The in vitro antiviral activity of Lacticaseibacillus casei MCJ protein-based metabolites on bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is a ubiquitous immunosuppressive etiological agent which is economically important for a wide host range in the livestock industry. Lactobacillus spp. has widely been using in the field of management and treatment of gastro-enteric disease for both humans and animals. The ability of Lacticaseibacillus casei MCJ protein-based metabolized to suppress BVDV infection in Madin-Darby Bovine Kidney cell line was demonstrated in this study. The protein-based metabolites were extracted from the cultured L. casei to obtain the safest and beneficial form of the probiotic bacteria. It is revealed that LPM have no cytotoxic effect and the cell viability remain more than 80% even after the cells are treated with 3000 µg/mL of LPM. The results of the plaque formation assay showed that LPM can reduce the viral infection rate. To know the mechanism of LPM for anti-BVDV activity, MDBK cells were exposed to LPM before, after and co-incubation of virus infection. The co-treatment of LPM with BVDV revealed the best results. The results suggest that the LPM has a potential anti-BVDV activity which could be a prospective candidate for the prevention and control of BVDV infection in an animal.

Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus.

Rotaviruses are rising as zoonotic viruses worldwide, causing the lethal dehydrating diarrhea in children, piglets, and other livestock of economic importance. A simple, swift, cost-effective, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of porcine rotavirus-A (PoRVA) by employing rabbit (capture antibody) and murine polyclonal antibodies (detector antibody) produced against VP6 of PoRVA (RVA/Pig-tc/CHN/TM-a/2009/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 protein and PoRVA. The detection limit of AC-ELISA was found 50 ng/ml of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from 295 porcine diarrhea samples, and results were compared with that of RT-PCR and TaqMan RT-qPCR. The relative sensitivity and specificity of AC-ELISA compared with those of TaqMan RT-qPCR were found as 94.4 and 99.2%, respectively, with the strong agreement (κ -0.58) between these two techniques. Furthermore, AC-ELISA could not detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pseudo rabies virus and porcine circovirus-2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples, which suggests that this technique can be used for large-scale surveillance and timely detection of rotavirus infection in the porcine farms.

In this study, we used a Chinese porcine rotavirus-A (PoRVA) strain containing the I5, a dominant VP6-genotype in pigs, for production of VP6 (most conserved) protein based polyclonal antibodies (pAb) in rabbits (as capture Ab) and mouse (as detector Ab) for development of simple, cost effective, highly specific and sensitive AC-ELISA for detection of PoRVA. Furthermore, there is no any previous published report on application of rabbit and mouse pAb against VP6 for developing an AC-ELISA against PoRVA.

Phentotypic, gentotypic antimicrobial resistance and pathogenicity of Salmonella enterica serovars Typimurium and Enteriditis in poultry and poultry products.

For detection and isolation of Salmonella enterica, 650 meat and tissue samples were processed using Rappaport-Vassiliadis Enrichment broth and Salmonella Chromogenic agar followed by confirmation through specific antisera and polymerase chain reaction (PCR) targeting their Specific Serovar Genomic Regions (SSGRS). Isolates were tested for 15 antibiotics (CRO, AMX, GEN, STR, TET, CHL, CLR, LVX, OFX, GAT, CIP, SXT, AMP, LIN and AZM) according to the disc diffusion method and antimicrobial resistant genes (tet(A), tet(B), tet(C), strA/strB, aadA, aac(3)IV), aadB, sul1, sul2 and sul3, blaCMY-2, blaTEM and blaSHV) using PCR. The overall prevalence of Salmonella enterica was 12%, being higher in markets (15%) as compared to poultry farms (37.2%). The MPN of all positive meat and tissue samples was found 3.6 MPN/gram (0.17-18). A total of 234 isolates were obtained, serovar Typimurium (139) and Enteridits (95) were the most prevalent. Antimicrobial resistance patterns were different in different serovars according to origin of Salmonella isolates. The overall isolates were highly resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%, 174/234), TET (64.5%, 151/234) and STR (64.5%, 151/234). Overall, the most common ARG was blaTEM (76%, 178/234), followed by blaSHV (71.7%, 168/234), tet(A) (64%, 151/234) and tet(B) (64%, 150/234), while the least ARG was aadB (7.2%, 17/234). Both Typimurium and Enteridits were tested in the Balb/C mice for pathogenicity. Both Typimurium and Enteridits were found to cause successful colonization, 100% morbidity but Enteriditis were found to cause 33% mortality.

Co-infection with porcine bocavirus and porcine circovirus 2 affects inflammatory cytokine production and tight junctions of IPEC-J2 cells.

