Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.     

Vaccination of older Bos taurus bulls against bovine babesiosis

Two separate groups of Bos taurus bulls, one of 106 and the second of 27 animals, imported to Israel from areas free of Babesia bovis and Babesia bigemina, were vaccinated against babesiosis with a bivalent live attenuated vaccine. In light of the fact that routine vaccination is recommended at the weaning age, these bulls--of highly susceptible breeds--were kept under close surveillance to prevent losses that might be caused by severe clinical reactions to their vaccination at the age of 16-18 months. Seven days after vaccination, about one-third of the 106 bulls in the first group developed clinical signs of B. bigemina infection, which peaked at day 9, and then diminished from day 11, when the patent period known for B. bovis infection was observed. Because of the severe clinical responses a total of 36% of the bulls required babesicidal treatment. Despite the treatment Babesia were not sterilized: 33 and 68% of the animals remained PCR positive for B. bigemina and B. bovis, respectively. To mitigate the severe responses to vaccination, the 27 bulls of the second group were vaccinated in two-steps: they were inoculated initially with avirulent culture-derived parasites and then vaccinated with the conventional donor-derived vaccine a month later. None of the bulls in the latter group developed clinical babesiosis, all were serologically positive to B. bigemina, and 67% showed seroconversion to B. bovis. In light of the experience described here, it is suggested that sensitive older cattle be vaccinated against babesiosis by priming them with avirulent in vitro-cultured parasites and then inoculating them with the conventional donor-derived vaccines.     

Live vaccines against bovine babesiosis

Bovine babesiosis is an important tick-borne disease caused by Babesia bovis, B. bigemina and B. divergens. The first steps taken in the development of an effective vaccination strategy against bovine babesiosis followed the observations that animals, recovered from natural infection with Babesia were strongly protected against subsequent challenge. Further investigation indicated that the use of donor blood from recovered animals to infect recipient animals did not produce the severe form of the disease. The past century has seen a refinement of this original carrier-donor system to one using attenuated less virulent strains with standardized doses of known parasite concentration to ensure reliability. With the implementation of good manufacturing practices further changes were necessary in the production of these vaccines, such as freezing for long-term storage to allow sufficient time for pre-release safety and effectivity testing. Regardless of these improvements the vaccines are not without problems and breakdowns and breakthroughs occur from time to time. Despite considerable research efforts into the development of alternative more consumer friendly vaccines, none is immediately forthcoming and the live attenuated babesiosis vaccines are still used in many countries.     

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.     

Babesia divergens (Phylum Apicomplexa) in vitro growth in the presence of calf serum

Resistance to severe babesiosis in young calves has frequently been ascribed to an unknown serum factor(s) which inhibits growth of Babesia bovis in vitro. Our experiments show that young calf sera are as suitable as adult bovine sera for the in vitro culture of Babesia divergens, indicating that in this species at least inverse age resistance is due to alternative mechanisms. The suitability of commercial foetal calf sera for B. divergens cultures seems highly variable.     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

Current status of bovine haemoparasitic diseases in Martinique (French West Indies)]

A serological survey using indirect immunofluorescence (IFAT) for bovine babesiosis (Babesia bovis and B. bigemina) and card test for anaplasmosis, indicates that these haemoparasites are widespread in Martinique. The high prevalences (62% for B. bovis, 52% for B. bigemina and 43% for Anaplasma marginale) lead to the hypothesis of an unstable epizootic situation for these three haemoparasitic diseases. However, the number of smears examined was too low to evaluate their clinical incidence. Both the American and Cuban card tests gave similar results in the detection of antibodies to A. marginale. Theileria mutans is described for the first time in Martinique. Trypanosomosis (Trypanosoma vivax) has disappeared from Martinique, on clinical and serological evidence.     

