Sequence conservation of the 12D3 gene in Mexican isolates of Babesia bovis.

The 12D3 antigen present in Babesia bovis has been evaluated as a recombinant vaccine candidate and the 12d3 coding sequence has been reported for an Australian and an USA (Texas) isolate of B. bovis. However, no approach has been conducted to perform analysis of 12d3 sequence conservation on a larger number of B. bovis isolates. This could provide important information to determine whether a recombinant vaccine containing this antigen could be widely used. This study reports the cloning and sequencing analysis of the 12d3 coding region in 20 different B. bovis isolates collected from various geographical regions in the tropics and subtropics of Mexico. Comparative analysis of the consensus nucleotide sequences obtained for each isolate revealed a high degree of conservation (94-99% sequence identity) among the 12d3 alleles present in the Mexican isolates when compared with the 12d3 ORF sequences from the Texan (T2Bo) B. bovis isolate. Similarly, BLASTX sequence homology search showed a high percent identity (93-99%) of the deduced amino acid 12D3 sequence as compared with the T2Bo isolate sequence. The high level of sequence conservation in 12d3 among the 20 B. bovis isolates collected from geographically distant locations in Mexico suggests that there exists a minimal bovine-host immunological pressure which could be translated into antigenic diversity or variation, and most probably this is reflected in the non-inmunodominant characteristic of the 12D3 antigen as it has been previously described in the literature. 12D3 antigen can be considered as a viable candidate for inclusion in a recombinant vaccine for cattle babesiosis caused by B. bovis in Mexico.

Immunization of Bos taurus steers with Babesia bovis recombinant antigens MSA-1, MSA-2c and 12D3.

The purpose of this research was to evaluate the recombinant proteins MSA-1, MSA-2c and 12D3 as a combined immunogen for cattle. Fifteen steers were randomly assigned into three groups of five animals each (I, II and III). On day 0, cattle in group I were injected with 50 microg each of rMSA-1, rMSA-2c and r12D3 with the adjuvant Montanide 75; cattle in Group II received adjuvant-PBS, and Group III were untreated controls. On day 14, cattle in Group I received a second injection of the three recombinant proteins in adjuvant and cattle in Group II again received adjuvant alone. On day 28, all groups of cattle were challenged with a field strain of Babesia bovis. After challenge, the experimental cattle were clinically and serologically monitored. Three of the five steers immunized with the combined recombinant B. bovis proteins seroconverted on day 14 post-immunization (P.I.) and the maximum titre was 1 : 1600. All five immunized steers presented strong seropositivity to B. bovis antigens at day 21 P.I. The prepatent periods of vaccinated cattle were delayed until day 10 post-challenge exposure versus 8 and 7 days in Groups II and III, respectively. Cattle in all groups had fever above 41 degrees C; the reduction in packed cell volume was not significantly different (P > 0.05) in vaccinated group I compared with Groups II and III (29% versus 26% and 31%, respectively). Treatment was required for one steer in the control group. During the period of the study, the weight of cattle in Groups I and II increased an average of 9 and 7 kg, whereas the weight of the control cattle was reduced on average 4 kg. Immunization with rMSA-1-rMSA-2c-r12D3 proteins was not sufficient to prevent clinical symptoms against challenge, but the immunologic response was sufficient to protect steers against a mild virulent strain of B. bovis.

Validation of an attenuated live vaccine against babesiosis in native cattle in an endemic area.

