Veterinary microbiology最新文献

Pneumonia caused by Mesomycoplasma hyopneumoniae (Mhp) is a respiratory disease with high morbidity and low mortality that typically presents in growing pigs. Although often subclinical, the disease can significantly affect the pig farming industry economically due to decreased growth rates and inefficient feed conversion. Effective control of Mhp depends on the detection of dominant strains prevalent in infected animals, which vary in virulence. However, traditional culture methods for diagnosing Mhp are laborious and slow, whereas multi-locus sequence typing, another possible method, requires identifying several genes. This study introduces a novel pair of polymerase chain reaction (PCR) primers for the rapid detection and genetic evolution analysis of Mhp strains to facilitate improved vaccine selection. The genetic evolutionary tree established using the PCR amplification fragment was highly similar to the genetic evolutionary tree established using whole-genome sequences. Analysis of 131 samples from Guangxi and Hunan slaughterhouses revealed a 30.53 % prevalence of Mhp. High-throughput sequencing has shown that Mhp has a diverse bacterial population in clinically collected samples. The prevalence of major strains may vary among regions. Additionally, the strains of Mhp vaccines sold may differ significantly from the strains prevalent on farms. In summary, this work has designed a pair of primers that will be useful for detecting the diversity of Mhp and for targeted prevention and control.

Porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome in piglets. Differences in the infectivity and horizontal transmissibility of different isolates of PCV2a, PCV2b, and PCV2d in pigs were evaluated by HE and IHC staining, PCR, virus titration, and IPMA to determine their clinical symptoms, pathological changes, levels of virus and antibody, and cohabitation infectivity. In the cohabitation infection experiment, weak viremia and low levels of antibodies were detected in the pigs challenged with PCV2a-CL, whereas no viremia or antibodies were detected in the corresponding cohabiting pigs. Furthermore, no PCV2 was isolated from any organ of pigs that were challenged with PCV2a-CL, as well as from those of their cohabiting pigs. In contrast, persistent viremia and pathological changes, including swollen inguinal lymph nodes, were detected in both the challenged and cohabiting pigs after PCV2b-BY or PCV2d-LNHC infection. Alive PCV2 was detected in the tonsils, inguinal lymph nodes, spleen, and kidneys of the experimental pigs by virus titration, and the highest viral titer was detected in the tonsils, followed by the inguinal lymph nodes. In a comparative analysis of the challenged and cohabiting pigs, a 1-week delay in viremia and specific antibodies was observed in the cohabiting pigs. Moreover, the number of viruses isolated from the tonsils and inguinal lymph nodes of the pigs cohabiting with PCV2d-LNHC-challenged pigs was significantly greater than those in the pigs that were directly challenged with PCV2d-LNHC in cohabitation infection experiment (P<0.05). Together, these results indicated that the infectivity and horizontal transmissibility of the strains PCV2b-BY and PCV2d-LNHC were much greater than those of the strain PCV2a-CL and provided some insights into PCV2 pathogenicity.

The complement system is the first defense line of the immune system. However, pathogens have evolved numerous strategies to evade complement attacks. Streptococcus suis is an important zoonotic bacterium, harmful to both the pig industry and human health. ApuA has been reported as a bifunctional amylopullulanase and also contributed to virulence of S. suis. Herein, we found that ApuA could activate both classical and alternative pathways of the complement system. Furthermore, by using bacterial two-hybrid, far-western blot and ELISA assays, it was confirmed that ApuA could interact with complement C3b. The interaction domain of ApuA with C3b was found to be its α-Amylase domain (ApuA_N). After construction of an apuA mutant (ΔapuA) and its complementary strain, it was found that compared to the wild-type strain (WT), ΔapuA had significantly increased C3b deposition and membrane attack complex formation. Additionally, ΔapuA showed significantly lower survival rates in human serum and blood and was more susceptible to engulfment by neutrophils and macrophages. Mice infected with ΔapuA had significantly higher survival rates and lower bacterial loads in their blood, lung and brains, compared to those infected with WT. In summary, this study identified ApuA as a novel factor involved in the complement evasion of S. suis and suggested its multifunctional role in the pathogenesis of S. suis.

