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DNA probes for the detection of Babesia caballi E. S. POSNETT and R. E. AMBROSIO* Molecular Biology Section, Veterinary Research Institute, Onderstepoort 0110, South Africa (Received 28 August 1990; revised 6 November 1990; accepted 6 November 1990) SUMMARY
A genomic library of Babesia caballi DNA was constructed in the plasmid vector pUCl 3. The specificity of the clones for B. caballi was established by the lack of hybridization to Babesia equi, Babesia bovis, Babesia bigemina and equine DNA. Two probes, pBCll and pBC191, were isolated that could detect 025 ng and 0125 ng of B. caballi DNA, corresponding to a parasitaemia of 0 1 2 % and 0 0 6 % respectively. pBC191 could detect B. caballi parasites in the blood of an experimentally infected horse as well as in naturally infected horses. Key words: equine babesiosis, Babesia caballi, DNA probe, tandem repeat.
sensitive than CF and does not produce many falsenegative results (Morzaria, Brocklesby & Harradine, 1977). However, this test cannot be standardized, Equine babesiosis is caused by the intra-erythrocytic and reading of results is time-consuming (Tenter & protozoan parasites Babesia equi and Babesia caballi Friedhoff, 1986). It is therefore difficult to detect the (Friedhoff, 1982). The disease is transmitted by ticks of the genera Dermacentor, Hyalomma and Rhi- low parasitaemias associated with B. caballi using these diagnostic procedures. Also, immunodiagpicephalus and is endemic in the tropics and nostic procedures are an indirect way of determining subtropics and also in an area from Northern infection and cannot distinguish between a past and France to the Soviet Union (Tenter & Friedhoff, a present infection. 1986; Friedhoff, 1982). B. caballi is the larger of the two species, and There is thus a need for a more rapid and sensitive development occurs in the vertebrate host exclustest for equine babesiosis. We have reported the ively in the erythrocytes. Red blood cells containing isolation and characterization of repetitive D N A more than two parasites (multiple infections) are rare probes for B. equi (Posnett & Ambrosio, 1989). Here with B. caballi, but do occur with B. equi. In we describe the isolation and characterization of B. caballi infections, usually less than O'l % of eryDNA probes for B. caballi. throcytes are infected compared to between 5 and 2 0 % in the case of B. equi. Demonstration of B. caballi parasites on blood smears is therefore MATERIALS AND METHODS difficult, even in acute cases. Characteristics of the DNA isolation disease include anaemia, fever and, unless treated, death (Purnell, 1981). After infection with B. caballi, B. caballi parasites (Warrenton isolate, Warrenton, animals may remain carriers of the parasites for up to South Africa) were isolated from the blood of 4 years. artificially infected horses. The average parasitaemia was 0-5%. Blood was centrifuged at 2000 £ for As a result of the increasing international trade in 15 min and the buffy coat removed. Centrifugation horses there is a considerable risk that the disease was repeated until the buffy coat was no longer may spread to disease-free areas such as North visible. The red blood cells were lysed with an equal America, Australia and the Far East (Tenter & volume of distilled water and the lysate passed Friedhoff, 1986). Importation of horses is restricted to those animals that are sero-negative by the through a Whatman CF-11 cellulose column (Ambrosio, Potgieter & Nel, 1986). complement fixation (CF) test (Wieland, 1986). Horses whose sera react positive to a 1:5 dilution The column eluate was incubated overnight at 37 may no longer be imported into the United States. °C in lysis buffer (032 M sucrose, 1 % Triton X-100, Even though the CF test rarely gives false-positive 5 mM MgCl 2 , 10 mM Tris-HCl, p H 7-4) containing results the test is relatively insensitive and yields a 2 % S D S and 100/
DNA probes for the detection of Babesia caballi E. S. POSNETT and R. E. AMBROSIO* Molecular Biology Section, Veterinary Research Institute, Onderstepoort 0110, South Africa (Received 28 August 1990; revised 6 November 1990; accepted 6 November 1990) SUMMARY
A genomic library of Babesia caballi DNA was constructed in the plasmid vector pUCl 3. The specificity of the clones for B. caballi was established by the lack of hybridization to Babesia equi, Babesia bovis, Babesia bigemina and equine DNA. Two probes, pBCll and pBC191, were isolated that could detect 025 ng and 0125 ng of B. caballi DNA, corresponding to a parasitaemia of 0 1 2 % and 0 0 6 % respectively. pBC191 could detect B. caballi parasites in the blood of an experimentally infected horse as well as in naturally infected horses. Key words: equine babesiosis, Babesia caballi, DNA probe, tandem repeat.
sensitive than CF and does not produce many falsenegative results (Morzaria, Brocklesby & Harradine, 1977). However, this test cannot be standardized, Equine babesiosis is caused by the intra-erythrocytic and reading of results is time-consuming (Tenter & protozoan parasites Babesia equi and Babesia caballi Friedhoff, 1986). It is therefore difficult to detect the (Friedhoff, 1982). The disease is transmitted by ticks of the genera Dermacentor, Hyalomma and Rhi- low parasitaemias associated with B. caballi using these diagnostic procedures. Also, immunodiagpicephalus and is endemic in the tropics and nostic procedures are an indirect way of determining subtropics and also in an area from Northern infection and cannot distinguish between a past and France to the Soviet Union (Tenter & Friedhoff, a present infection. 1986; Friedhoff, 1982). B. caballi is the larger of the two species, and There is thus a need for a more rapid and sensitive development occurs in the vertebrate host exclustest for equine babesiosis. We have reported the ively in the erythrocytes. Red blood cells containing isolation and characterization of repetitive D N A more than two parasites (multiple infections) are rare probes for B. equi (Posnett & Ambrosio, 1989). Here with B. caballi, but do occur with B. equi. In we describe the isolation and characterization of B. caballi infections, usually less than O'l % of eryDNA probes for B. caballi. throcytes are infected compared to between 5 and 2 0 % in the case of B. equi. Demonstration of B. caballi parasites on blood smears is therefore MATERIALS AND METHODS difficult, even in acute cases. Characteristics of the DNA isolation disease include anaemia, fever and, unless treated, death (Purnell, 1981). After infection with B. caballi, B. caballi parasites (Warrenton isolate, Warrenton, animals may remain carriers of the parasites for up to South Africa) were isolated from the blood of 4 years. artificially infected horses. The average parasitaemia was 0-5%. Blood was centrifuged at 2000 £ for As a result of the increasing international trade in 15 min and the buffy coat removed. Centrifugation horses there is a considerable risk that the disease was repeated until the buffy coat was no longer may spread to disease-free areas such as North visible. The red blood cells were lysed with an equal America, Australia and the Far East (Tenter & volume of distilled water and the lysate passed Friedhoff, 1986). Importation of horses is restricted to those animals that are sero-negative by the through a Whatman CF-11 cellulose column (Ambrosio, Potgieter & Nel, 1986). complement fixation (CF) test (Wieland, 1986). Horses whose sera react positive to a 1:5 dilution The column eluate was incubated overnight at 37 may no longer be imported into the United States. °C in lysis buffer (032 M sucrose, 1 % Triton X-100, Even though the CF test rarely gives false-positive 5 mM MgCl 2 , 10 mM Tris-HCl, p H 7-4) containing results the test is relatively insensitive and yields a 2 % S D S and 100/
