METHODS: A total of 646 blood specimens from cattle, dairy cattle and yaks from 14 provinces were collected and tested for the presence of the three Babesia species via a specific nested PCR assay based on the rap-1 and ama-1 genes. The PCR results were confirmed by DNA sequencing. Gene sequences and the genetic characterization were determined for selected positive samples from each sampling area. RESULTS: Of a total of 646 samples, 134 (20.7 %), 60 (9.3 %) and 10 (1.5 %) were positive for B. bovis, B. bigemina and B. ovata infections, respectively. Mixed infections were found in 7 of 14 provinces; 43 (6.7 %) samples were infected with B. bovis and B. bigemina. Three samples (0.5 %) exhibited a co-infection with B. bovis and B. ovata, and 6 (0.9 %) were infected with all three parasites. The rap-1a gene of B. bovis indicated a high degree of sequence heterogeneity compared with other published rap-1a sequences worldwide and was 85-100 % identical to B. bovis rap-1a sequences in Chinese isolates. B. bigemina rap-1c and B. ovata ama-1 genes were nearly identical, with 97.8-99.3 % and 97.8-99.6 % sequence identity, respectively, in GenBank. CONCLUSIONS: Positive rates of B. bovis and B. bigemina infection are somewhat high in China. The B. bovis infection in yaks was first reported. The significant sequence heterogeneity in different variants of the rap-1a gene from Chinese B. bovis isolates might be a great threat to the cattle industry if RAP-1a protein is used as immunological antigen against Babesia infections in China. The data obtained in this study can be used to plan effective control strategies against babesiosis in China.

Differentiation between Israeli B. bovis vaccine strain and field isolates.

The present study demonstrated for the first time the ability to distinguish between the Israeli Babesia bovis vaccine strain and field isolates. The existence of an additional EcoRI restriction site in the rhoptry-associated protein-1 (rap-1) gene, which is unique to the Israeli vaccine strain, and the abolition of one of the HaeIII restriction sites in the rap-1 gene of the vaccine strain enabled distinction between the Israeli B. bovis vaccine strain and field isolates, and this was the basis for polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) development. ClustalW sequence alignment of RAP-1-deduced amino acids of the Israeli B. bovis strains and of field isolates showed that the total sequence identity among the RAP-1 amino acid sequences ranged from 97.5% to 100%. However, comparison between amino acids of RAP-1 of the Israeli vaccine strain and of field isolates, on the one hand, and B. bovis strains from Argentina, Mexico, Brazil, and USA, on the other hand, revealed 90% identity. The PCR-RFLP assay offered the great advantage of being able to distinguish between vaccine and field isolates in mixtures and provide new insight into the molecular epidemiology of B. bovis infections in Israel.

An epidemiological survey of bovine Babesia and Theileria parasites in cattle, buffaloes, and sheep in Egypt.

Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood-DNA samples sourced from cattle (n=439), buffaloes (n=50), and sheep (n=105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3-100% and 95.3-100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.

Genotypic diversity in Babesia bovis field isolates and vaccine strains from South Africa.

Genotypic diversity in Babesia bovis (cause of Asiatic redwater in cattle) vaccine strains and field isolates from South Africa were investigated using the Bv80 gene as well as microsatellites. The S11 vaccine strain possessed both A and B alleles of the Bv80 gene, as well as genotypic diversity within each allele type as defined by repeat variation resulting in different amplicon sizes. Rapid serial passage of vaccine strain from passage S10 to S24 resulted in loss of genotypic diversity that yielded a single allele A genotype with an amplicon size of 558 bp. This suggested that clonal selection occurred during rapid passaging. Extensive genotypic diversity exists in 44 field isolates characterized with both Bv80 A and B alleles, but can be readily distinguished from the S24 vaccine strain using either the Bv80 allele specific PCR assays or using multi-locus micro-satellite typing. This indicated that no recent documented clinical cases of Asiatic redwater were caused by the reversion to virulence of the current vaccine strain.

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.

Evidence for extensive genetic diversity and substructuring of the Babesia bovis metapopulation.

