Search

BackgroundIntestinal infections with Escherichia coli (E. coli) are mostly occur in piglets between 1~10 days old, which major lead to diarrhea and edema in newborn piglets. These diseases caused by E. coli can increase mortality, morbidity and growth delay of piglets, which are responsible for economic losses. In this study was to investigate the prevalence of antibiotic resistance, transmission mechanisms, and molecular epidemiology of E. coli strains isolated from pig farms in Hubei province. Furthermore, clonal and genetic diversity of isolates were identified. ResultsA total of 29 E. coli isolates were obtained from fecal of weaned piglets from Hubei province. The E. coli isolates in different regions demonstrate different genetic diversity. Multilocus sequence typing (MLST) presented that ST165 was the common sequence type, accounting for 27.6% of all E. coli isolates, followed by ST744, ST1081, ST101 and ST10. All of the isolates were resistant to the tested antibiotics to vary degrees, and more than 80% of E. coli isolates presented high resistance rates to ampicillin, lincomycin, doxycycline, tetracycline, sulfaisoxazole and ampicillin. There was one E. coli strain that was resistant to the fifteen antimicrobial agents tested. Overall, most of the isolates were conferring resistance to 5-7 antimicrobial agents tested.ConclusionsOur study reported E. coli isolates with high antimicrobial resistance and explores the genetic diversity of E. coli isolated from swine-origin. From the results obtained it can be concluded that these isolates present high prevalent multi-drug resistance. These data provide a greater understanding of the genetic diversity and antimicrobial resistance of E. coli.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP) and responsible for major economic losses in global swine industry. After colonization of the respiratory epithelium, M. hyopneumoniae elicits a general mucociliary clearance loss, prolonged inflammatory response, host immunosuppression and secondary infections. Until now, the pathogenesis of M. hyopneumoniae is not completely elucidated. This present study explores the pathogenicity of mhp390 (P68, a membrane-associated lipoprotein) by elucidating its multiple functions. Microtitrer plate adherence assay demonstrated that mhp390 is a new cilia adhesin that plays an important role in binding to swine tracheal cilia. Notably, mhp390 could induce significant apoptosis of lymphocytes and monocytes from peripheral blood mononuclear cells (PBMCs), as well as primary alveolar macrophages (PAMs), which might weaken the host immune response. In addition, mhp390 contributes to the production of proinflammatory cytokines, at least partially, via the release of IL-1β and TNF-α. To the best of our knowledge, this is the first report of the multiple functions of M. hyopneumoniae mhp390, which may supplement known virulence genes and further develop our understanding of the pathogenicity of M. hyopneumoniae.

Japaneses encephalitis (JE) is most common zoonoses caused by Japanese encephalitis virus (JEV) with a high mortality and disability rate. To take timely preventive and control measures, early and rapid detection of JE RNA is necessary. But due to characteristic brief and low viraemia, JE RNA detection remains challenging. In this study, a real-time nucleic acid sequence-based amplification (RT-NASBA) was developed for rapid and simultaneous detection of JEV. Four pairs of primer were designed using a multiple genome alignment of all JEV strains from GenBank. NASBA assay established and optimal reaction conditions were confirmed by using primers and probe on ns1 gene of JEV. The specificity and sensitivity of the assay were compared with RT-PCR by using serial RNA and virus cultivation dilutions. The results showed that JEV RT-NASBA assay was established, and robust signals could be observed in 10 min with high specificity. The limit of dectetion of RT-NASBA was 6 copies per reaction. The assay was thus 100 to 1, 000 times more sensitive than RT-PCR. The cross-reaction was performed with other porcine pathogens, and negative amplification results indicated the high specificity of this method. The novel JEV RT-NASBA assay could be used as an efficient molecular biology tool to diagnose JEV, which would facilitate the surveillance of reproductive failure disease in swine and would be beneficial for public health security.

