Genotype diversity of Mycoplasma Hyopneumoniae in Chinese swine herds based on multilocus sequence typing

Background Between 2018 and 2020, 989 clinical specimens from pigs showing clinical signs of a variety of swine diseases in 27 provinces in China were sampled and submitted for further testing. Nested PCR targeting the 16S rRNA gene of Mycoplasma hyopneumoniae and subsequent sequencing were used to analyse these specimens. Mycoplasma hyopneumoniae-positive samples were assayed by multilocus sequence typing (MLST). The aim of the study was to reveal the distribution of M. hyopneumoniae and determine the genotypes of M. hyopneumoniae in pig herds in China based on MLST. Results Among these 989 samples, 199 samples were M. hyopneumoniae-positive. The M. hyopneumoniae positivity rate was 7.2% (35/494) in 2018, 18.4% (38/207) in 2019, and 43.8% (126/288) in 2020. In total, 47 samples were successfully assayed by MLST. Sixteen new M. hyopneumoniae sequence types from 9 provinces were recorded in the present study. Conclusions This is the first report on sample positivity rates and molecular typing results for M. hyopneumoniae in swine herds in China. MLST has revealed high genotype diversity among M. hyopneumoniae from different provinces of China.

Prevalence of Swine Diseases in Ijebu Division of Ogun State

This study was carried out by visiting 20 towns/ villages in Ijebu-division of Ogun State, where a total of 5,043 pigs were observed for various disease conditions. Blood and faecal samples and skin scrapings were collected from 137 sick animals, of which 3.65% were positive for the presence of Trypanosoma brucei. The following gastrointestinal nematode eggs were found in the faecal samples evaluated: Ascaris sp. (45.26%), Strongyles (10.22%), Strongyloides sp. (5.11%) and Trichuris sp. (2.92%). Sarcoptes scabiei which was the only ectoparasite of pigs encountered in this study accounted for 12.41% of the 137 skin scrapings collected. A total of 606 out of 5,043 animals were found to be clinically sick. Identified disease conditions include wound infection (33.50%), diarrhea (22.11%), piglet anaemia (15.84%), mange (10.89%), tail sore (6.44%), foot rot (4.95%), mastitis (2.97%), metritis (1.82%) and pneumonia (1.49%).

Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in PDB, which showed classic and non-classical types were mainly determined by the C-terminal β-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd=15.6μM) and with some selectivity (~2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

A novel multi-epitope recombinant protein for diagnosis of African swine fever

Background: African swine fever(ASF) is an acute, severe and highly fatal infectious disease of pigs. The disease spreads rapidly, causing huge economic losses to the pig industry in infected areas. The structural proteins p30 and p54 in African swine fever virus(ASFV) have been verified as diagnostic antigens.Methods: In this study, we constructed a novel multi-epitope fusion antigen gene based on P30 and P54 proteins, induced expression in a prokaryotic expression system and analyzed the reactivity of the recombinant fusion protein. The purified recombinant protein m35 was used as the coating antigen to establish an indirect enzyme-linked immunosorbent assay (ELISA) detection method for ASFV. 116 serum samples and positive sera of other swine diseases were detected by indirect ELISA.Results: Our results indicate that the m35 gene fragment with a length of 558bp was successfully constructed. SDS-PAGE and Western Blotting analysis showed that the protein had a band at 22kDa, proving its good reactogenicity. ROC analysis was performed to validate the assay, the area under the ROC curve is 0.9738 (95% confidence interval, 0.9336 to 1.014), and does not cross-react with other swine diseases.Conclusion: Our results show that its sensitivity and specificity were highly accurate. It is feasible to use this recombinant protein as a diagnostic antigen to distinguish ASFV infection.

