Optimization of the method for detecting Cryptosporidium spp. DNA in fecal samples from calves using PCR and comparison of the obtained results with the results of microscopy

Intestinal infections cause significant damage to cattle farms; one of the most acute problem is cryptosporidiosis. But the early diagnostics can significantly reduce the incidence of cryptosporidiosis in neonatal calves. The most sensitive are molecular methods based on polymerase chain reaction (PCR), but an inappropriate DNA preparation protocol can reduce it. Currently, this problem prevents the spread of the PCR in the routine diagnosis of parasitosis. In the present study, for the first time in Russia various methods of DNA extraction from feces were compared and the most optimal one was selected: with preliminary bead-beating and DNA sorption on the spin-column. Further this method was used as basic for real-time PCR developed for diagnosing cryptosporidiosis. Our new system was tested on real samples from calves and compared with classical microscopy; that was our target. 20 fecal samples from calves (Holstein breed) were collected directly from rectum on farms located in Vologda region, in 2023, after cryptosporidiosis outbreak. Each sample was divided in two aliquots, first for microscopy detection by Ziehl-Neelsen staining using Diakhim kit («Abris+», Russia), second was fixed in 70% ethanol for molecular detection. Stained sliders were observed with Olympus cx33 light microscope (Olympus, Japan) at x1000 magnification using oil immersion. The degree of infection was assessed. To develop a PCR-based test-system primers for 18S rRNA Cryptosporidium spp. Crypt-F 5´-TAATCAAGAACGAAAGTTAGGGGA-3´ Crypt-R 5´-GAGTAAGGAACAACCTCCAATCT-3´, probe Crypt-P R6G-5´-ATGGTTAAGACTACGACGGTATCTGA-3´-BHQ1 were designed allow detection of all species of Cryptosporidium . A few modified DNA extraction protocols of fecal samples were evaluated: using conventional concentrates HEM (LLC HEM, Russia), concentrates HEM without formaldehyde or washing by phosphate buffer without concentration. Then QIAamp PowerFecal Pro DNA Kit (QIAgen, Germany) were used to extract DNA from the received fecal sediment, including a stage of DNA purification on spin columns. Clarified fecal extract was obtained in accordance with the guidelines MU 1.3.2569-09, DNA was extracted by precipitation method using AmpliTest RIBO-prep kit (Centre for Strategic Planning and Management of Biomedical Health Risks, of the Federal medical and biological agency, Russia). As a result sample preparation using HEM concentrators had no advantages, and sample preparation in the presence of formalin had a negative effect on PCR. Obtaining the supernatant of the clarified fecal extract and then DNA extraction using AmpliTest RIBO-prep kit allow only poor detection of Cryptosporidium due to the high loss of oocytes in the sedimentary fraction of feces. Extraction of DNA from crude feces using bead-beating and purification on spin columns proved to be effective for receiving of parasitic DNA. Homogenization of feces, cell disruption and release of nucleic material are carried out during bead-beating on a TissueLyser LT homogenizer (QIAgen, Germany). Cryptosporidium oocysts were detected in 15 of 20 samples, but 4 of them have extremely low concentration of oocysts. Using the most effective PCR-based assay, 15 of 20 samples also were positive. Accordingly, all samples with high and medium level of Cryptosporidium oocysts were PCR positive. But two samples with low level of oocyst have different results of microscopy and PCR, one was determined to be weakly positive by PCR (Ct> 40) although microscopy was negative, and conversely, the second one was weakly positive by microscopy (“+”), but determined as negative by PCR. Additionally commercially kit for DNA extraction and PCR detection DNA Cryptosporidium spp. FBioNucleo (Fractal Bio, Russia) was tested. Cryptosporidium spp. was detected in 9 of 20 samples, all weakly positive and two medium positive samples were determined as negative. Thus, the FBioNukleo kit showed lower sensitivity than the assay proposed. Finally, an effective assay has been designed including bead-beating and purification on spin columns, which provides the sensitivity of PCR similar to the microscopy with Ziehl-Neelsen staining.