The objective of this study was to evaluate in native cattle the use of an in vitro derived attenuated live vaccine (Babesia bovis-Babesia bigemina). Three commercial farms located in a tropical region in Chiapas State, Mexico were included. For each ranch, 40 animals were selected as negative to Babesia spp. by using an immunofluorescent antibody test (IFAT) and PCR. Animals were distributed in four groups with 10 animals each: (i) <9 months, (ii) 9-18, (iii) 18-36 and (iv) >36 months old. From each group, two subgroups were formed with five animals each; one subgroup was vaccinated and the other served as control without vaccination. Monitoring and sampling were carried out initially at vaccination (day 0), at day 7 and then every 4 weeks for 12 months. During the study rectal temperature ( degrees C), packed cell volume (Ht %) and percentage of erythrocytes parasitized were registered, furthermore IFAT and PCR were performed. Prevalence rate at the beginning of the study was 83% by IFAT. During the survey, 26 non-vaccinated of the 120 selected animals (43%) showed clinical symptoms of babesiosis, confirmed by stained smears versus only four (6.6%) of the vaccinated ones. All Babesia-affected animals required specific treatment. Vaccinated cattle showed titres of up to 1 : 1840 and 1 : 1027 for B. bovis and B. bigemina, respectively by IFAT. Protection conferred by vaccination was about 93%. We propose that this vaccine should not only be used in cattle coming from babesiosis free zones, but also in native cattle kept in hyperendemic areas.

Advances in the development of molecular tools for the control of bovine babesiosis in Mexico.

The severe negative impact that bovine babesiosis has in the Mexican cattle industry has not been ameliorated basically due to the lack of safe and effective commercially available vaccines and sensitive and reliable diagnostic tests. In recent years, the Bovine Babesiosis Laboratory at the National Center for Disciplinary Research in Veterinary Parasitology-INIFAP in Morelos State, Mexico has been directing efforts towards three main research areas: (1) The development of in vitro culture-derived, improved and safer live vaccines. This has been done in two ways: using gamma-irradiated bovine serum and erythrocytes for the in vitro culture of vaccine strains, which reduces the risk of contaminating pathogens, and improving the immune response, by the addition of L. casei, a strong stimulant of the innate immune system. (2) The study of antigens considered as vaccine candidates with the goal of developing a recombinant vaccine that suits the country's needs. Knowing their degree of conservation or variation in Mexican isolates, their phylogenetic relationship and their protective, immuno-stimulatory properties, are first steps towards that goal. (3) The development of new tools for diagnosis, detection and discrimination of bovine babesiosis is the third area. Developing variants of ELISA, which are more reliable than the currently used IFAT, are a priority, and finally, taking advantage of the genomes of Babesia bigemina, and B. bovis, we are identifying genes than allow us to discriminate isolates using molecular tools.

Cloned lines of Babesia bovis differ in their ability to induce cerebral babesiosis in cattle.

Clones of a Babesia bovis isolate known to cause particularly severe cerebral babesiosis were tested for virulence phenotype by inoculation of cattle. Clones were selected for phenotyping by two criteria - rate of growth in culture and hybridization of a virulence-related probe to Southern blots. Largely on the basis of associated mortality, B. bovis clones were judged to vary in their pathogenic potential.

Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review.

Ticks play an important role in human and veterinary medicine, in particular due to their ability to transmit a wide spectrum of pathogenic micro-organisms of protozoal, rickettsial, bacterial and viral origin. Pathogens in ticks can be identified by conventional methods such as indirect immunofluorescence, isolation in cell culture or by using histological staining techniques. However, the advent of the polymerase chain reaction (PCR) has resulted in tremendous improvements in the specific and sensitive detection of pathogen DNA in ticks. In this paper, literature on DNA extraction methods, PCR protocols, primers and probes, which are in use for the successful detection and identification of pathogens in ticks, are critically reviewed. Some recommendations are also given towards the end of this review.

Bovine babesiosis and anaplasmosis follow-up on cattle relocated in an endemic area for hemoparasitic diseases.