Surveillance results of communicable swine diseases in breeding pig farms near Ulaanbaatar city

We have done laboratory-based analyses of highly contagious and infectious swine diseases commonly occurring in household and pig farms on 200 swine serum samples collected from Bayanzurkh, Songinokhairkhan, Nalaikh, Khan-Uul district of Ulaanbaatar city, to establish the rate of infection, to gather information on farming and bio-security, to further detect this infectious diseases, for the purpose of risk assessment and preventive research. According to ELISA test results Classical swine fever 27 (13.5%), Parvovirus 50 (25%), Influenza 16 (8%), Circovirus 112 (56%) samples had shown positive results. Presence of classical swine fever antibody in 27/200 samples are reaction of vaccine immunization. Also antibody presence of Swine parvovirus, Influenza, circovirus in samples indicates necessity of make it clear that whether symptomless cases around farms. But antibody absence of porcine food and mouth disease, porcine reproductive and respiratory syndrome virus, swine mycoplasmosis transmissible gastro-enteritis, coronovirus, leptospirosis, brucellosis indicates such disease free in farms. Antibody absence of African swine fever done by surveillance this time shows no further infection however it was occurred in Mongolia early in 2019. Fundamental surveying is a key tool for monitoring health status of pig farm and formulating eradication program for swine infectious diseases as well as help farmers prevent economic losses by implementing prophylactic measures against sudden cases of such diseases. Thus we suggest that above mentioned surveillance study need to be continued as a tool for monitoring because of it is importance of keep pig farmers sustainable, furthermore crucial for nation`s economy. Улаанбаатар хотын ойролцоо үржүүлж буй гахайд зонхилон тохиолдох халдварт өвчнүүдийг тандан судалсан дүн Улаанбаатар хотын Баянзүрх, Налайх, Сонгинохайрхан, Хан-Уул зэрэг 4 дүүргийн өрхийн болон аж ахуйн гахайд зонхилон тохиолддог гоц болон халдварт өвчнүүдийн лабораторид суурилсан шинжилгээг хийх, халдварлалтын түвшинг тогтоох, аж ахуй эрхлэлт, биоаюулгүй байдлын талаар мэдээлэл цуглуулах, цаашид халдварт өвчнийг эрт илрүүлэх, эрсдэлийг үнэлэх, урьдчилан сэргийлэх судалгааны ажлын зорилгын хүрээнд нийт 200 дээжинд эсрэгбием илрүүлэх ЭЛИЗА урвалаар шинжилгээг хийхэд гахайн сонгомол мялзан 27 (13.5%), парвовирус 50 (25%), томуу 16 (8%), цирковирус 112 (56%) дээж эерэг дүн үзүүлэв. Гахайн сонгомол мялзан өвчний эсрэгбием (27/200) илэрсэн нь урьдчилан сэргийлэх зорилгоор тарьсан вакцины урвал юм. Гахайн парвовирус, гахайн томуу, гахайн цирковирус зэрэг өвчний вирусын эсрэгбием илэрч байгаа нь ил шинж тэмдэггүй, халдварын тархалт байгаа эсэхийг баталгаажуулах шаардлагатайг харуулж байна. Харин гахайн шүлхий, микоплазм, гахайн үржил, амьсгалын замын хам шинж, гахайн ходоод гэдэсний замын халдварт үрэвсэл, коронавирус, лептоспироз, бруцеллёз зэрэг өвчний эсрэгбием илрээгүй нь халдваргүйг харуулж байна. Гахайн африкийн мялзан өвчин Монгол оронд 2019 оны эхээр гарч байсан ч энэ удаагийн тандалтаар эсрэгбием илрээгүй нь өвчлөл дахиж гараагүй болохыг харуулж байна. Тандан судалгаа нь гахайн эрүүл мэндийг хянах, эрүүлжүүлэх хөтөлбөрийг боловсруулах гахайн аж ахуйд тохиолдох эдийн засгийн болзошгүй эрсдэлээс урьдчилан хянахад чухал ач холбогдолтой. Тиймээс энэхүү суурь судалгааг жил бүр тогтмол хийх нь гахайн аж ахуйг тогтвортой байлгах төдийгүй улс орны эдийн засагт чухал ач холбогдолтой тул гахайн эрүүл мэндийг хянах зорилгоор үргэлжлүүлэн хийж байх шаардлагатай байна. Түлхүүр үг: Гахайн халдварт өвчнүүд, ЭЛИЗА, тандан судалгаа, эсрэгбием

