engleski

Genotyping of piroplasms detected in canine stained peripheral blood smears and postmortem tissue imprints

Piroplasmosis is a common infectious haemolytic anaemia in Croatian dogs caused by different species of Babesia genus. Based on morphology of merozoites, Babesia species are divided into large and small piroplasms. This morphological criterion is insufficient for species differentiation ; therefore, use of molecular methods for genotyping of piroplasms from animals is necessary. Routinely, genotyping has been performed from peripheral blood samples, but in some forensic, second opinion requests and archival retrospective analyses genotyping from stained tissue imprints and blood smears is needed. Twelve archival stained cytologic samples from dogs (7 tissue imprints and 5 blood smears) were microscopically analyzed for merozoite presence, parasitemia level and morphology. Genotyping was performed from scrapings of cytological samples using three different protocols ; 1. simple PCR, 2. nested PCR, and 3. simple PCR with purified and concentrated DNA. In all samples merozoites were detected. Parasitemia levels were scored as low, moderate and high. In blood smears, merozoites morphology was piriform measuring 3, 75-1, 88 μm. Round shrunken merozoite form measuring 1, 31-0, 87 μm was detected in erythrocytes from postmortal tissue imprints. With no correlation to parasitemia level, DNA was amplified in 33%, 75% and 67% of samples in the 1, 2 and 3 protocol, respectively. All PCR products amplified were sequenced successfully revealing presence of B. canis. In current study we have shown that B. canis merozoites can have a typical large form in peripheral blood smears and they post mortally became round, small and shrunken like small Babesia species. DNA concentration and nested PCR protocols showed higher sensitivity then simple PCR and can be used for amplification of DNA from archival cytological samples. In the light of obtained results it seems that determination of Babesia species based on classical morphological methods is doubtful and genotyping should be use as confirmatory method.

Genotyping ; blood smears ; organ imprints ; Babesia canis

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