Ectromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method. In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement. In conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

Background: Cryptosporidium spp. and Enterocytozoon bieneusi are important enteric diarrheal pathogens commonly found in various animals and humans and have significant implications for public health. The purpose of present study was to investigate the prevalence, species and genotypes and zoonotic evaluation of the two parasitic pathogens in dogs and cats in Wenzhou, southern of China. Methods: 89 fecal samples collected from 51 dogs and 38 cats were used to investigate the positive rates of Cryptosporidium spp. and Enterocytozoon bieneusi using the nested polymerase chain reaction amplification on methods, species and genotypes of them were conformed by sequencing and alignment. Result: The positive rates of Cryptosporidium spp. and Enterocytozoon bieneusi were 7.8%(4/51), 17.6%(9/51) in dogs and 7.9%(3/38) ,10.5%(4/38) in cats, respectively. Sequence analysis revealed that Cryptosporidium canis, genotypes PtEb IX, CD9 and NED3 of E. bieneusi were confirmed in dogs, Cryptosporidium felis, Type IV in cats, respectively. C canis, C. felis and Type IV of E. bieneusi genotype belong to the zoonotic pathogens.The zoonotic species/genotype of these two pathogens identified in pets in this study indicated the potential zoonotic risk in pets-to- humans transmission. The effective interventions should be taken to prevent the spread of these enteric zoonotic pathogens.

Enterocytozoon bieneusi is a major zoonotic pathogen causing human microsporidiosis, with small ruminants serving as crucial reservoir hosts. Hu sheep and local goats are pillar livestock breeds in Zhejiang Province, yet no parallel comparative epidemiological data on E. bieneusi exist for sympatric small ruminants in southern Zhejiang, and the infection status in Hu sheep remains entirely uncharacterized. Here, a total of 297 fecal samples (159 Hu sheep, 138 goats) were collected from four administrative regions in southern Zhejiang from 2021 to 2023 and screened via nested PCR targeting the ribosomal internal transcribed spacer (ITS) region of the E. bieneusi genome. Overall, the total E. bieneusi prevalence reached 18.18%, and Hu sheep exhibited a significantly higher infection rate (24.53%, 39/159) than sympatric goats (10.87%, 15/138; χ² = 9.27, P = 0.002). Obvious geographical and age-dependent disparities were observed in Hu sheep, with pre-weaning lambs (< 3 months) showing the highest prevalence (60.42%), while adult sheep (> 1 year) had the lowest rate (4.48%). No significant age or regional differences were detected in goats. Five genotypes were identified from Hu sheep: CM7 (n = 25), BEB6 (n = 10), CHG1 (n = 2), Type IV (n = 1), and CHG5 (n = 1). Five genotypes were detected from goats: CHG1 (n = 9), BEB6 (n = 3), Type IV (n = 1), CHG3 (n = 1), and CYG-2 (n = 1). BEB6, Type IV, and CHG1 were shared by both hosts. All genotypes belonged to zoonotic Group 1 and Group 2. This study represents the first systematic parallel survey of E. bieneusi in cohabiting Hu sheep and goats in eastern China, identifies distinct host-specific infection patterns linked to divergent rearing systems, provides molecular evidence for potential cross-species transmission, and underscores the need for breed-tailored biosecurity strategies under a One Health framework.

Enterocytozoon bieneusi is a major zoonotic pathogen causing human microsporidiosis, with small ruminants serving as crucial reservoir hosts. Hu sheep and local goats are pillar livestock breeds in Zhejiang Province, yet no parallel comparative epidemiological data on E. bieneusi exist for sympatric small ruminants in southern Zhejiang, and the infection status in Hu sheep remains entirely uncharacterized. Here, a total of 297 fecal samples (159 Hu sheep, 138 goats) were collected from four administrative regions in southern Zhejiang from 2021 to 2023 and screened via nested PCR targeting the ribosomal internal transcribed spacer (ITS) region of the E. bieneusi genome. Overall, the total E. bieneusi prevalence reached 18.18%, and Hu sheep exhibited a significantly higher infection rate (24.53%, 39/159) than sympatric goats (10.87%, 15/138; χ² = 9.27, p = 0.002). Obvious geographical and age-dependent disparities were observed in Hu sheep, with pre-weaning lambs (1 year) had the lowest rate (4.48%). No significant age or regional differences were detected in goats. Five genotypes were identified from Hu sheep: CM7 (n = 25), BEB6 (n = 10), CHG1 (n = 2), Type IV (n = 1), and CHG5 (n = 1). Five genotypes were detected from goats: CHG1 (n = 9), BEB6 (n = 3), Type IV (n = 1), CHG3 (n = 1), and CYG-2 (n = 1). BEB6, Type IV, and CHG1 were shared by both hosts. All genotypes belonged to zoonotic Group 1 and Group 2. This study provides the first systematic parallel survey of E. bieneusi in sympatric Hu sheep and goats in eastern China. It identifies distinct host-specific infection patterns potentially associated with divergent rearing systems, provides molecular data consistent with potential inter-species circulation and shared environmental exposure, and highlights the need for breed-tailored biosecurity strategies within a One Health framework.

Porcine reproductive and respiratory syndrome virus (PRRSV) employs complex strategies to evade host immunity, yet its interplay with the pyroptotic cell d