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As a zoonotic parasite, Cryptosporidium spp. could cause severe diarrhea mainly in calves and children globally. Monitoring and prevention of Cryptosporidium spp.’s prevalence is of great significance in both economy and public health aspects. In this study, specific primers and probes were designed within the conserved region of 18S rRNA gene for Cryptosporidium spp. and recombinase polymerase amplification assays based on the fluorescence monitoring (real-time RPA) as well as combined with a lateral flow strip (LFS RPA) were developed. Both of the two RPA assays allowed the exponential amplification of the target fragment within 20 min. After incubation on a metal bath at 42 °C, the LFS RPA results were displayed on the lateral flow strip within 5 min while real-time RPA allowed the real-time observation of the results in Genie III at 39 °C. The RPA assays showed high specificity for Cryptosporidium spp. without any cross-reaction with other tested pathogens causing diarrhea in cattle. With the recombinant plasmid DNA containing the 18S rRNA gene of Cryptosporidium spp. serving as a template, the limit of detection for real-time RPA and LFS RPA assays were 14.6 and 12.7 copies/reaction, respectively. Moreover, the RPA assays were validated by testing diarrheic cattle fecal samples and compared with a real-time PCR. The positive ratio of Cryptosporidium spp. was 24.04 % (44/183) and 26.23 % (48/183) in both RPA assays and real-time PCR assay, respectively, and the kappa coefficient value was 0.942. The diagnostic specificity and diagnostic sensitivity of both RPA assays were 100 % and 91.67 %, respectively. Forty-one of 48 positive samples were successfully sequenced and four Cryptosporidium species were detected, including C. parvum (n = 20), C. andersoni (n = 17), C. bovis (n = 3) and C. ryanae (n = 1). The developed RPA assays are easy to operate and faster to obtain the detection results, and they are suiting for the point-of-care detection and facilitating the prevention and control of Cryptosporidium spp. infections.

Abstract Background Bovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals. Results An RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 102 copies per reaction and 1.4 × 101 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively. Conclusions In this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.

Duck is often used in meat fraud as a substitute for more expensive meats. Rapid detection of duck ingredient in meat products is of great significance for combating meat fraud and safeguarding the interests of consumers. Therefore, we aim to develop duck-specific recombinase polymerase amplification (RPA)-based assays for the rapid detection of duck ingredient in animal-derived foods. Using Cytb gene as target, the real-time RPA and RPA combined with lateral flow strips (LFS RPA) were developed successfully for the rapid detection of ducks in 20 min at 39 °C and 40 °C, respectively. The assays did not show cross-reactions with 6 other livestock and poultry. The developed RPA assays could detect 10 pg duck genomic DNA per reaction and 0.1 % (w/w) duck ingredient in duck and mutton mixed powder within 30 min, including a rapid nucleic acid extraction. Furthermore, duck ingredient could be detected in 30 different actual foods including heat-processed meats and blood products. Therefore, duck-specific real-time RPA and LFS RPA assays were successfully developed with good specificity and sensitivity, which could enable rapid detection of duck ingredient in the field and provide technical support for combating the meat fraud.

Hepatitis E virus (HEV) is a zoonotic pathogen that causes global hepatitis E. Outbreaks of hepatitis E are directly linked to the consumption of pork liver products. Herein reverse transcription recombinase polymerase amplification assays targeting the ORF2 gene were developed for the rapid detection of HEV by integrating the fluorescence detection platform (qRT-RPA) and the visible lateral flow biosensor by naked eyes (LFB RT-RPA). The qRT-RPA assay effectively detected HEV RNA with a limit of detection (LOD) of 154 copies/μl (95%CI: 126–333 copies/µl) in Genie III at 41°C for 20 min. Besides this, the LFB RT-RPA detected the HEV RNA with a LOD of 24 copies/μl (95%CI: 20–57 copies/µl) in an incubator block at 41°C for 20 min. The developed RT-RPA assays also showed good specificity for HEV, with no cross-reactions with any of the other important swine pathogens examined in this work. The performance of the developed RT-RPA assays was validated on 14 HEV RNA-positive and 66 HEV RNA-negative raw pork liver samples identified by a previously described qRT-PCR. Consequently, 11 and 12 samples were HEV RNA-positive as detected by the qRT-RPA and the LFB RT-RPA, respectively. Compared to qRT-PCR, the qRT-RPA and LFB RT- RPA assays revealed a coincidence rate of 96.3 and 97.5% as well as a Kappa value of 0.858 and 0.908, respectively. These results ascertain that the developed RT-RPA assays are effective diagnostic tools for the point-of-care detection of HEV in resource-limited settings.

Bovine kobuvirus (BKV) is a novel kobuvirus considered to be closely related to calf diarrhea and has become a worldwide epidemic. Currently, the BKV lacks an efficient and convenient detection method to assist the research on BKV prevalence. In this study, a new and specific TaqMan-based real-time RT-PCR for the detection of BKV was developed using the conserved region of the 3D gene. The assay was highly specific for BKV, without cross-amplification with other non-targeted pathogens. The limit of detection of this assay was 102 copies. Standard curves showed a strong linear correlation from 102 to 106 copies of BKV standard RNA per reaction, and the parameters revealed as a slope of −3.54, efficiency of 91.64%, and regression coefficients (R2) of 0.998. The assay was also reproducible, with the intra-assay and inter-assay coefficient of variation

Bone immunity regulates osteoclast differentiation and bone resorption and is a potential target for the treatment of postmenopausal osteoporosis (PMOP). The molecular network between bone metabolism and the immune system is complex. However, the molecular mechanism underlying the involvement of the major histocompatibility complex class II (MHC-II) molecule protein presentation pathway in PMOP remains to be elucidated. The MHC-II molecule is a core molecule of the protein presentation pathway. It is combined with the processed short peptide and presented to T lymphocytes, thereby activating them to become effector T cells. T-cell-derived inflammatory factors promote bone remodeling in PMOP. Moreover, the MHC-II molecule is highly expressed in osteoclast precursors. MHC-II transactivator (CIITA) is the main regulator of MHC-II gene expression and the switch for protein presentation. CIITA is also a major regulator of osteoclast differentiation and bone homeostasis. Therefore, we hypothesized that the MHC-II promotes osteoclast differentiation, providing a novel pathogenic mechanism and a potential target for the treatment of PMOP.

ABSTRACT: In recent years, ostrich disease characterized by paralysis and diarrhea has been circulating in some regions of China, causing huge economic losses to the ostrich breeding industry. In our study, clinical samples from diseased ostriches were collected, and only parvovirus was detected. The virus distribution analysis by histopathology and quantitative real-time PCR assays indicated that the virus had a wide range of tissue tropisms. The full-length genome of the ostrich parvovirus (OsPV) was sequenced and comprehensively analyzed. Interestingly, the phylogenetic and alignment results indicated that the OsPV and the goose parvovirus (GPV) form a separate branch. In contrast to GPV strains, OsPV showed 2 new 14 nucleotide deletions in the inverted terminal repeat (ITR) region. Furthermore, recombination analysis indicated that OsPV was a recombination strain between the vaccine strain SYG61v and the virulent strain B strain, with the major parent of OsPV as the SYG61v strain and the minor parent as the B strain. The 14 nucleotide deletions in the ITR region as well as recombination may be some of the reasons for the cross-species transmission of parvovirus from goose to ostrich. The above data will contribute to a better understanding of the molecular biology of the novel OsPV and help to develop the vaccine candidate strain.