Detection and molecular characterization of a novel large Babesia species in a dog

Babesia canis has generally been considered the only large Babesia to infect dogs. Here we describe the molecular characterization of a large Babesia species that was detected in the blood and bone marrow of a dog with clinical and hematological abnormalities consistent with babesiosis. Analysis of the 18S rRNA genes revealed a unique sequence that shared 93.9% sequence identity with B. bigemina and 93.5% sequence identity with B. caballi, compared to 91.2-91.6% identity with B. canis canis, B. c. vogeli, and B. c. rossi. Cross-reactive antibodies against B. canis, B. gibsoni (Asian genotype), or B. gibsoni (California genotype) antigens were not detected in acute or convalescent serum samples. The dog was treated with imidocarb diproprionate, which resulted in the resolution of clinical signs, and subsequently Babesia DNA was not detectable by PCR in post-treatment samples. The organism described in this report represents a genetically unique large Babesia sp. and is the eighth genetically distinct piroplasm capable of infecting the domestic dog.     

Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method

Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with considerable worldwide economic, medical, and veterinary impact. Bovine babesiosis and other TBDs were considered responsible for 50% of the deaths of cattle that occurred in Mozambique in the first year after importation from neighbouring countries. Here, we present the detection of Babesia bigemina and Babesia bovis in cattle from Mozambique using two distinct PCR methods. For this study, blood samples were collected in one farm located near Maputo city. The DNA samples were analyzed using a previously described nested PCR and a novel hot-start PCR method. Primers were selected for the hot-start PCR based on the putative gene of an undescribed aspartic protease named babesipsin, present in both B. bovis and B. bigemina. The combination of hot-start polymerase and long primers (29-31 bp) were in this study determinant for the successful amplification and detection in only one PCR. With a seminested approach the sensitivity was further increased. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. A total of 117 field samples were tested by seminested hot-start PCR, and 104 were positive for B. bigemina (90%), 97 were positive for B. bovis (82%), 86 were mixed infections (52%) and only 2 were negative for both Babesia species (1.7%). The results confirm that this area of Mozambique is endemic for babesiosis, and that this TBD should be regarded as a threat for imported cattle.     

Progression towards endemic stability to bovine babesiosis in cattle introduced onto a game ranch

An opportunity to study progression toward endemic stability to Babesia bigemina arose when cattle were reintroduced onto a game ranch in 1999 after an absence of three years. The study was conducted between August 2000 and June 2001. The unvaccinated breeding cows were sampled only once. Calves born during October 1999 were initially vaccinated against B. bigemina and Babesia bovis at the age of 4 months and were then bled at 10, 17 and 20 months of age. Calves born during 2000 were bled at 7 and 8 months of age. Sera were collected from all the cattle sampled and later tested for antibodies against B. bigemina and B. bovis using the indirect fluorescent antibody (IFA) test. Although endemic stability to B. bigemina had not been achieved at Nooitgedacht 2 years after resumption of cattle ranching, the high seroprevalence in the unvaccinated 8-month-old calves suggested that the situation was approaching stability and that calf vaccination against bovine babesiosis was not required. Tick control should therefore be restricted to prevent excessive tick worry. Only vaccinated cattle were positive to B. bovis and it was concluded that the parasite was absent from the ranch.     

Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B bigemina transmitted by Boophilus microplus

OBJECTIVE: To assess the effect of breed of cattle on the transmission rates of and innate resistance to Babesia bovis and B bigemina parasites transmitted by Boophilus microplus ticks. DESIGN: Groups of 56 purebred B indicus and 52 B indicus cross B taurus (50%, F1 generation) steers were placed in a paddock seeded with and also naturally infested with B microplus which were the progeny of females ticks fed on B taurus cattle specifically infected with a virulent isolate of B bovis. The cattle were placed in the infested paddock 50 days after seeding had started. PROCEDURE: Cattle were inspected from horseback daily for 50 days. Clinically ill cattle were brought to yards and assessed by monitoring fever, depression of packed-cell volume, parasitaemia and severity of clinical signs. Any animals that met preset criteria were treated for babesiosis. Blood samples were collected from all cattle on day 28, 35 and 42 after exposure and antibodies to Babesia spp and packed cell volume measured. RESULTS: All steers, except for one crossbred, seroconverted to B bovis and B bigemina by day 35 and 75% of the crossbred steers showed a maximum depression in packed cell volume of more than 15% due to infection with Babesia spp compared with only 36% of the B indicus group. Ten of the 52 crossbreds and 1 of the 56 B indicus steers showed severe clinical signs. Two of the crossbreds required treatment of which one died 2 weeks after initial treatment. CONCLUSIONS: Pure-bred B indicus cattle have a high degree of resistance to babesiosis, but crossbred cattle are sufficiently susceptible to warrant the use of preventive measures such as vaccination. Transmission rates of B bovis and B bigemina to B indicus and crossbred cattle previously unexposed to B microplus were the same.     

