Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA

Pseudorabies (Aujeszky disease) virus (PRV) was eliminated from domestic swine in many countries using glycoprotein E (gE)-deleted vaccines and serum antibody gE ELISAs, but PRV continues to circulate in some regions and in most feral swine populations in the world. We created a dual-matrix (serum and oral fluid) indirect IgG gE ELISA (iELISA) and evaluated its performance using samples from 4 groups of 10 pigs each: negative control (NC), vaccination (MLV), PRV inoculation (PRV), and vaccination followed by challenge (MLV-PRV). All serum and oral fluid samples collected before PRV challenge and all NC samples throughout the study were negative for gE antibodies by commercial blocking ELISA (bELISA) and our iELISA. Nasal swab samples from 9 of 10 animals in the PRV group were gB quantitative PRC (qPCR) positive at 2 days post-inoculation (dpi). The oral fluid iELISA detected a significant S/P response in the PRV ( p = 0.03) and MLV-PRV ( p = 0.01) groups by 6 dpi. ROC analyses of serum bELISA ( n = 428), serum iELISA ( n = 426), and oral fluid iELISA ( n = 247) showed no significant differences in performance ( p > 0.05). Our data support the concept of PRV surveillance based on oral fluid samples tested by an indirect gE ELISA.

Molecular detection and clinical aspects of porcine circovirus 3 infection in pigs from Brazil

ABSTRACT Porcine circovirus 3 (PCV-3) DNA has been detected in serum samples from apparently healthy pigs as well as pigs with different clinical conditions. Molecular detection of PCV-3 was observed in swine serum samples from Southeastern - Brazil using a nested PCR designed specifically for this study. The epidemiology and clinical aspects of PCV-3 infection were evaluated. The samples originated from 154 pigs of both genders from different production phases and with different clinical presentations, sampled from 31 pig farms visited between 2013 and 2018. In this study, PCV-3 was detected in 26.7% of samples from all populations across varying ages. Statistical association (P=0.0285) was observed only between animals with respiratory signs and PCV-3; no PCV-3-positive animal had diarrhea. No statistical association was observed between PCV-3 and age, or gender of the pigs. Because PCV-3 is a newly discovered virus, there is very little information about its epidemiology. We hope that these data can help in future studies investigating PCV-3 epidemiology.

Streptococcus suis Uptakes Carbohydrate Source from Host Glycoproteins by N-glycans Degradation System for Optimal Survival and Full Virulence during Infection

Infection with the epidemic virulent strain of Streptococcus suis serotype 2 (SS2) can cause septicemia in swine and humans, leading to pneumonia, meningitis and even cytokine storm of Streptococcal toxic shock-like syndrome. Despite some progress concerning the contribution of bacterial adhesion, biofilm, toxicity and stress response to the SS2 systemic infection, the precise mechanism underlying bacterial survival and growth within the host bloodstream remains elusive. Here, we reported the SS2 virulent strains with a more than 20 kb endoSS-related insertion region that showed significantly higher proliferative ability in swine serum than low-virulent strains. Further study identified a complete N-glycans degradation system encoded within this insertion region, and found that both GH92 and EndoSS contribute to bacterial virulence, but that only DndoSS was required for optimal growth of SS2 in host serum. The supplement of hydrolyzed high-mannose-containing glycoprotein by GH92 and EndoSS could completely restore the growth deficiency of endoSS deletion mutant in swine serum. EndoSS only hydrolyzed a part of the model glycoprotein RNase B with high-mannose N-linked glycoforms into a low molecular weight form, and the solo activity of GH92 could not show any changes comparing with the blank control in SDS-PAGE gel. However, complete hydrolyzation was observed under the co-incubation of EndoSS and GH92, suggesting GH92 may degrade the high-mannose arms of N-glycans to generate a substrate for EndoSS. In summary, these findings provide compelling evidences that EndoSS-related N-glycans degradation system may enable SS2 to adapt to host serum-specific availability of carbon sources from glycoforms, and be required for optimal colonization and full virulence during systemic infection.

Identification of Porcine circovirus type (PCV2) and type 3 (PCV3), Porcine 2 parvovirus (PPV) in swine by multiplex PCR test

Background : Aiming to simultaneously detect three important viruses known to be involved in reproductive problems of sows, a multiplex PCR (mPCR) test was developed to provide rapid diagnosis of porcine circovirus type 2 and 3 (PCV2, PCV3) and to illustrate parvovirus (PPV) prevalence in sow herds. Methods : Three pairs of specific primers were designed to target PCV2 Cap gene, PCV3 Cap gene and PPV NS1 gene, with predicted mPCR products of 702 bp, 267 bp and 380 bp, respectively. Results : The detection limit of mPCR was 100 copies/ reaction per target gene. Sequencing of mPCR products performed with clinical serum samples accurately confirmed results. The mPCR was run against a panel of 94 swine serum samples whose infection status had been pre-determined by commercial real-time PCR kits. Overall, the mPCR results matched 100% with the real-time PCRs. Conclusions : The developed mPCR test functions successfully and can be used in routine rapid diagnosis of PCV2, PCV3 and PPV.