A multiplex PCR/DNA probe assay was used to monitor Babesia bovis, B. bigemina and Anaplasma marginale infection in cattle introduced to a Boophilus microplus-infested area in Veracruz, Mexico. Eight intact, 18-month-old, cross-bred beef cattle (four naive, Group A; four Babesia species--premunized, Group B) were immediately exposed to ticks after arrival and were clinically monitored from day 6 to day 98 post-exposure (PE) to ticks. Blood sample analysis for DNA detection by the MPCR/DNA probe assay showed that Group A animals were infected with B. bovis from day 11 up to day 22 PE, requiring treatment on days 17-20. Group B animals were detected positive to B. bovis on days 17-20, did not require treatment and remained persistently infected from days 70 to 84 PE. Treatment of Group A animals delayed the infection with B. bigemina. These animals became positive to the parasite on days 63-77 PE. In contrast, Group B animals (untreated) showed B. bigemina infection on days 21-26 and 63-84 PE. One animal was positive for A. marginale infection on days 63-66 PE, the rest of the animals became so on days 80-98 PE. All infected animals required treatment with oxytetracycline. Monitoring the triple hemoparasite infection with the MPCR/DNA probe assay provided important epidemiological information. Thus, precautionary measures can be established when cattle are moved to a babesiosis/anaplasmosis risk area.

Use of a duplex PCR/DNA probe assay to monitor Babesia bovis and Babesia bigemina in cattle during a vaccination trial.

A Duplex Polymerase Chain Reaction (DPCR)/DNA probe assay was used to detect Babesia bovis and B. bigemina DNA in cattle undergoing immunization trials. Blood samples were collected from 15 non-splenectomized, 1-2 years old bulls, inoculated with 1 x 10(7) each of culture-derived B. bovis- and B. bigemina-infected erythrocytes. 15 bulls inoculated with normal erythrocytes served as a control group. All cattle were field exposed to tick-transmitted Babesia 21 days (20 animals, Group I) and 60 days (10 animals, Group II) post-inoculation (PI). After immunization, the DPCR/DNA probe assay detected B. bigemina and B. bovis parasite DNA in all inoculated animals from days 4 to 14 PI. At challenge, B. bovis DNA was detected in all control animals as early as day 8 (Group I), or day 11 (Group II) post-introduction to a tick-infested area. The immunized bulls showed B. bovis positive PCR/DNA probe signals from day 0 (Group II) and day 8 (group I), up to day 32 post-exposure to ticks. Positive B. bigemina signals were detected from day 0 (Group I) and day 8 (Group II), up to day 36 post-exposure to ticks. During challenge, it was not possible to clearly define whether the PCR/DNA probe signals detected in the blood from immunized cattle were a result of amplified DNA from the culture-derived parasites, from the tick-transmitted parasites, or both.

Comparative sensitivity of two tests for the diagnosis of multiple hemoparasite infection of cattle.

The purpose of this study was to evaluate the efficacy of light microscopy (LM) examination of blood smears and a multiplex polymerase chain reaction (MPCR) assay, in terms of their ability to detect cattle experimentally infected with Babesia bovis, Babesia bigemina, and Anaplasma marginale. Blood samples were collected from 32 intact, 1-2 year old, Holstein bulls, previous to and after simultaneous inoculation of culture-derived or field isolates of B. bovis- and B. bigemina-infected erythrocytes. To establish the triple hemoparasite infection, 16 of the bulls were also inoculated with a calf-derived isolate of A. marginale. The results showed that both tests had 100% specificity. In contrast, the sensitivities of the MPCR assay against the LM test were 93.5% and 70.9%; 96.7% and 100%; and 93.8% and 93.8% for B. bovis, B. bigemina, and A. marginale infection, respectively. The advantages and disadvantages of the MPCR assay to differentially diagnose cattle with multiple hemoparasite infection are discussed.

Use of polymerase chain reaction in bovine babesiosis research.

The polymerase chain reaction (PCR) in babesiosis research was originally developed to detect Babesia bovis or Babesia bigemina in blood samples containing infected erythrocytes. These preliminary studies led to development of a sensitive PCR/DNA probe assay to detect the following hemoparasites: B. bigemina and B. bovis in a single sample. This modified procedure, referred to as a duplex PCR/ nonradioactive probe assay, has an analytic sensitivity of 0.00001% for B. bigemina, and 0.00001% infected erythrocytes for B. bovis. This procedure has been modified to detect Babesia DNA in tick tissue and hemolymph. The above procedures can be performed in central laboratories that have access to a thermocycler and quality reagents. Precautions must be observed to prevent cross-contamination of samples. At the present time the procedure has application in epidemiology studies to detect carriers and the species of Babesia in the bovine population. Preliminary studies are in progress by various research groups to utilize this technique in studying the biology of the Babesia protozoans in tick vectors. The applications, advantages, and disadvantages of the technique are presented.