Bacillus cereus (B. cereus) is widely distributed in the environment. It is one of the most common opportunistic food-borne pathogens associated with food poisoning, not only being majorly reported to cause fatal infections of the gastrointestinal tract, but also responsible for abdominal distress and vomiting. The current study was undertaken to evaluate the biological characteristics and the genetic evolution of B. cereus isolated from infected organs of dead Elaphurus davidianus (E. davidianus). B. cereus was characterized through antibiotic sensitivity tests, mouse lethality assay, whole genome sequencing analysis, and genome annotation. The results revealed that the isolated B. cereus strain was highly resistant to rifampicin, lincomycin, sulfamethoxazole, erythromycin, and ampicillin, with a high pathogenicity phenotype. KEGG annotation revealed that "metabolic pathways" had the largest number of unigenes, followed by "biosynthesis of secondary metabolites" and "biosynthesis of antibiotics". GO analysis resulted in 8039 unigenes categorized. Meanwhile, 54,779 unigenes were annotated and grouped into 23 categories based on COG functional classifications. Moreover, one gene (codY) was found to be related to the host in conformity with the analysis done on PHI-base. Other tests led to the identification of 16 B. cereus virulence factor genes and five resistance types, with potential resistance against bacitracin, penicillin, and fosfomycin. We isolated a highly drug-resistant and pathogenic B. cereus strain from E. davidianus, showing that a variety of antimicrobial drugs should be avoided in clinical treatments. Furthermore, to the best of our knowledge, this is the first study to report whole genome sequencing of a emergence of food-borne B. cereus strain isolated from E. davidianus deer; it will be helpful to extensively investigate the genetic and molecular mechanisms of drug resistance and pathogenesis about B. cereus in both humans and animals.

Porcine circovirus type 2 (PCV2) can cause various clinical diseases in pigs, resulting in huge losses for the pig farms all over the world. In order to develop a new strategy to control PCV2, it is essential to understand its mechanisms firstly, especially PCV2 interferes with the host's innate immunity. In the present study, lncRNA and mRNA expression profiles in porcine lymphnode response to PCV2 infection were deeply sequenced and analyzed. 3271 novel lncRNAs were identified in all. 1898 mRNAs and 282 lncRNAs showed differential expression between control and PCV2-infected groups. The bioinformatics analysis including lncRNA-mRNA co-expression network construction, as well as GO and KEGG pathway analysis focused on the DEGs was carried out. The results indicated that lncRNAs might participate in PCV2 infection-induced the pathogenesis of immunosuppression through regulating the host's immune responses, biological regulation, response to stimulus, cellular component organization or biogenesis and metabolism. And these differentially expressed lncRNAs might play important roles in response to PCV2 infection in the host's innate immune system. These findings provided a large-scale survey of dysregulated lncRNAs after PCV2 infection, especially the lncRNAs responded to host's innate immune within the lymphnode. This study will provide a novel insight into the lncRNAs' functions and the possible immunosuppressive mechanism induced by PCV2 infection. However, further research will be required to verify the characteristic function of the dysregulated lncRNAs.

Diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) causes high levels of morbidity and mortality in neonatal piglets. Owing to the abuse of antibiotics and emergence of drug resistance, antibiotics are no longer considered only beneficial, but also potentially harmful drugs. Supplements that can inhibit the growth of bacteria are expected to replace antibiotics. Tea polyphenols have numerous important biological functions, including antibacterial, antiviral, antioxidative, anti-inflammatory, and antihypertensive effects. We investigated the role of tea polyphenols in ETEC K88 infection using a mouse model. Pretreating with tea polyphenols attenuated the symptoms induced by ETEC K88. Furthermore, in a cell adherence assay, tea polyphenols inhibited ETEC K88 adherence to IPEC-J2 cells. When cells were infected with ETEC K88, mRNA and protein levels of claudin-1 were significantly decreased compared with those of control cells. However, when cells were pretreated with tea polyphenols, claudin-1 mRNA and protein levels were higher than those in cells without pretreatment upon cell infection with ETEC K88. TLR2 mRNA levels were also higher following cell infection with ETEC K88 when cells were pretreated with tea polyphenols. These data revealed that tea polyphenols could increase the barrier integrity of IPEC-J2 cells by upregulating expression of claudin-1 through activation of TLR2. Tea polyphenols had beneficial effects on epithelial barrier function. Therefore, tea polyphenols could be used as a novel strategy to control and treat pig infections caused by ETEC K88.