Attainment of endemic stability to Babesia bigemina in cattle on a South African ranch where non-intensive tick control was applied

The seroprevalence of Babesia bigemina and Babesia bovis antibodies in non-vaccinated cattle was monitored on a South African ranch. The main objective was to assess the endemic stability to bovine babesiosis in cattle maintained under relaxed tick-control measures. Cattle were bled at the age of 7, 8, 10, 17, 20 and 30-120 months and the sera tested for the presence of antibodies using the indirect fluorescent antibody (IFA) test. None of the animals were positive to B. bovis. Seroprevalence of B. bigemina antibodies was 46, 70, 90, 92, 54 and 82% in the various age classes, respectively. Endemic stability was therefore reached by the time the calves were 9 months old. The high seroprevalence of B. bigemina was probably due to the high vector tick population on the ranch, which would have encouraged frequent transmission of B. bigemina. An endemically stable situation to B. bigemina could therefore be achieved merely by adopting a tick-control method that allows a reasonable number of ticks on cattle rather than relying entirely on intensive tick control and vaccination.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and Babesia bigemina and is characterized by significant morbidity and mortality worldwide. The disease is widespread in the northeastern region of Thailand, where an increasingly large part of the livestock is composed of water buffaloes. The present study was therefore conducted to investigate the epidemiological distribution of B. bovis and B. bigemina in water buffaloes in the northeastern region of Thailand. A total of 305 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the nested PCR (nPCR) assay, ELISA, and IFAT techniques. The overall prevalence of B. bovis and B. bigemina was 11.2% and 3.6% by nPCR, 14.7% and 5.9% by ELISA, and 16.8% and 5.6% by IFAT, respectively. The high concordance between the molecular and the serological detection tests revealed the specificity and sensitivity of the diagnostic assays used for the detection of infection as well as the endemic stability status of the parasites in the surveyed areas. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location but not gender. Our data provide valuable information regarding the epidemiology of B. bovis and B. bigemina infection in water buffaloes in the northeastern region of Thailand which will likely be very beneficial for management and control programs of this disease.     

Seroepidemiological studies of bovine babesiosis on Pemba Island, Tanzania.

Serological evidence of infection with Babesia bovis and Babesia bigemina at a number of sites in Pemba was obtained using an enzyme-linked immunosorbent assay (ELISA) capable of detecting the appropriate parasite-specific antibody. Overall, 96% of animals were found to be positive for B. bovis, 88% were positive for B. bigemina and 88% were positive for both Babesia species. Antibody to B. bovis and B. bigemina was detected early in life in a number of calves born on Pemba, and was considered to be of maternal origin. The amount of maternal antibody in the serum of individual animals fell throughout the first 3 months of life. Later in life, antibody levels increased, probably in response to Babesia infection from natural tick challenge. These results suggest that infection with both Babesia parasites is widespread throughout Pemba and that both parasites probably exist in an enzootically stable situation.     

Development of two immunochromatographic tests for the serodiagnosis of bovine babesiosis

In this study, we developed two immunochromatographic tests (ICTs), which are nitrocellulose membrane-based immunoassays for the convenient and rapid serodiagnosis of bovine babesiosis caused by Babesia bovis (BoICT) and Babesia bigemina (BiICT). The efficacy of two ICTs was evaluated using 13 positive sera from experimentally infected cattle with B. bovis or B. bigemina. Clear results showed that the BoICT and ELISA detected antibodies in sera collected from 14 to 93 days post-infection, while BiICT and ELISA detected from 13 to 274 days post-infection. In additon, non-infected cattle, Neospora caninum, and Cryptosporidium parvum were negative in two ICTs. To evaluate the field utility of the ICTs, we tested 186 field bovine sera collected from cattle living in Yanbian (China) and Mato Grosso do Sul (Brazil). The results of ICTs were compared to those of classical serodiagnostic methods, enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence assay (IFAT). The overall concordances of BoICT were determined as 92.5 and 90.3% when the results of ELISA and IFAT were set as the reference standards, respectively. In contrast, those of BiICT showed 96.8 and 92.5% relative to the results of standard ELISA and IFAT, respectively. Conventional and rapid diagnosic devices for bovine babesiosis may provide a valuable tool in clinical and field applications.     

