A SpeI-AvaI fragment (0.3 kbp) from pBbil6 (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 IL1 of blood with a parasitemia of as low as 1 in 108 cells and which contained an estimated 30 B. bigemina-infected erythrocytes. By a direct PCR method, B. bigemina DNA was amplified from 20 ,ul of packed erythrocytes with a calculated parasitemia of 1 in 109 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. Babesia bigemina is one of several Babesia species known to cause bovine babesiosis. The disease is clinically manifested by anemia, fever, hemoglobinuria, and the presence of parasites in the host erythrocytes (20). Recovery from babesiosis is followed by the apparent elimination of parasites from the peripheral blood. However, subclinical infection may last for several years (14). The serological techniques used to diagnose bovine babesiosis do not consistently detect carrier animals and do not specifically eliminate cross-reactions between B. bigemina and Babesia bovis, another important hemoparasite (10). Subclinically infected cattle can be proved to be carriers by subinoculation of blood into susceptible splenectomized calves (14). The use of specific DNA probes and nucleic acid hybridization to detect B. bigemina directly in blood from carrier cattle has several advantages over conventional microscopic, serologic, and subinoculation techniques. Although a radioactively labeled probe derived from cloned segments of genomic B. bigemi...
A procedure for cloning Babesia bovis was developed. The procedure was used to establish and cultivate homogeneous populations of parasites and to isolate B. bovis from carrier animals. Three different clone lines of B. bovis based on in vitro growth rates were established.
SummaryThe most efficient procedure for cryopreserving viable Babesia bovis organisms for in vitro cultivation consists of freezing extracellular parasites in a solution of 10% (w/v) polyvinylpyrrolidone (PVP) using a cooling rate of 20 °C/min. Although cultures can be established from thawed infected erythrocytes, the plating efficiency is relatively low. Freezing extracellular parasites resulted in plating efficiency up to 25%, when thawed and placed in culture. Glycerin or dimethyl sulphoxide (Me2SO) can be used successfully in the cryopreservation of B. bovis, but apparent toxic effects greatly decrease their efficiency. B. bovis parasites have been kept at − 196 °C for 60 days with no appreciable reduction in plating efficiency.
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