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.

Prospective study for the detection of Anaplasma marginale Theiler, 1911 (Rickettsiales: Anaplasmataceae) in Costa Rica.

A prospective study was conducted to assess the dynamics of the infection and host response to Anaplasma marginale in one closed herd in the dry tropical forest of Costa Rica. The study subjects were the dams and their calves born during 1 breeding season (1995-1996). All cows were sampled at 3 month intervals for antibody detection using a competitive ELISA (cELISA) and for antigen detection using PCR/nonradioactive probe assay. All 24 calves born during the study were individually identified at birth and subsequently sampled each month for PCR and cELISA. Ticks were identified from all animals throughout the entire study period. The results from this study confirmed that the cELISA is a reliable assay for identifying new and carrier infections and that carrier infections can exist at levels below that detectable by PCR. In addition, it was demonstrated that calves born in this region will most likely be exposed to Anaplasma within the first 6 months of age.

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.

Seasonal incidence and hemoparasite infection rates of Ixodid ticks (Acari: Ixodidae) detached from cattle in Costa Rica.

To determine the tick species hindering the cattle industry in Costa Rica and to assess infection rates of ticks with three important hemoparasite species, cattle were monitored during a period of six months (October 1992-March 1993). Four farms were located in the dry pacific region of the canton of Tilarán and a fifth farm on the slopes of the Poás volcano in a cool tropical cloud-forest ecosystem. On each farm 3 to 5 animals of 6 to 24 months of age were selected at random. All ticks were removed on a monthly basis from the right half side of each animal, while the site of attachment was recorded. Ticks were counted and differentiated according to species, developmental stage and sex. Moreover, engorged female ticks were assayed for the presence of Babesia bigemina, Babesia bovis and Anaplasma marginale using the polymerase chain reaction (PCR) multiplex system. Two species of ticks, Amblyomma cajennense and Boophilus microplus, were encountered on the cattle in the Tilarán region and one species, B. microplus, was detected in the Poás region. Two to ten times as many ticks were encountered in the Tilarán region than in the Poás region, which is in accordance with a stable enzootic protozoan disease situation in the former region and an unstable epizootic situation in the latter region. Nymphal and adult stages of both tick species were present in largest numbers on the ventral parts of the animals. PCR analysis of entire ticks indicated very high infection rates with hemoparasites of veterinary importance. This was in accordance with high seroprevalence rates in the hosts.

Seasonal incidence and hemoparasite infection rates of Ixodid ticks (Acari: Ixodidae) detached from cattle in Costa Rica

To determine the tick species hindering the cattle industry in Costa Rica and to assess infection rates of ticks with three important hemoparasite species, cattle were monitored during a period of six months (October 1992-March 1993). Four farms were located in the dry pacific region of the canton of Tilar n and a fifth farm on the slopes of the Po s volcano in a cool tropical cloud-forest ecosystem. On each farm 3 to 5 animals of 6 to 24 months of age were selected at random. All ticks were removed on a monthly basis from the right half side of each animal, while the site of attachment was recorded. Ticks were counted and differentiated according to species, developmental stage and sex. Moreover, engorged female ticks were assayed for the presence of Babesia bigemina, Babesia bovis and Anaplasma marginale using the polymerase chain reaction (PCR) multiplex system. Two species of ticks, Amblyomma cajennense and Boophilus microplus, were encountered on the cattle in the Tilarán region and one species, B. microplus, was detected in the Poás region. Two to ten times as many ticks were encountered in the Tilarán region than in the Poás region, which is in accordance with a stable enzootic protozoan disease situation in the former region and an unstable epizootic situation in the latter region. Nymphal and adult stages of both tick species were present in largest numbers on the ventral parts of the animals. PCR analysis of entire ticks indicated very high infection rates with hemoparasites of veterinary importance. This was in accordance with high seroprevalence rates in the hosts

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.

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.

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.

The effect of neutrophils, tumor necrosis factor, and granulocyte macrophage/colony stimulating factor on Babesia bovis and Babesia bigemina in culture.