Streptococcus suis serotype 2 is a major zoonotic pathogen, and the two-component system plays an important role in bacterial pathogenesis. The present study targeted the 1910HK/RR two-component system of S. suis 2. A 1910HK/RR deletion mutant (Δ1910HK/RR) and the corresponding complementation strain (CΔ1910HK/RR) were constructed in S. suis 2 strain 05ZYH33. 1910HK/RR deletion had no effect on S. suis 2 growth, but significantly inhibited the adherence and invasion of S. suis 2 to HEp-2 cells. Analysis of the role of 1910HK/RR in murine and pig infection models demonstrated that 1910HK/RR played a distinct role in the virulence of S. suis 2. In addition, deletion of 1910HK/RR significantly impaired the survival of 05ZYH33 in human blood. These data provided important insights into the pathogenesis of S. suis 2.

Pulmonary arterial hypertension (PAH), also known as broilers ascites syndrome, is characterized by hypoxia, pulmonary artery pressure, and right heart failure. However, less information is available about the molecular mechanisms of PAH. We evaluated the mediation of calcium-sensing receptor by inducing hypoxia for the possible proliferation of pulmonary artery smooth muscle cells via the G protein pathway. For this purpose, we used an in vitro trial of chicken cell culture and confirmed our results by using immunohistochemistry, immunofluorescence staining, quantitative real-time polymerase chain reaction assay, and Western blotting analysis. Our results showed that the mRNA and protein expression levels of calcium-sensing receptor (CaSR) were significantly upregulated in cells when co-incubated with CaCl2. However, the levels of mRNA and protein were obviously decreased when supplemented with blocking agents (NiCl2, 2-APB, and D609). Furthermore, the experimentally induced hypoxia also upregulated the expression of CaSR gene as compared to CaSR gene expression in control cells. Together, these results indicate that hypoxia plays an important role in the expression of CaSR gene in pulmonary artery smooth muscle cells and reveals new targets for the CaSR excited hypothesis to prevent and control PAH in chickens.

In order to detect and simultaneously discriminate PCV1, PCV2 and PCV3, a multiplex PCR assay was developed and used to detect clinical samples in this study. Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 and PCV3. The tissue samples from eight pig farms were detected using the multiplex PCR assay. The results showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and Real-time PCR methods. It proved that this multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei Province, Central China.

The znuA gene is known to be important for growth and survival in Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida under low Zn(2+) conditions. This gene is also present in Actinobacillus pleuropneumoniae serotype 1; therefore, the aim of this study was to investigate the existence of a similar role for the znuA gene in the growth and virulence of this organism. A precisely defined ΔznuA deletion mutant of A. pleuropneumoniae was constructed based on the sequence of the wild-type SLW01 using transconjugation and counterselection. This mutation was found to be lethal in low-Zn(2+) medium. Furthermore, the ΔznuA mutant strain exhibited attenuated virulence (≥22-fold) as well as reduced mortality and morbidity in a murine (Balb/C) model of infection. The majority of the bacteria were cleared from the lungs within 2 weeks. The ΔznuA mutant strain caused no adverse effects in pigs at doses of up to 1.0×10(9) CFU/mL. The ΔznuA mutant strain induced a significant immune response and conferred 80% and 100% protection on immunised pigs against challenge with A. pleuropneumoniae strains belonging to homologous or heterologous serovars, respectively, compared to the blank controls. The data obtained in this study indicate the potential of the mutant ