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To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3), specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested. Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies.L-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181), the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181). The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181). The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.
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Here, we report the development of an indirect ELISA antibodies detection method for African swine fever virus (ASFV). Two purified monoclonal antibodies (mAbs) against ASFV p30 and p54 protein were used as targets and a phage-displayed 12-mer peptide library was used to conduct four rounds of biopanning to screen peptide epitopes, then amino acids GGG was used as a linker to synthesize tandem-epitope peptide of ASFV p30 and p54 protein which was used as coating antigen. The optimum reaction conditions of indirect ELISA were determined by chessboard titration, and clinical serum samples were used to evaluate the specificity, sensitivity, stability and conformity of this method. The biopanning experiment indicated that 146PAEPYTT152 was a core domain of the B cell linear epitope of p54 protein. The optimization results of ELISA reaction conditions showed that the tandem-epitope peptide coupled with ovalbumin (OVA) at N-terminal had low background of non-specific serum reaction. And the optimum reaction effect was obtained when the polypeptide antigen was coated with carbonate buffer in 2 mug.mL-1, the serum was diluted 100-fold with blocking solution (1% gelatin solution), and the HRP-antibody was diluted 5 000 times with 0.05% PBST solution. The cut-off value was determined to be 0.339. Furthermore, the results of specificity, sensitivity and stability tests showed that there is no cross-reaction in positive serum samples of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV), the detection limit of ASFV positive sera is 1:1 600, and the method had high repeatability. Finally, Total 320 swine serum samples were detected simultaneously by the present established method and commercial ASFV antibody detection kit. The results showed that the relative specificity and sensitivity of the two methods were 97.6% and 97.3%, respectively. And the coincidence rate was 97.5%. In conclusion, this method showed good specificity, sensitivity, repeatability and coincidence rate, that had the potential value of developing clinical diagnostic kit.Copyright © 2022 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.
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Objective: The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. Method: The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value. The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. Result: Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1..102 copies/L. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. Conclusion: In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.
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Viral infections are known as one of the major factors causing death. Ginseng is a medicinal plant that demonstrated a wide range of antiviral potential, and saponins are the major bioactive ingredients in the genus Panax with vast therapeutic potential. Studies focusing on the antiviral activity of the genus Panax plant-derived agents (extracts and saponins) and their mechanisms were identified and summarized, including contributions mainly from January 2016 until January 2022. P. ginseng, P. notoginseng, and P. quinquefolius were included in the review as valuable medicinal herbs against infections with 14 types of viruses. Reports from 9 extracts and 12 bioactive saponins were included, with 6 types of protopanaxadiol (PPD) ginsenosides and 6 types of protopanaxatriol (PPT) ginsenosides. The mechanisms mainly involved the inhibition of viral attachment and replication, the modulation of immune response by regulating signaling pathways, including the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway, phosphoinositide-dependent kinase-1 (PDK1)/ protein kinase B (Akt) signaling pathway, c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. This review includes detailed information about the mentioned antiviral effects of the genus Panax extracts and saponins in vitro and in vivo, and in human clinical trials, which provides a scientific basis for ginseng as an adjunctive therapeutic drug or nutraceutical.
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In order to build a specific, sensitive and rapid detection method for PAstV3 detection, the PAstVB gene sequences in Genbank were used and the conserved region in ORFlb was selected to design specific primers and TaqMan probe. Clinical stool samples were collected and preliminary detected by this newly established real-time RT-PCR method after reaction systems and conditions optimization. This detection method established in this study has a good linear relationship with the standard curve, with R2 value up to 0.9971. The sensitivity is 100 times higher than conventional PCR method, The variation co-efficient of in-batch and inter-batch repeatability test is less than 2.0%, indicating good repeatability. The detection results of Clinical samples showed that the positive rate of this method is higher than conventional PCR method. The establishment of this method provides a rapid detection means for PAstV3 laboratory diagnosis and epidemiological investigation.
