Biotin-labeled DNA probe in a PCR-based assay increases detection sensitivity for the equine hemoparasite Babesia caballi.

A DNA probe from Babesia caballi (Bc1) was selected by antibody screening of a genomic library. The Bc1 probe hybridized specifically to B. caballi genomic DNA. A polymerase-chain-reaction-based assay for B. caballi DNA was developed from primers deduced from the probe nucleotide sequence. An amplified product of 1.6 kb was detected from as little as 500 fg B. caballi template DNA. Sensitivity increased 1000-fold when the biotin-labeled Bc1 probe was hybridized to the amplicons in a Southern blot.

Monitoring Babesia bovis infections in cattle by using PCR-based tests.

The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.

Babesia equi erythrocytic stage continuously cultured in an enriched medium.

Babesia equi was continuously cultured through 90 passages in an enriched chemically defined basal medium (HL-1) supplemented with 20% fetal bovine serum and serum replacement factors, including lipid-rich bovine serum albumin, bovine insulin, and human transferrin. Cryopreservation and subsequent recovery of B. equi were easily achieved. Inoculation of a splenectomized and an intact horse with cultured infected erythrocytes resulted in parasitemias and B. equi in vitro reisolation from both animals. In vitro forms of the parasite resembled in vivo forms. After establishment, parasitemias of 10-15% were commonly observed.

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.

Evaluation of agar gel immunodiffusion and indirect fluorescent antibody assays as supplemental tests for dourine in equids.

The agar gel immunodiffusion (AGID) and indirect fluorescent antibody (IFA) assays were evaluated as supplemental tests to the complement-fixation (CF) test, the official US importation certification test for dourine in equids. The American stabilate (n = 10 animals) or the Canadian stabilate (n = 6 animals) of Trypanosoma equiperdum cultured in rat blood was administered by catheterization and infusion in the urogenital tract of 16 equids. To assess parasitemia and serologic responses by use of the CF, AGID, and IFA tests, a total of 787 serum and blood samples were obtained from equids before exposure and 3 times a week after exposure to T equiperdum. Results of the IFA and AGID tests were compared with the CF test results. The disease was diagnosed earlier by the IFA test than by the AGID test, regardless of antigen preparation or exposure group. The mean number of days between exposure and positive result by the CF and IFA tests was the same when either homologous or heterologous antigen was used in the IFA test. In general, the IFA test was more sensitive than the AGID test in diagnosing dourine, regardless of the antigen preparation used in the test or exposure group. Differences in test specificity were observed among both groups of exposed equids when either antigen was used (P < 0.05). The AGID test, using the American antigen, was more specific than the IFA test for sera from both groups of equids. When the Canadian antigen was used, the IFA test was a more specific test than the AGID test (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Culture confirmation of the carrier status of Babesia caballi-infected horses.

Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.

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.

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.