Nucleic acid probes as a diagnostic method for tick-borne hemoparasites of veterinary importance.

An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts of Anaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isotopic detection system, probes have allowed scientists to test for--originally in samples collected from experimentally infected animals and later in samples collected in the field--the presence of hemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures.

Antibody response to a Babesia bigemina rhoptry-associated protein 1 surface-exposed and neutralization-sensitive epitope in immune cattle.

Protective immunity against Babesia bigemina is hypothesized to involve antibodies directed against merozoite surface-exposed epitopes. Levels of antibody against a rhoptry-associated protein 1 (RAP-1) B-lymphocyte epitope, defined by surface-reactive and inhibitory monoclonal antibodies, in immune cattle sera were determined. All cattle produced antibodies to the epitope; however, there was limited correlation between immune protection induced by infection or RAP-1 immunization and the level of antibody to the neutralization-sensitive B-lymphocyte epitope examined.

Bovine babesiosis: a review of recent advances.

Bovine babesiosis, caused by parasites of the genus Babesia, is one of the world's most severe tick-borne problems of cattle in temperate to tropical areas. In the Americas Babesia bovis and B. bigemina are the causative agents, with the former considered to produce the greatest economic impact. The great complexity of the relationships causal agent-vector-host has severely hindered the efforts towards the production of a safe, long-lasting, solid-protection inducing vaccine. Recent important contributions that have encouraged the study of these agents include the development of in vitro cultivation systems, procedures for the isolation of single infected-erythrocytes, density gradient-based centrifugation systems for the isolation and concentration of both infected erythrocytes and merozoites, isozyme detection and differentiation systems that help discriminate between parasite species, and development of DNA-based diagnostics and characterization protocols. Currently, the study of the cellular immune responses against these parasites is taking new endeavors in order to discern the relationship between B cells, T cells, macrophages and their products and parasites leading to the establishment of solid, long-lasting protection. In an attempt to design a rational vaccine, T cell lines and clones are being established, and phagocytosis of infected erythrocytes and their antigens studied to try to pinpoint relevant epitopes.

Polymerase chain reaction-based diagnostic assay to detect cattle chronically infected with Babesia bovis.

From a B. bovis gene sequence coding for a 60 kDa merozoite surface protein previously published, two sets of primers were designed for the Polymerase Chain Reaction (PCR) assay. Primer set BoF/BoR was used to prime Taq Polymerase DNA amplification of a 350 bp fragment of the target B. bovis DNA. Primer set BoFN/BoRN was used to prepare a PCR-synthesized, Digoxigenin-dUTP-labeled probe (291 bp) which would hybridize to a sequence within the PCR-amplified parasite target DNA. PCR amplification of target DNA obtained from in vitro-cultured B. bovis and nucleic acid hybridization of amplified product with the nonradioactive DNA probe showed that a 350 bp fragment could be detected when as little as 10 pg of genomic parasite DNA was utilized in the assay. A fragment of similar size was amplified from genomic DNA from four other B. bovis isolates but not from B. bigemina, Anaplasma marginale, or bovine leukocyte DNA. The PCR product was detected in blood samples containing approximately 3 B. bovis-infected erythrocytes (20 microliters of packed cells with a parasitemia of 0.000001%). By using the PCR/DNA probe assay, 16 out of 20 animals experimentally inoculated with B. bovis were detected positive, whereas no PCR product was observed in bovine blood samples collected from 20 B. bigemina-infected, and 20 uninfected cattle tested. The PCR-DNA probe assay was shown to be sensitive in detecting some cattle with B. bovis-chronic infection. The specificity and high analytical sensitivity of the test provides a valuable tool to apply in conducting epidemiological studies.

Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood.

A highly sensitive polymerase chain reaction (PCR) based method was developed to detect, in the same blood sample, DNA of hemoparasites frequently found together infecting cattle in tropical and subtropical areas. Bovine blood containing equal parasitemias of Babesia bigemina, B. bovis and Anaplasma marginale infected erythrocytes was mixed to standardize the test. Twenty microliters of 10-fold dilutions from the pooled blood sample were resuspended in PCR mixture buffer containing each of the species-specific sets of primers. Group I primers (BiIA/IB, BoF/R and Am9/10) which specifically bind B. bigemina, B. bovis and A. marginale DNA were used to amplify a fragment of DNA from genomic parasite DNA. Group II nested primers (BiIAN/IBN, BoFN/RN and Am11/12) were used to prepare, via incorporation of digoxigenin-11-dUTP by PCR, nonradioactive probes specific for internal sequences present in DNA amplified with Group I primers. Agarose gel electrophoresis and Southern blot hybridization studies showed that by using Group I primers, DNA fragments of 278 bp, 350 bp and 200 bp were specifically amplified in samples containing B. bigemina, B. bovis and A. marginale DNA, respectively. The analytical sensitivity of the multiple PCR test, as evaluated by nucleic acid hybridization with the nonradioactive probe, was 0.00001%, 0.00001% and 0.0001% infected erythrocytes for B. bigemina, B. bovis and A. marginale, respectively. Blood collected from cattle previously inoculated with B. bovis (4 years), A. marginale (2 years) and B. bigemina (1 year) was demonstrated to be latently infected by using the Multiplex PCR test.

Use of multiplex polymerase chain reaction-based assay to conduct epidemiological studies on bovine hemoparasites in Mexico.

A study was conducted to test the applicability of a Polymerase Chain Reaction (PCR)-based approach for the simultaneous detection of the bovine hemoparasites Babesia bigemina, B. bovis and Anaplasma marginale. Bovine blood samples from cattle ranches of a previously determined enzootic zone in the Yucatan Peninsula of Mexico, were collected from peripheral blood and processed for PCR analysis. Blood samples were subjected to DNA amplification by placing an aliquot in a reaction tube containing oligonucleotide primers specific for DNA of each hemoparasite species. The PCR products were detected by Dot-Blot nucleic acid hybridization utilizing nonradioactive, species-specific, digoxigenin PCR-labeled DNA probes. Four hundred twenty one field samples analyzed by the multiplex PCR-DNA probe assay showed 66.7%, 60.1% and 59.6% prevalence rates for B. bigemina, B. bovis and A. marginale, respectively. The multiplex PCR analysis showed that animals with single, double or triple infection could be detected with the parasite specific DNA probes. The procedure is proposed as a valuable tool for the epidemiological analysis in regions where the hemoparasite species are concurrently infecting cattle.

Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification.