Babesia bovis is a tick-transmitted haemoprotozoan and a causative agent of bovine babesiosis, a cattle disease that causes significant economic loss in tropical and subtropical regions. A panel of nineteen micro- and minisatellite markers was used to estimate population genetic parameters of eighteen parasite isolates originating from different continents, countries and geographic regions including North America (Mexico, USA), South America (Argentina, Brazil), the Middle East (Israel) and Australia. For eleven of the eighteen isolates, a unique haplotype was inferred suggesting selection of a single genotype by either in vitro cultivation or amplification in splenectomized calves. Furthermore, a high genetic diversity (H = 0.780) over all marker loci was estimated. Linkage disequilibrium was observed in the total study group but also in sample subgroups from the Americas, Brazil, and Israel and Australia. In contrast, corresponding to their more confined geographic origin, samples from Israel and Argentina were each found to be in equilibrium suggestive of random mating and frequent genetic exchange. The genetic differentiation (F(ST)) of the total study group over all nineteen loci was estimated by analysis of variance (Θ) and Nei's estimation of heterozygosity (G(ST')) as 0.296 and 0.312, respectively. Thus, about 30% of the genetic diversity of the parasite population is associated with genetic differences between parasite isolates sampled from the different geographic regions. The pairwise similarity of multilocus genotypes (MLGs) was assessed and a neighbour-joining dendrogram generated. MLGs were found to cluster according to the country/continent of origin of isolates, but did not distinguish the attenuated from the pathogenic parasite state. The distant geographic origin of the isolates studied allows an initial glimpse into the large extent of genetic diversity and differentiation of the B. bovis population on a global scale.

Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes.

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4-26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

[Molecular characterization of Babesia bovis msa-2c gene]. / Babesia bovis'in msa-2c geninin moleküler karakterizasyonu.

OBJECTIVE: This study was carried out to determine the molecular characterization of msa-2c gene of one Babesia bovis isolate from cattle in the Aegean Region and to compare identities with similar isolates from the World and Turkey. METHODS: Between 2008-2010 blood samples were collected from a total of 235 cattle localized in 9 provinces of the Marmara and Aegean Regions. Smears were prepared, genomic DNA's were extracted from the blood samples and investigated for Babesia species by RLB. PCR was performed on one sample determined as B. bovis, the obtained amplicon was purified, sequenced and deposited to GenBank. Identities with similar isolates from Turkey and the World were investigated. RESULTS: Bovine babesiosis was not determined in the microscopic examination. According to the RLB results there was no B. bovis positivity in cattle from the Marmara Region, while only one B. bovis positivity was detected in cattle from the Aegean Region. The molecular prevalence of B. bovis was determined as 0.42% in the total of the examined 235 cattle. The sequenced B. bovis isolate shared 91-92% and 89-96% identities with the isolates from Turkey and the World, respectively. CONCLUSION: Molecular characterization of msa-2c gene region of B. bovis detected from cattle in the Aegean Region was carried out in this study.

Population genetic analysis and sub-structuring in Babesia bovis.

The tick-borne protozoan parasite, Babesia bovis is one of the causes of bovine babesiosis, an economically important disease of cattle in tropical and sub-tropical countries. Using the recently published genome sequence of the parasite, we developed a panel of eight mini- and micro-satellite markers and used these to investigate the role of genetic exchange in the population structure and diversity of the parasite using isolates from Zambia and Turkey. This population genetic analysis showed that genetic exchange occurs and that there are high levels of genetic diversity, with geographical sub-structuring quantified using Wright's F Index. Linkage disequilibrium was observed when isolates from both countries were treated as one population, but when isolates from Zambia were analysed separately linkage equilibrium was observed. The Turkish isolates were sub-structured, containing two genetically distinct sub-groups, both of which appeared to be in linkage equilibrium. The results of the Zambian study suggest that a sub-set of the parasite population is responsible for the westward spread of babesiosis into the previously disease-free central region of the country. The Zambian isolates had a significantly higher number of genotypes per sample than those from Turkey and age was found to be a significant predictor of the multiplicity of infection. The high levels of diversity seen in the Zambian and Turkish B. bovis populations have implications in the development of subunit vaccines against the disease and the spread of drug resistance.

Molecular and serologic evidence for Babesia bovis-like parasites in white-tailed deer (Odocoileus virginianus) in south Texas.

The current study was undertaken to determine if white-tailed deer in south Texas harbor Babesia bovis, a causative agent of bovine babesiosis. Blood samples from free-ranging white-tailed deer (Odocoileus virginianus) on two ranches in LaSalle and Webb Counties were screened for B. bovis and other hemoparasites by the polymerase chain reaction (PCR) to detect the piroplasm 18S rDNA. Serology was conducted on selected samples to detect antibody activity to B. bovis by the immunofluorescent antibody test (IFAT). PCR revealed that 16% of the LaSalle County samples and 4% of the Webb County samples were positive for B. bovis. Five of the LaSalle County and the two Webb County B. bovis 18S rDNA amplicons were cloned and sequenced. The resulting clones shared 99% identity to B. bovis 18S rRNA gene sequences derived from cattle isolates. Weak seroreactivity to B. bovis was shown by the IFAT. The samples were also screened for additional hemoparasites of deer including Theileria cervi, Babesia odocoilei and other Babesia spp. A genotypically unique Theileria sp. was found, along with T. cervi and B. odocoilei. The finding of putative B. bovis in white-tailed deer necessitates further study to determine if deer may act as a transient host or even a reservoir of infection for B. bovis pathogenic to cattle.

Genotypic diversity of merozoite surface antigen 1 of Babesia bovis within an endemic population.

Multiple genetically distinct strains of a pathogen circulate and compete for dominance within populations of animal reservoir hosts. Understanding the basis for genotypic strain structure is critical for predicting how pathogens respond to selective pressures and how shifts in pathogen population structure can lead to disease outbreaks. Evidence from related Apicomplexans such as Plasmodium, Toxoplasma, Cryptosporidium and Theileria suggests that various patterns of population dynamics exist, including but not limited to clonal, oligoclonal, panmictic and epidemic genotypic strain structures. In Babesia bovis, genetic diversity of variable merozoite surface antigen (VMSA) genes has been associated with disease outbreaks, including in previously vaccinated animals. However, the extent of VMSA diversity within a defined population in an endemic area has not been examined. We analyzed genotypic diversity and temporal change of MSA-1, a member of the VMSA family, in individual infected animals within a reservoir host population. Twenty-eight distinct MSA-1 genotypes were identified within the herd. All genotypically distinct MSA-1 sequences clustered into three groups based on sequence similarity. Two thirds of the animals tested changed their dominant MSA-1 genotypes during a 6-month period. Five animals within the population contained multiple genotypes. Interestingly, the predominant genotypes within those five animals also changed over the 6-month sampling period, suggesting ongoing transmission or emergence of variant MSA-1 genotypes within the herd. This study demonstrated an unexpected level of diversity for a single copy gene in a haploid genome, and illustrates the dynamic genotype structure of B. bovis within an individual animal in an endemic region. Co-infection with multiple diverse MSA-1 genotypes provides a basis for more extensive genotypic shifts that characterizes outbreak strains.

Babesia bovis: a comprehensive phylogenetic analysis of plastid-encoded genes supports green algal origin of apicoplasts.

Apicomplexan parasites commonly contain a unique, non-photosynthetic plastid-like organelle termed the apicoplast. Previous analyses of other plastid-containing organisms suggest that apicoplasts were derived from a red algal ancestor. In this report, we present an extensive phylogenetic study of apicoplast origins using multiple previously reported apicoplast sequences as well as several sequences recently reported. Phylogenetic analysis of amino acid sequences was used to determine the evolutionary origin of the organelle. A total of nine plastid genes from 37 species were incorporated in our study. The data strongly support a green algal origin for apicoplasts and Euglenozoan plastids. Further, the nearest green algae lineage to the Apicomplexans is the parasite Helicosporidium, suggesting that apicoplasts may have originated by lateral transfer from green algal parasite lineages. The results also substantiate earlier findings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separate secondary endosymbiotic event likely originating from a red algal lineage.

Phylogenetic analysis of Mexican Babesia bovis isolates using msa and ssrRNA gene sequences.

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2), and msa-2b). Small subunit ribosomal (ssr)RNA gene is subject to evolutive pressure and has been used in phylogenetic studies. To determine the phylogenetic relationship among B. bovis Mexican isolates using different genetic markers, PCR amplicons, corresponding to msa-1, msa-2c, msa-2b, and ssrRNA genes, were cloned and plasmids carrying the corresponding inserts were sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 12 geographically different B. bovis isolates and a reference strain. Overall sequence identities of 47.7%, 72.3%, 87.7%, and 94% were determined for msa-1, msa-2b, msa-2c, and ssrRNA, respectively. A robust phylogenetic tree was obtained with msa-2b sequences. The phylogenetic analysis suggests that Mexican B. bovis isolates group in clades not concordant with the Mexican geography. However, the Mexican isolates group together in an American clade separated from the Australian clade. Sequence heterogeneity in msa-1, msa-2b, and msa-2c coding regions of Mexican B. bovis isolates present in different geographical regions can be a result of either differential evolutive pressure or cattle movement from commercial trade.