Porcine bocavirus (PBoV) has a high prevalence in both healthy and diseased swine around the world. It was recently reported that PBoV and porcine circovirus type 2 (PCV2)-which contribute to porcine diarrheal disease-have a high rate of co-infection. To clarify the pathogenesis of PBoV, we examined the co-infection rate and effects of these two pathogens in IPEC-J2 porcine intestinal enterocytes. Both single and co-infection had cytopathic effects in IPEC-J2 cells. The apoptosis and proliferation rates of cells infected with both viruses did not differ significantly from those of cells infected with either one alone. PBoV and PCV2 induced the upregulation of inflammatory cytokines and the downregulation of the tight junction proteins occludin and claudin 1 in the early stage of infection, leading to destruction of epithelial barrier integrity and enhanced cytotoxicity. These findings provide insight into the pathogenic mechanisms of PBoV and PCV2 and a basis for developing effective strategies to prevent the spread of gastrointestinal diseases in pigs and other livestock.

The construction of recombinant Lactobacillus casei expressing BVDV E2 protein and its immune response in mice.

Bovine viral diarrhea virus (BVDV) is the etiological agent of BVD causes substantial economic losses and endemic in world-wide cattle population. Mucosal immunity plays an important role in protection against BVDV infection and Lactobacillus casei is believed as an excellent live vaccine vector for expressing foreign genes. In this study, we have constructed a novel recombinant L. casei/pELX1-E2 strain expressing the most immunogenic E2 antigen of BVDV; using growth phage dependent surface expression system pELX1. The expression of E2 protein was verified by SDS-PAGE, Western blotting, and Immunofluorescence microscopic analysis. The immune responses triggered by the E2 producing recombinant L. casei were evaluated in BALB/c mice revealed that oral and intranasal (IN) administration of the recombinant strain was able to induce a significantly higher level of specific anti-E2 mucosal IgA and serum IgG as well as the greater level of cellular response by IFN-γ and IL-12 than those of intramuscular (IM) and control groups of mice. However, IN inoculation was found the most potent route of immunization. The ability of the recombinant strain to induce serum neutralizing antibody against BVDV and reduced viral load after viral challenge indicated better protection of BVDV infection. Therefore, this recombinant L. casei expressing E2 could be a safe and promising mucosal vaccine candidate against BVD.

Prevalence, quantification and isolation of pathogenic shiga toxin Escherichia coli O157:H7 along the production and supply chain of pork around Hubei Province of China.

Shiga toxin Escherichia coli (STEC) O157:H7 is an important zoonotic food borne pathogen causing gastroenteritis that may lead to life threatening hemorragic colitis (HC) and hemorrhagic uremic syndrome (HUS). 325 meat and tissue samples were tested for enumeration of O157:H7 strains using most probable number (MPN)-PCR targeting their specific genes flicH7 and rfbO157 followed by isolation, sereotyping and pathogenicity testing. The overall prevalence of O157:H7 was 41.3% (134/325) along the production and supply chain of pork (PSCP), being higher in supply chain (59%, 118/200) as compared to pig farms (12.8%, 16/125). Along the PSCP, the highest prevalence was found in slaughter houses (86.25%, 69/80) followed by wet- (53.3%, 32/60) and super-markets (28.3%, 17/60). The MPN values ranged from 3 to 1100 MPN/g in overall positive samples, being higher in slaughter houses followed by wet and super markets. Except from intestine and meat samples of slaughter house, the MPN was found higher in summer as compared to winter samples. Eight STEC O157:H7 isolated from meat and liver samples were tested in Balb/C mice for pathogenicity. After development of clinical signs and symptoms, 50-83.3% mortality was produced in the infected mice. Histopathological investigations revealed visible necrosis of intestinal epithelial cells, shedding of cellular debris in the intestine, while in the kidney, necrosis of renal cortical portion of tubular epithelial cells was observed. STEC O157:H7 is prevalent along PSCP around Hubei of China in different proportions being alarmingly higher in supply chain and markets which is a matter of concern for public health.

Genotype distribution of Chinese Mycoplasma bovis isolates and their evolutionary relationship to strains from other countries.