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In order to establish a method for rapid differential identification of Senecavirus A (SVA) and en-cephalomyocarditis virus (EMCV), two pairs of corresponding specific primers were designed based on the highly conserved 3D genes of SVA and EMCV. And two different fluorescent labeled TaqMan probes were used to establish a dual TaqMan real-time PCR method for simultaneous detection of these two viruses, and we also optimize the reaction conditions. The results showed that the minimum detection of the method was 760 copies/ micro L and 98 copies/ micro L for SVA and EMCV. respectively, and it can specifically detect SVA and EMCV, and there was no cross reaction with CSFV, PRRSV and PEDV. The established standard curves showed good linear relationship. Repeated experimental group and inter-group coefficient of variation were less than 5%. The results indicated that the dual-quantitative PCR established in this study has the advantages of convenience, rapidity, good specificity. high sensitivity and good repeatability .and can be used for simultaneous detection of SVA and EMCV.
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The informal poultry and pig sector in the Eastern Cape Province (ECP) of South Africa is of significant socio-economic importance as it sustains livelihoods and ensures food security;yet little is known about the distribution and prevalence of infectious and zoonotic diseases in this region. This paper reviews data published for pig and poultry diseases in the province during the last 20 years (2000-2020). The review included relevant published papers identified by a computerised literature search from Web of Science;provincial animal health reports;the national database from the Department of Agriculture, Land Reform and Rural Development (DALRRD);animal health reports submitted by DALRRD to the World Organisation for Animal Health (OIE) via the World Animal Health Information Database (WAHID) interface and laboratory records. A publication was considered eligible if it included qualitative or quantitative information on any disease affecting pigs and poultry including zoonosis. The search retrieved 174 publications, of which 26 were relevant. The review found that Newcastle disease (ND), coccidiosis and fowl pox (FP) were the most reported avian diseases in the national database, whereas avian infectious bronchitis (AIB), ND and highly pathogenic avian influenza (HPAI) were the most reported diseases in the OIE database. Classical swine fever (CSF) was the most reported pig disease in both databases. The retrieved literature on pig and poultry diseases was scarce and no longer up to date, providing decision makers with little information. The review identified important zoonotic diseases that require further studies yet failed to find information on important neglected diseases like leptospirosis.
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This proceedings contains 10 papers on risk management policy of the ministry of health, labour and welfare for ensuring safe wild game meat, prospective of application of food safety risk assessment for game meat, coronavirus disease (COVID-19) for animal owners, shelter medicine and COVID-19, the characteristics of bats as natural reservoirs of the novel coronavirus, chalkbrood in honey bees and its control measures, the economic impact of classical swine fever in Japan, benzalkonium chloride resistance in Listeria monocytogenes isolated in Japan, COVID-19 outbreak and epidemiological research in Japan and the amendment of the act on domestic animal infectious diseases control.
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The inhibitory effects of 5-aminolevulinic acid phosphate (5-ALA), an important amino acid for energy production in the host, against viral infections were previously reported. Here, the antiviral effects of 5-ALA against classical swine fever virus (CSFV) belonging to the genus Pestivirus in the Flaviviridae family and its possible mechanisms were investigated. CSFV replication was suppressed in swine cells supplemented with 5-ALA or its metabolite, protoporphyrin IX (PPIX). The infectivity titer of CSFV was decreased after mixing with PPIX extracellularly. In addition, the activities of the replication cycle were decreased in the presence of PPIX based on the CSFV replicon assay. These results showed that PPIX exerted antiviral effects by inactivating virus particles and inhibiting the replication cycle. To evaluate the in vivo efficacy of 5-ALA, pigs were supplemented daily with 5-ALA for 1 week before virus inoculation and then inoculated with a virulent CSFV strain at the 107.0 50% tissue culture infectious dose. The clinical scores of the supplemented group were significantly lower than those of the nonsupplemented group, whereas the virus growth was not. Taken together, 5-ALA showed antiviral effects against CSFV in vitro, and PPIX played a key role by inactivating virus particles extracellularly and inhibiting the replication cycle intracellularly.