Bovine neutrophils, human recombinant tumor necrosis factor-alpha (TNF), and bovine recombinant granulocyte macrophage/colony stimulating factor (GM/CSF) were added to microaerophilic cultures of Babesia bovis and Babesia bigemina to determine if those substances could inhibit growth. Incorporation of [3H]hypoxanthine by the Babesia spp. was utilized as an indirect measure of parasite growth. When neutrophils were added to cultures of B. bovis and B. bigemina, the highest percentage inhibition of growth was attained. There was no significant enhancement of neutrophil killing when TNF or GM/CSF or both were added to either Babesia spp. Addition of TNF or GM/CSF or both substances (without neutrophils) resulted in an increase in growth of B. bovis and B. bigemina. For B. bovis, the group that contained neutrophils only and the group that contained neutrophils and TNF resulted in significantly higher growth inhibitions than the treatment group which contained neutrophils and GM/CSF or the group that contained neutrophils, TNF, and GM/CSF. No significant differences in inhibition were observed for the same treatment groups between B. bovis and B. bigemina.

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.

Screening of a Babesia bigemina cDNA library with monoclonal antibodies directed to surface antigens.

A Babesia bigemina cDNA library prepared in lambda ZAP bacteriophage vector was immunoscreened to detect clones expressing surface-exposed epitopes of B. bigemina. A nonradioactive indirect plaque-lift immunoassay was used to detect the positive clones. The primary antibody consisted of a pooled sample of six monoclonal antibodies (mAb) specific for B. bigemina that recognizes various parasite surface antigens of different molecular mass. Screening of approximately 300,000 plaque-forming units from the lambda ZAP cDNA expression library resulted in the identification of five positive clones. The five recombinant clones were immunoscreened individually with each of the six mAb. All five independently obtained clones consisted of lambda ZAP recombinants expressing B. bigemina components recognized by mAb C2F3G3 and B1B3C4. Restriction enzyme digests of rescued recombinant phagemids showed that only four clones contained B. bigemina cDNA. One clone (lambda ZAP Bbi1) contained an insert of approximately 0.6 kBp whereas the other three clones (lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5) carried a cDNA insert of approximately 1.7 kBp. Immunoblotting of protein extracts from recombinants lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5 with mAb C2F3G3 and B1B3C4 demonstrated the expression of a recombinant B. bigemina polypeptide of 55 kDa in E. coli.

Babesia bigemina: quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe.

Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia bigemina-specific DNA probe. DNA from progeny of female Boophilus microplus infected with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3+) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microplus demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately 10(6) infective forms. This number of infective forms is suitable for isolation and further characterization.

Absorption of an alternate source of immunoglobulin in pups.

Newborn pups from 4 large litters were alloted to 6 groups to determine effect of time and route of administration on absorption of an alternate source of immunoglobulin. Selective absorption of specific classes of immunoglobulins was also investigated. The alternate source of immunoglobulin consisted of pooled serum that was administered either PO or SC. Control groups were either left with the dam (group C1) or fed milk replacer (group C2). Blood samples were collected from pups at birth and 24 hours. Immunoglobulin (IgA, IgG, IgM) concentrations were determined by use of radial immunodiffusion on samples of pooled serum, colostrum, and pups' serum (birth and 24 hours). Serum IgA concentration was less than the sensitivity of the procedure and was not included in the statistical analysis. Pups fed 8 ml of pooled serum at birth and 12 hours later (group T1) absorbed more (P less than 0.05) IgG and IgM than did group-C2 pups, but less (P less than 0.05) than did group-C1 pups. Pups fed 8 ml of pooled serum at 12 hours only had significant (P less than 0.05) increase of IgG concentration, but no absorption of IgM (P greater than 0.05) at 24 hours, compared with control pups (group C2). Pups administered 8 ml of pooled serum SC at birth (group SC1) had similar (P greater than 0.05) absorption of IgG and higher (P less than 0.05) absorption of IgM than did pups of group T1.(ABSTRACT TRUNCATED AT 250 WORDS)