This study was undertaken to determine the genotypic distribution of Chinese M. bovis strains and their similarity to isolates from other countries. Two multilocus sequence typing (MLST) schemes (MLST-1 and MLST-2) and pulsed field gel electrophoresis (PFGE) were used to compare 44 Chinese strains and the M. bovis type strain PG45. The results showed a high genetic homogeneity of Chinese isolates; 43 of 44 (97.7%) Chinese isolates were identified as ST-10 and as ST-34 by MLST-1, while for MLST-2 42 of 44 (95.5%) were identified as ST-10 with the two remaining isolates of ST-32 and ST43. PFGE clustered 42 of 44 (95.5%) of the Chinese isolates into PT-I. The overall agreement rate between the three typing methods was 97.8% (95% CI:86.8-99.9%). The type strain PG45 was identified as a unique type by all three methods. When the MLST-2 scheme was further used to analyze 16 isolates of Australian and Israeli origin ST-10 was more dominant among Australian isolates (7/8), compared with those from Israel (3/8). The evolutionary relationship of the 60 isolates typed in this study assessed together with 206 additional isolates retrieved from pubmlst/mbovis database analyzed by geoBURST Minimum spanning tree (MST) confirmed that the Chinese, Israeli and Australian M. bovis isolates typed in this study that were predominantly ST-10, were clustered in CC3 with isolates originating from the USA. Our results suggest that ST-10 is an emerging clone of M. bovis population. We hypothesized that the widespread distribution of this type is a result of global livestock movements. These findings will help further the understanding of the global evolution of M. bovis and development of novel vaccines against M. bovis.

Development and Validation of Monoclonal Antibody-Based Antigen Capture ELISA for Detection of Group A Porcine Rotavirus.

Porcine rotavirus-A (PoRVA) is one of the common causes of mild to severe dehydrating diarrhea, leading to losses in weaning and postweaning piglets. A rapid, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of PoRVA, by using VP6 (a highly conserved and antigenic protein of group-A rotavirus)-directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). The detection limit of AC-ELISA was found to be equal to that of conventional reverse transcription-polymerase chain reaction (RT-PCR; about 102.5 TCID50/mL). For validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR and TaqMan RT-quantitative PCR (qPCR). The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa = 0.972) between these two techniques. Total detection rate with AC-ELISA, conventional RT-PCR, and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without any statistical significant difference. Moreover, AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudorabies virus, and porcine circovirus-2. These results suggested that our developed method was rapid, highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.

Porcine Epidemic Diarrhea Virus Induces Autophagy to Benefit Its Replication.

The new porcine epidemic diarrhea (PED) has caused devastating economic losses to the swine industry worldwide. Despite extensive research on the relationship between autophagy and virus infection, the concrete role of autophagy in porcine epidemic diarrhea virus (PEDV) infection has not been reported. In this study, autophagy was demonstrated to be triggered by the effective replication of PEDV through transmission electron microscopy, confocal microscopy, and Western blot analysis. Moreover, autophagy was confirmed to benefit PEDV replication by using autophagy regulators and RNA interference. Furthermore, autophagy might be associated with the expression of inflammatory cytokines and have a positive feedback loop with the NF-κB signaling pathway during PEDV infection. This work is the first attempt to explore the complex interplay between autophagy and PEDV infection. Our findings might accelerate our understanding of the pathogenesis of PEDV infection and provide new insights into the development of effective therapeutic strategies.

Genetic characteristics of porcine epidemic diarrhea virus in Chinese mainland, revealing genetic markers of classical and variant virulent parental/attenuated strains.

Since October 2010, porcine epidemic diarrhea (PED) caused by variant porcine epidemic diarrhea virus (PEDV) has led great economic losses to the global pig industry, especially in China. To study the genetic characteristics of PEDV strains in Chinese mainland, a total of 603 clinical samples from nine provinces/districts of Chinese mainland from January 2014 to December 2015 were collected for RT-PCR detection and 1-1323bp of S gene of 91 isolates and ORF3 gene of 46 isolates were sequenced. The results showed that the variant PEDV were the dominant pathogens of viral diarrhea diseases in these areas. Six novel variant PEDV strains (FJAX1, FJAX2, HeNPDS1, HeNPDS2, HeNPY3, and HeNPY4) with two amino acids (aa) deletion at the 56-57 aa of S protein were identified. A total of 405 Chinese PEDV strains were subjected to phylogenetic and phylogeographic analysis. The results revealed that the subgroup Va in variant PEDV group were the dominant subgroup and the spread trend of variant PEDV strains seemed to be from the southeast coastal districts to other coastal districts and interior districts. The N-terminal of S gene (1-750bp), to some extent, could represent S1 or full length S gene for phylogenetic, similarity, antigen index, hydrophilicity plot, and differentiation analyses. The 404-472bp of S gene contained the three genetic markers, i.e., "TAA" insertion at 404-405bp, "ACAGGT" deletion at 430-435bp, and "ATA" deletion at 455-457bp can be used to differentiate the classical and variant virulent parental/attenuated PEDV strains and help us to learn the infectious and genetic characteristics of PEDV strains more convenient and cheaper. This study has important implication for understanding the infectious, genetic, and evolutionary aspects of PEDV strains in Chinese mainland.