A Multiplex PCR Assay for Simultaneous Detection of Giardia duodenalis , Cryptosporidium parvum , Blastocystis spp. and Enterocytozoon bieneusi in Goats.

Simple Summary: Gastrointestinal parasitic infections are highly prevalent among goat populations, and field samples commonly display mixed infections. Giardia duodenalis, Cryptosporidium parvum, Blastocystis spp., and Enterocytozoon bieneusi are four common zoonotic intestinal parasites with similar symptoms, including diarrhea, vomiting, and dehydration. In this study, a multiplex PCR method was developed for the differential detection of these four zoonotic protozoans. Genomic DNA extracted from 130 goat stool samples obtained from four farms in Zhanjiang city, Guangdong Province, China, was tested with both the single-target PCRs and the multiplex PCR assay developed. The positive rates of G. duodenalis, C. parvum, Blastocystis spp., and E. bieneusi were 23.08% (30/130), 24.62% (32/130), 41.54% (54/130), and 12.31% (16/130), respectively. Furthermore, the detection results showed a high prevalence of mixed infections of goat parasites, predominantly involving two parasite species. The findings of the multiplex PCR were consistent with those of single-target PCRs. This multiplex PCR method showed high specificity, sensitivity, accuracy, and cost-effectiveness, and it was suitable for the rapid large-scale screening of goat stool samples for infections caused by G. duodenalis, C. parvum, Blastocystis spp., and E. bieneusi. It thus provided a reliable and efficient tool for animal health management and disease monitoring. Giardia duodenalis, Cryptosporidium parvum, Blastocystis spp. and Enterocytozoon bieneusi are four common zoonotic parasites associated with severe diarrhea and enteric diseases. In this study, we developed a multiplex PCR assay for the simultaneous detection of these four zoonotic protozoans in goat stool samples and assessed its detection efficiency. Specific primers were designed from conserved gene sequences retrieved from GenBank, and the PCR conditions were optimized. Genomic DNA from 130 samples was subjected to both single-target PCR and multiplex PCR. The multiplex PCR assay successfully amplified specific gene fragments (G. duodenalis, 1400 bp; C. parvum, 755 bp; Blastocystis spp., 573 bp; E. bieneusi, 314 bp). The assay sensitivity was ≥10² copies of pathogenic DNA clones with high specificity confirmed by negative results for other intestinal parasites. The detection rates were 23.08% (30/130) for G. duodenalis, 24.62% (32/130) for C. parvum, 41.54% (54/130) for Blastocystis spp., and 12.31% (16/130) for E. bieneusi, matching the single-target PCR results. The sensitivity and predictive values were 100.00%. This multiplex PCR provided a rapid, sensitive, specific, and cost-effective approach for detecting these four parasites. It also provided essential technical support for the rapid detection and epidemiological investigation of G. duodenalis, C. parvum, Blastocystis spp., and E. bieneusi infections in goat fecal samples. [ABSTRACT FROM AUTHOR]