Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal

Abstract

Background: Tick-borne protozoan parasites (TBPPs) cause significant problems for domestic animals' health in Nepal. TBPPs are routinely diagnosed by labor-intensive blood smear microscopy. In Nepal, there are some reports of Babesia and Theileria in cattle, although species identification is rarely performed. Therefore, we performed conventional nested PCR (nPCR) followed by sequence analysis to identify TBPP species infecting cattle in Nepal.

Methods: One hundred and six blood samples were collected from cattle in the Kathmandu Valley. Thin blood smears were prepared for microscopic examination. Parasite DNA was extracted from the blood, and nPCR and sequencing were performed to identify the TBPPs present.

Results: Among the 106 samples, 45 (42.5%) were positive for piroplasm (Babesia spp. and Theileria spp.) via microscope observation and 56 (52.8%) samples were positive via nPCR. The obtained PCR products were used for direct sequencing, and we identified the species as B. bigemina, B. bovis, T. annulate and T. orientalis. Phylogenetic analyses showed that the B. bovis, B. bigemina and T. orientalis sequences from this study belonged to each species clade. On the other hand, T. annulate was divided into two clades in the analysis, and our T. annulate sequences were also divided in these two clades. The piroplasm-positive cattle showed lower hemoglobin and red blood cells than healthy cattle.

Conclusions: To the best of our knowledge, this study is the first to apply molecular detection and species determination of TBPPs in cattle in Nepal. The results of this study may be used as a starting point for the development of successful TBPP surveillance and prevention programs in Nepal.

Keywords: Babesia; Nepal; PCR; Theileria; cattle.

Figure 1

Map of Kathmandu Valley showing the origins of the samples.

Figure 2

Trend of Babesia spp. and Theileria spp. reported in Kathmandu Valley.

Figure 3

Agarose gel electrophoresis of nPCR products using 18s rRNA primers for piroplasm. Left to right: M, DNA ladder; B, blank; S1–S5, samples; NC, negative control.

Figure 4

Phylogenetic relationship of piroplasm sequences from Nepalese cattle. The sequences were aligned and indels or undetermined nucleotides were excluded. (a) The resulting 858 bp (LC762262, LC762265) was used for the maximum likelihood analysis. The bootstrap values and length of the substitutions/site (0.04) are indicated. (b) The resulting 866 bp (LC762266–LC762269) sequence was used for the maximum likelihood analysis. The bootstrap values and length of the substitutions/site (0.04) are indicated. * Indicates reference genome sequences.

Figure 5

Hematological parameters (hemoglobin, RBC and WBC) shown by dot-plot for healthy (black round symbols) and piroplasm-positive (black box symbols) cattle.