An update of the isolation and characterization of culture-derived soluble antigens of Babesia bovis

In this paper recent progress concerning the identification of soluble antigens from cultures of Babesia bovis parasites is reviewed. Soluble antigens present in the supernatant of B. bovis cultures have been shown to be efficient immunogens for induction of protective immunity against bovine babesiosis. Immunochemical analysis of culture supernatants has demonstrated that at least 3 parasite antigens are released in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that at least 2 of these antigens have molecular weights within the range of 37 000-40 000 daltons. Crossed immunoelectrophoretic studies have further revealed an antigenic spectrum consisting of 1 major and 2 minor antigens with mobilities in the albumin and alpha 1 regions. Within the infected erythrocyte, 2 antigens have been localized on or near the erythrocytic membrane, while the third antigen appears to be directly associated with the parasite.     

The inability of a South African Babesia bovis vaccine strain to infect Boophilus microplus

A strain of Babesia bovis that had been attenuated by rapid syringe passage through a series of 23 splenectomized calves was unable to infect its vector Boophilus microplus. An attempt to transmit the attenuated Australian Babesia bigemina G strain with a South African strain of B. microplus was likewise unsuccessful. The epidemiological implication of these observations in terms of babesiosis control is discussed.     

Infectivity of cryopreserved Babesia bovis, Babesia bigemina and Anaplasma centrale for cattle after thawing, dilution and incubation at 30 degrees C

Blood containing either Babesia bovis, Babesia bigemina or Anaplasma centrale was mixed with an equal volume of 3 M glycerol in phosphate-buffered saline with or without glucose and then stored in liquid nitrogen for 2-30 days. After being thawed, the parasitized blood was subjected to various procedures, including dilution up to 1000-fold followed by incubation at 30 or 4 degrees C for 8 h, before infectivity of the parasites was tested in a total of 70 cattle. The results showed that the blood cryopreserved with glycerol remained highly infective after thawing, despite dilution and incubation for 8 h at 30 degrees C. The results have practical application in the use of frozen, live vaccines against bovine babesiosis and anaplasmosis.     

In silico predicted conserved B-cell epitopes in the merozoite surface antigen-2 family of B. bovis are neutralization sensitive

The merozoite surface antigens MSA-2 of Babesia bovis constitute a family of polymorphic GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes. These are therefore putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-2 antigens of the biologically cloned Mo7 strain are encoded by four tandemly organized genes: msa-2a₁, a₂, b and c, and (ii) at least one allele of each of these genes is present in the Argentine R1A strain with a moderate degree of polymorphism. The present work was aimed at defining neutralization-sensitive B-cell epitopes in the MSA-2 family, that are conserved among different B. bovis geographical isolates. To this end, msa-2a, b and c alleles from different isolates from Argentina, USA and Mexico were amplified by PCR, cloned and sequenced. Bioinformatic analysis by ClustalW alignments and B-cell epitope prediction algorithms performed on these sequences allowed the identification of several regions containing putative conserved B-cell epitopes. Four peptides representing these regions: (KDYKTMVKFCN from msa-2a₁; YYKKHIS, from msa-2b; and THDALKAVKQLIKT and ELLKLLIEA from msa-2c) were chemically synthesized, conjugated to keyhole limpet hemocyanin and used to inoculate mice to obtain immune sera. Anti-peptide antibodies recognized B. bovis merozoite extracts in all cases in ELISA tests. In addition, these sera reacted with the surface of merozoites of an Argentine and a Mexican B. bovis strains in immunofluorescence assays, and sera against two of the selected peptides inhibited invasion of erythrocytes by in vitro cultured merozoites. Taken together, the results show that the peptide sequences selected by bioinformatic analysis represent expressed and geographically conserved B. bovis B-cell epitopes that might be strong candidates for development of subunit vaccines.