An optimized assay for detecting Encephalitozoon intestinalis and Enterocytozoon bieneusi in dairy calf feces using polymerase chain reaction technology

Abstract

The purpose of this study was to optimize primary and nested polymerase chain reaction (PCR) assays for detecting the microsporidia Encephalitozoon intestinalis and Enterocytozoon bieneusi in fecal samples from dairy calves. PCR for these microsporidia were compared to immunofluorescence assays (IFA) based on commercially available monoclonal antibodies specific for outer wall proteins of Enc. intestinalis or Ent. bieneusi. Fecal samples were collected from 15 dairy calves and processed by molecular sieving followed by salt floatation to recover Enc. intestinalis and Ent. bieneusi spores. An aliquot of the final supernatant was applied to glass slides for IFA testing; another aliquot was extracted for total DNA using a QIAamp Stool Mini-Kit for primary and nested Enc. intestinalis- and Ent. bieneusi-specific PCR analysis. Internal standards were generated for both Enc. intestinalis and Ent. bieneusi PCR assays to control for false negative reactions due to the presence of inhibitors commonly found in fecal samples. Using the commercial MicrosporIFA (Waterborne, Inc.) as the gold standard, the optimized Enc. intestinalis PCR method provided 85.7% sensitivity and 100% specificity with a kappa value = 0.865. Likewise, using the commercial BienusiGlo IFA (Waterborne, Inc.) as the gold standard, the optimized Ent. bieneusi PCR method provided 83.3% sensitivity and 100% specificity with a kappa value = 0.857. Sequencing of amplicons from both PCR assays confirmed the presence of Enc. intestinalis or Ent. bieneusi. In conclusion, our optimized assays for recovering and detecting Enc. intestinalis or Ent. bieneusi in feces from dairy calves provides a valuable alternative to traditional IFA methods that require expertise to identify extremely small microsporidia spores (~ 2.0 µm). Our assays also improve upon existing molecular detection techniques for these microsporidia by incorporating an internal standard to control for false negative reactions.

Keywords: Encephalitozoon intestinalis; Enterocytozoon bieneusi; Immunofluorescence assay; Internal standard; PCR.

Fig. 1

Immunofluorescence staining of fecal smears from dairy calves containing high (a), low (c), or negligible (e) numbers of Encephalitozoon intestinalis spores with commercial MicrosporFA monoclonal antibodies or fecal smears from dairy calves containing containing high (b), low (d), or negligible (f) numbers of Enterocytozoon bieneusi spores with commercial BienusiGlo monoclonal antibodies. Arrows point to representative reactive spore

Fig. 2

a Primary and nested Encephalitozoon intestinalis-specific PCR assay of cell culture-derived Enc. intestinalis (Enc) or spore DNA obtained from Enc. intestinalis positive or negative and Ent. bieneusi positive or negative calves; b Primary and nested Ent. bieneusi-specific PCR analysis of Enc. intestinalis positive or negative and Ent. bieneusi positive or negative calves. MrS, molecular mass markers. Observed size of amplication products: 600-internal standards, 950-Enc. intestinalis amplicon from primary PCR, 450-Enc. intestinalis amplicon from nested PCR, 435-Ent. bieneusi amplicon from primary PCR, 390-Ent. bieneusi amplicon from nested PCR