A SpeI-AvaI fragment (0.3 kbp) from pBbi16 (a pBR322 derivative containing a 6.3-kbp Babesia bigemina DNA insert) was subcloned into the pBluescript phagemid vector and was sequenced by the dideoxy-mediated chain termination method. Two sets of primers were designed for the polymerase chain reaction (PCR) assay. Primer set IA-IB was used to amplify a 278-bp DNA fragment, and primer set IAN-IBN was used to prepare a probe directed to a site within the PCR-amplified target DNA. Digoxigenin-dUTP was incorporated into the probe during the amplification reaction. PCR amplification of target DNA obtained from in vitro-cultured B. bigemina and nucleic acid hybridization of amplified product with the nonradioactive DNA probe showed that a 278-bp fragment could be detected when as little as 100 fg of parasite genomic DNA was used in the assay. A fragment of similar size was amplified from genomic DNAs from several B. bigemina isolates but not from DNAs from Babesia bovis, Anaplasma marginale, or six species of bacteria or bovine leukocytes. Similarly, the PCR product could be detected in DNA samples purified from 200 microliters of blood with a parasitemia of as low as 1 in 10(8) cells and which contained an estimated 30 B. bigemina-infected erythrocytes. By a direct PCR method, B. bigemina DNA was amplified from 20 microliters of packed erythrocytes with a calculated parasitemia of 1 in 10(9) cells. With the analytical sensitivity level of the PCR-DNA probe assay, six cattle with inapparent, 11-month chronic B. bigemina infection were found to be positive. No PCR product was observed in bovine blood samples collected from a splenectomized, A. marginale-infected bovine, a 4-year chronic B. bovis-infected animal, or 20 uninfected cattle from Missouri which were subjected to amplification. The PCR-DNA probe assay was shown to be sensitive in detecting latently infected cattle. The specificity and high analytical sensitivity of the test provide valuable tools for performing large-scale epidemiological studies.

Antibody response to Babesia bigemina infection in calves measured by ELISA and immunoblotting techniques.

To measure the antibody response to Babesia bigemina with an ELISA test, three groups of cattle were experimentally infected with two isolates of the parasite. It was possible to demonstrate specific antibody binding directed against the parasite as early as the 7 days postinfection (PI). The highest level of antibody was obtained around day 1 to 23 and remained detectable for 260 days. Challenge of the animals 260 days PI with a tick-induced B. bigemina infection depicted that homologous strain-challenged calves did not show an increase of IgG antibody levels, where as those challenged with the heterologous isolate did. In the latter groups the resulting level of antibodies was even higher than after the primary infection. The immunoblotting technique showed that the antibody response is probably directed against groups of B. bigemina components with a relative mobilities of 68-64 kDa, 62-54 kDa and 52-42 kDa, which appear to be major components of the protozoa. By observing the cross-reacting antigenicity among seven B. bigemina isolates, it was demonstrated that these components are not isolate-restricted.

Evaluation of a DNA-based probe for the detection of cattle experimentally infected with Babesia bigemina.

A digoxigenin-labeled probe was used for hybridization to various preparations of Babesia bigemina-infected erythrocyte extracts. Dot blot hybridization and immunological detection of DNA hybrids revealed that the probe was specific for B. bigemina DNA because it did not hybridize to the DNA of B. bovis, a closely related species. Studies of sensitivity showed that the probe would bind to as little as 1 ng of B. bigemina DNA, but not to 1 microgram of the B. bovis DNA. The probe reacted with equal intensity against seven B. bigemina isolates from different geographic areas. The lowest percentage of B. bigemina-infected erythrocytes detected was 0.001%, a level of parasitemia not usually detected with the light microscope. Six intact, mixed-breed steers, approximately 3 years old, were inoculated with a blood stabilate containing B. bigemina-infected erythrocytes. Blood samples collected from day -1 to day 86 postinoculation (PI) and prepared for DNA extraction were analyzed in a dot blot hybridization assay using a nonradioactive DNA probe. Hybridization reaction (HR) signals were compared to results obtained by light microscopy (LM) examination of Giemsa-stained blood smears and to antibody titers of serum samples assayed with the complement fixation test (CFT) and indirect fluorescent antibody test (IFAT). Four of six inoculated steers became infected with B. bigemina as assessed by LM. The parasitemias were low (less than 0.01-0.05) at day 10 PI. Only three steers were serologically positive by CFT (titer 1:40-1:160) and IFAT (1:1280). All four infected steers had positive HR signals in the dot blot assay. The HR signals were observed from day 10 to day 77 PI and were usually correlated with the presence of parasites in blood as observed by LM. The HR signals varied in intensity for different blood samples from the experimental animals and with day of blood sample collection. Although the signal intensity did not correlate with the parasitemia level estimated by LM, the nucleic acid hybridization assay was more sensitive than LM, CFT, or IFAT for the detection of B. bigemina-infected cattle.