The objectives of this study were to evaluate mucosal immune responses in peripartum Holstein cows, to assess the impact of intranasal modified live viral (MLV) vaccination on mucosal immunity, and to explore the relationship between genotype and peripartum immune responses. Eighty multiparous Holstein cows were randomized to receive either: 1) intranasal MLV tri-valent viral vaccine 18–24 days prior to expected calving (DC); 2) the same vaccine within twelve hours after parturition (F); 3) vaccine at both time points (DCF), or 4) no vaccine (CON). Nasal secretions and sera were collected from all cattle pre-vaccination and on multiple days before and after calving to determine concentrations of interferon beta (IFN-beta) and IFN-gamma and bovine herpesvirus-1 (BHV-1-) and bovine respiratory syncytial virus (BRSV-) specific IgA in nasal secretions, and BHV-1 and BRSV serum neutralizing (SN) titers. Cows were genotyped by bead-based microarray, genotypes were used to categorize previously established health traits, and relationships between immune responses and genotype were evaluated. There was no significant effect of vaccination on immune responses, although all vaccinated groups demonstrated numerically increased IFN-gamma within four days post vaccination. There was a significant (P <0.0001) time effect on nasal IgA in CON, F, and DCF groups, with the highest nasal IgA titers measured post calving. There was a significant (P <0.0001) time effect on nasal IFN-beta in all groups. Significant relationships between genotype and immune response were not detected. Contrary to previous reports of systemic immunosuppression, bovine mucosal responses appear to be intact in the peripartum period.

The Porcine reproductive and respiratory syndrome (PRRS) causes severe financial losses to the global swine industry. Due to continuous virus evolution, the protection against the PRRS provided by current vaccines is limited. In order to find new antiviral strategies, this study investigated the antiviral potential of antimicrobial peptides (AMPs) against PRRSV. Given the diversity of PRRSV strains and the limited effectiveness of existing vaccines in controlling PRRSV, this study evaluated the inhibitory effects of KLAK, Cecropin B, Piscidin1, and Caerin1.1 on 3 strains of PRRSV (lineage 5 classical strain, lineage 8 highly pathogenic strain, and lineage 1 NADC30-like strain). Caerin1.1 exhibited significant dose-dependent antiviral activity, with an effective concentration (EC₅₀) of 7.5 μM. Caerin1.1 effectively inhibited PRRSV replication when added before or in early infection but showed reduced effectiveness when added in late infection, indicating its potential involvement in targeting early transcription mechanisms of viral RNA polymerase and significantly upregulating cytokine gene expression. In the NADC30 strain-based animal infection model, Caerin1.1 treatment significantly reduced lung viral loads and inflammation in the lungs of PRRSV-infected pigs, with a mortality rate of 0 % (0/5) in the treated group compared to 66.67 % (4/6) in the untreated group, indicating a reduction in the mortality rate. Additionally, compared with the untreated group, the Caerin1.1-treated group showed significant improvements, such as lighter fever, more daily weight gain, less clinical symptoms, less viral load in blood, and less virus oral shedding (P < 0.05). These findings reveal the potential of antimicrobial peptides as PRRSV therapeutic agents and suggest that Caerin1.1 is a promising candidate for a novel anti-PRRSV drug.

Porcine deltacoronavirus (PDCoV), a cross-species transmissible enterovirus, frequently induces severe diarrhea and vomiting symptoms in piglets, which not only pose a significant menace to the global pig industry but also a potential public safety risk. In a previous study, we isolated a vaccine candidate, PDCoV CZ2020-P100, by passaging a parental PDCoV strain in vitro, exhibiting attenuated virulence and enhanced replication. However, the factors underlying these differences between primary and passaged strains remain unknown. In this study, we present the transcriptional landscapes of porcine kidney epithelial cells (LLC-PK1) cells infected with PDCoV CZ2020-P1 strain and P100 strain using the RNA-sequencing. We identified 105 differentially expressed genes (DEGs) in P1-infected cells and 295 DEGs in P100-infected cells. Enrichment analyses indicated that many DEGs showed enrichment in immune and inflammatory responses, with a more and higher upregulation of DEGs enriched in the P100-infected group. Notably, the DEGs were concentrated in the MAPK pathway within the P100-infected group, with significant upregulation in EphA2 and c-Fos. Knockdown of EphA2 and c-Fos reduced PDCoV infection and significantly impaired P100 replication compared to P1, suggesting a novel mechanism in which EphA2 and c-Fos are highly involved in passaged virus replication. Our findings illuminate the resemblances and distinctions in the gene expression patterns of host cells infected with P1 and P100, confirming that EphA2 and c-Fos play key roles in high-passage PDCoV replication. These results enhance our understanding of the changes in virulence and replication capacity during the process of passaging.

