Diagnosis of acute bovine sarcocystosis using genomic DNA probes

Abstract

DNA probes were prepared from a Sarcocystis cruzi sporozoite genomic library constructed in bacteriophage lambda gt 10. Of the 96 clones examined, 14 showed strong hybridisation to parasite DNA and were further analyzed. The 14 clones were amplified in Escherichia coli strain LE392, the DNA was sized on 0.8% agarose gel, and Southern blots prepared. Two clones of approximately 1.4 kbp and 4.3 kbp, respectively, which showed strong hybridisation with merozoite DNA were selected as probes. The probes hybridized to S. cruzi, and S. hirsuta but not to bovine or canine cellular DNA. The probes detected as little as 27 pg of S. cruzi merozoite DNA, equivalent to DNA extracted from 12.9 merozoites. Four 3-month-old calves were orally administered approximately 200,000 S. cruzi sporocysts; a fifth calf served as a control. Subsequently, blood fractions (buffy coats, granulocytes fraction (polymorphonuclear cells), plasma) were collected from the calves twice weekly during a 3.5 month period, in order to detect infection by DNA probes and by conventional diagnostic tests. Total cellular DNA from the fractions was extracted by phenol/chloroform/isoamyl alcohol method and dot blotted on nylon membranes. Samples were then hybridised to the selected probes radiolabelled with [a_32P]dATP. Indirect fluorescent antibody technique using an anti-merozoite monoclonal antibody was done on buffy coat smears while direct microscopy was used to identify merozoites in blood films stained with Wright's stain. The probes detected merozoites intermittently from day 22-39 post infection in the buffy coat and granulocyte fractions, in the infected calves. This was earlier and longer than diagnosis with either the indirect fluorescent antibody technique which detected merozoites in buffy coats from day 28-35 post infection or the direct microscopy which detected merozoites in blood films from day 25-28 post infection. In conclusion, the present study was able to develop genomic DNA probes that were highly sensitive for Sarcocystis cruzi and can be used in the diagnosis of acute bovine sarcocystosis. The probes detected the presence of merozoite DNA in granulocyte fraction. Whether this indicates that polymorphonuclear cells are involved in the pathogenesis of Sarcocystis remains to be determined.