Multiplex and On-site PCR detection of swine diseases based on the microfluidic chip system

Background: At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process takes a rather long time, has the risks of degradation of biological samples and generation of pathogenic microorganisms, and does not meet the rapid on-site detection demand. Therefore, development of a technology for rapid and high-throughput detection of pathogenic microorganisms at the customs port is of great significance. This study was to develop a microfluidic chip to be applied to rapid high-throughput detection for swine disease with higher accuracy and lower risk of spreading pathogenic microorganisms during transportation. Results: PCR technology has the advantages of high accurate and sensitivity in disease detection, clinical testing and food quarantine, so it plays an important role in customs inspection. However, the traditional PCR detection instrument has a large size, is time-consuming and has strict requirements on the experimental environment, which greatly limit its application in on-site testing. In this paper, the positive nucleic acid of four swine diseases were detected by a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Eight clinical samples were detected together on the microfluidic chip in the system, and the detection results were obtained in about an hour. The detection limit of this microfluidic PCR detection system was as low as 1 copies/μL. The results show that the high sensitivity and specificity of the microfluidic PCR detection system in disease detection will play an important role in customs inspection and quarantine during customs clearance. Conclusion: The microfluidic PCR detection system established in this study could meet the requirements for rapid detection of samples at the customs port The new method can avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reducing the inspection cycle, and would enable parallel inspections on one chip which greatly raising the efficiency of inspection.

Multiplex and On-site PCR detection of swine diseases based on the microfluidic chip system

Background：At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process takes a rather long time, has the risks of degradation of biological samples [32] and generation of pathogenic microorganisms [33], and does not meet the rapid on-site detection demand [34]. Therefore, development of a technology for rapid and high- throughput detection of pathogenic microorganisms at the customs port is of great significance. This study was to develop a microfluidic chip to be applied to rapid high-throughput swine disease detection with higher accuracy and lower risk of spreading pathogenic microorganisms during transportation.Results: PCR technology has the advantages of high accurate and sensitivity in disease detection, clinical testing and food quarantine, so it plays an important role in customs inspection. However, the traditional PCR detection instrument has a large size, is time-consuming and has strict requirements on the experimental environment, which greatly limit its application in on-site testing. In this paper, the clinical samples of four swine diseases were detected by a portable and rapid microfluidic PCR system, which could achieve a on- site real-time quantitative PCR detection. Eight clinical samples were detected together on the microfluidic chip in the system, and the detection results were obtained in about an hour. The detection limit of this microfluidic PCR detection system was as low as 1 copies/μL. The results show that the high sensitivity and specificity of the microfluidic PCR detection system in disease detection will play an important role in customs inspection and quarantine during customs clearance.Conclusion: The microfluidic PCR detection system established in this study could meet the requirements for rapid detection of samples at the customs portThe new method can avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reducing the inspection cycle, and would enable parallel inspections on one chip which greatly raising the efficiency of inspection.

SPDB: a specialized database and web-based analysis platform for swine pathogens

The rapid and accurate diagnosis of swine diseases is indispensable for reducing their negative impacts on the pork industry. Next-generation sequencing (NGS) is a promising diagnostic tool for swine diseases. To support the application of NGS in the diagnosis of swine disease, we established the Swine Pathogen Database (SPDB). The SPDB represents the first comprehensive and highly specialized database and analysis platform for swine pathogens. The current version features an online genome search tool, which now contains 26 148 genomes of swine, swine pathogens and phylogenetically related species. This database offers a comprehensive bioinformatics analysis pipeline for the identification of 4403 swine pathogens and their related species in clinical samples, based on targeted 16S rRNA gene sequencing and metagenomic NGS data. The SPDB provides a powerful and user-friendly service for veterinarians and researchers to support the applications of NGS in swine disease research. Database URL: http://spdatabase.com:2080/