Many cattle infected with Mycoplasma bovis remain healthy while others develop severe chronic respiratory disease. We hypothesized that inflammatory stimuli such as co-pathogens worsen disease outcomes in M. bovis-infected calves. Calves (n=24) were intrabronchially inoculated with M. bovis and either killed bacterial lysate, transient M. haemolytica infection, or saline. Caseonecrotic lesions developed in 7/7 animals given M. haemolytica and M. bovis compared to 2/8 given M. bovis with no inflammatory stimulus, and 6/9 animals given bacterial lysate and M. bovis (P=0.01). Animals receiving M. haemolytica and M. bovis had more caseonecrotic foci in lungs than those receiving M. bovis with no inflammatory stimulus (median = 21 vs 0; P = 0.01), with an intermediate response (median = 5) in animals given bacterial lysate. In addition to caseonecrotic foci, infected animals developed neutrophilic bronchiolitis that appeared to develop into caseonecrotic foci, peribronchiolar lymphocytic cuffs that were not associated with the other lesions, and 4 animals with bronchiolitis obliterans. The data showed that transient lung inflammation at the time of M. bovis infection provoked the development of caseonecrotic bronchopneumonia, and the severity of inflammation influenced the number of caseonecrotic foci that developed. In contrast, caseonecrotic lesions were few or absent in M. bovis-infected calves without a concurrent inflammatory stimulus. These studies provide insight into how caseonecrotic lesions develop within the lung of M. bovis-infected calves. This and other studies suggest that controlling co-pathogens and harmful inflammatory responses in animals infected with M. bovis could potentially minimize development of M. bovis caseonecrotic bronchopneumonia.

Herpesviruses-encoded microRNAs (miRNAs) have been discovered to be essential regulators in viral life cycle, participating in viral replication, latent or lytic infection, and immunological escape. However, the roles of miRNAs encoded by duck plague virus (DPV) are still unknown. Dev-miR-D28-3p is a miRNA uniquely encoded by DPV CHv strain. The aim of this study was to explore the effect of dev-miR-D28-3p on DPV replication and explore the potential mechanisms involved. Our findings demonstrated that transfection of dev-miR-D28-3p mimic into duck embryo fibroblasts (DEFs) effectively suppressed viral copies, viral titers and viral protein expressions during DPV infection, while the results above were reversed after transfection with dev-miR-D28-3p inhibitor. Subsequently, we further discovered that dev-miR-D28-3p specifically bound to DPV-encoded UL27 and inhibited its expression, suggesting that UL27 was the target gene of dev-miR-D28-3p. Finally, we investigated the role of UL27 in DPV replication and found the overexpression of UL27 increased viral copies, viral titers, and viral protein expressions; whereas the opposite results appear when knockdown of UL27. Our findings illustrated a novel mechanism that DPV regulated itself replication via dev-miR-D28-3p, paving the way for exploring the role of DPV-encoded miRNAs.

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic orthoflavivirus causing human encephalitis and reproductive disorders in pigs. Cell-intrinsic antiviral restriction factors are the first line of defense that prevent a virus from establishing a productive infection, while the molecular mechanism of the virus-host interaction is still not fully understood. Our in vitro experiments demonstrated that the Solute Carrier Family 25 Member 12 (SLC25A12) interacted with the JEV nonstructural protein 1 (NS1) and inhibited JEV replication. Furthermore, we showed that knockdown or knockout of SLC25A12 promoted JEV replication, while overexpression of SLC25A12 repressed viral replication. Finally, we demonstrated that SLC25A12 increased IRF7 mRNA levels, which promoted IFN-β expression and subsequently induced antiviral effects. Collectively, our study revealed that SLC25A12 interacted with NS1, inhibiting viral RNA synthesis and transcription and enhancing type I interferon induction for antiviral effects.

Porcine epidemic diarrhea virus is attenuated upon adaptation to cell culture. Exclusively genomic mutations have been traced to the ORF3 gene of the laboratory strains. Previous attempts to express the protein were unsuccessful. We sought to express the ORF3 protein in both mammalian and bacteria cells as a prerequisite for investigation of the protein’s role. For prokaryotic expression, two vector systems, pET28-a(+) and pGEX-4T-1 were constructed and expressed in Escherichia coli cells. For eukaryotic analyses, ORF3/pEGFP-C1 vector constructs were expressed in human embryonic, green monkey kidney and mouse fibrous cells. Intriguingly, there was minimal expression of the ORF3 gene. Following a documented hint that truncated ORF3 revealed higher expression, ORF3 gene was truncated. The simple modular architecture research tool analysis predicted two transmembrane domains between amino acid (aa) 41–63 and aa 76–98. Consequently, we generated two fragments; ORF-N (aa 1–98) inclusive of transmembrane domains and ORF3-C (aa 99–224). These truncated sequences were constructed as the whole gene here referred to as ORF3 wild type (wt). Coomassie blue stained gels revealed bands of ORF3-C expressed as a fusion protein of 17.5 and 39 kDa in pET28-a(+) and pGEX-4T-1 vectors, respectively. In contrast, ORF3-N was not. Additionally, ORF3-N induction decreased total cellular proteins suggesting inhibition of protein synthesis or metabolism. Solubility tests carried out at 30 °C, 25 °C and 18 °C showed that ORF3 formed inclusion bodies. Similar findings were observed in mammalian cells. Noteworthy, morphological distortions appeared in mammalian cells expressing ORF3 protein or its truncated mutants suggesting significance in host viability.