Self-assembly into virus–like particles of the recombinant capsid protein of porcine circovirus type 3 and its application on antibodies detection

PCV3 capsid protein (Cap) is an important antigen for diagnosis and vaccine development. To achieve high-level expression of recombinant PCV3 Cap in Escherichia coli (E. coli), the gene of wild-type entire Cap (wt-eCap) was amplified from clinical samples, and three optimized entire Cap (opti-eCap) and one optimized Cap deleted nuclear location signal (NLS) (opti-dCap) gene fragments encoding the same amino acid sequence with wt-eCap were synthesized based on the codon bias of E. coli. Those gene fragments were inserted into the pET30a expression vector. One recombinant strain with the highest expressed soluble eCap from four entire Cap (one wt-eCap and three opti-eCap) and one recombinant strain expressed opti-dCap were selected for further purification. The purified eCap and dCap were identified by transmission electron microscopy (TEM), a large number of round hollow particles with a diameter of 10 nm virus-like particles (VLPs) were observed in eCap, whereas irregular aggregation of proteins observed in dCap. After formation the VLPs were applied as a coating antigen to establish an indirect ELISA (I-ELISA) for detection of PCV3-specific antibody in swine serum. 373 clinical swine serum samples from China collected in 2019 were tested utilizing the VLP-based I-ELISA method under optimized conditions. To the best of our knowledge, this is the first report of self-assembly into VLPs of PCV3 recombinant Cap. Our results demonstrated that the VLP-based I-ELISA will be a valuable tool for detecting the presence of PCV3 antibodies in serum samples and will facilitate screening of large numbers of swine serum for clinical purposes.

Study of leptospirosia in animal and livestock

Leptospirosis is a zoonotic bacterial disease that affects vulnerable populations such as rural subsistence farmers and urban slum dwellers. Leptospirosis is an infectious disease caused by pathogenic organisms belonging to the genus leptospira, that are transmitted directly or indirectly from animals to humans. In Mongolian human cases of leptospirosis has not been reported yet and this zoonotic disease not well study. We had use the kit in order to detect Pomona, Tarassovi, Hebdomadis, Icterohaemorrhagiae, Grippotyphosa, Sejroe and Canicola serotype by Enzyme-linked immunosorbent assay (ELISA) test. We analyzed 397 samples of bovine serum by indirect ELISA. The bovine serum samples were collected from Selenge province, and out of 101 samples 2 were positive. And, out of 171 bovine 1 serum was positive in Tov province. In contrast, no positive samples detected in 137 swine serum. Linnodee leptospira /ireland/- ELISA kit can detect a swine Bratislava serotype and cattle Hardjo serotype. To detect Hardjo serotype, 397 samples of cattle serum and 137 samples of swine serum were investigated. The 29 serum of cattle were positive, which has 7.3% infection rate, and, only one swine sera was positive out of 137, that has 0.73% infection rate. The 137 swine serum were tested by ELISA, which can detect Leptospira Bratislava serotype; and 12 out of that were positive. This indicates 8.7% of all sample are positive. Total of 397 bovine sera were examined by ELISA and specific antibody against Pomona, Tarassovi, Hebdomadis, Icterohaemorrhagiae, Grippotyphosa, Sejroe and Canicola serotype detected in 0,75 % (3 sera samples). Hardjo serotype detected in 7.3 % (29 sera) of bovine samples and 7.6 % (1 serum) of 13 swine sera samples and furthermore, bratislava serotype antibody detected in 8.7% (12 sera) of the pigs included in our study. Our study indicates that risk of human leptospirosis infection through animal derived food consumption, soil and water contamination is present due to prevalence of hardjo and bratislava serotype in cattle and pig farms. These results correlates with study conducted by Odontsetseg N. PhD in 2005 which stated that Hardjo serotype of Leptospira interrogans was detected in cattle herd in our country and these suggest that leptospirosis is prevalent in certain regions of our country. Мал амьтны лептоспирозийн тандан судалгааны дүн Хураангуй: Мал амьтны гаралтай хүнсний бүтээгдэхүүн ус, хөрсөөр дамжин хүнд халдварладаг зооноз өвчин болох Лептоспирозийн танадан судалгааг Булган, Орхон, Сэлэнгэ, Төв аймгууд болон Улаанбаатар хот орчмоос цуглуулсан үхрийн 721, гахайн 169, зарим мэрэгч амьтдын 108, усны 22 нийт 1020 сорьцыг ийлдэс судлал болон молекул биологи, нян судлалын аргаар шинжлэв. Судалгааны дүнд шинжилгээнд хамрагдсан нийт 397 үхрийн сорьцны 32 буюу 8.06%, 137 гахайн сорьцны 13 буюу 9.4%-д нь L. pomona, L. tarassovi, L. hebdomadis, L. icterohaemorrhagiae, L. grippotyphosa, L. sejroe, L. canicola, L. hardjo, L. bratislava хэвшлүүдийн эсрэг үүссэн өвөрмөц эсрэгбием тус тус илрэв. Лептоспирозийн байгалийн дамжуулагч болох мэрэгчийн 108-н сорьцонд Полимериазан Гинжин Урвал (ПГУ) тавихад 8 сорьц буюу 7.4%-д нь лептоспирозийн G1, G2 генийн өвөрмөц бүтээгдэхүүн илэрсэн болно. Ийлдэс судлал, ПГУ- аар эерэг гарсан сорьцуудад үүсгэгч илрүүлэх нян судлалын шинжилгээ хийсэн боловч үүсгэгч өсгөвөрлөгдсөнгүй. Манай орны Орхон, Сэлэнгийн сав газар, Архангай, Төв аймаг, Улаанбаатар хот орчмын үхэр, гахай, мэрэгчидийн сорьц лептоспирозоор эерэг дүн үзүүлсэн нь манай орны мал амьтан, мэрэгчидэд өвчний халдварлалт байгааг харуулж байна. Түлхүүр үг: Leptospira, үүсгэгч, өвөрмөц эсрэгбием, ийлдсийн хэвшил, ген