Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves

Abstract

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified.

Keywords

• Nested PCR
• Oocysts
• Protozoan
• Ziehl-Neeslsen.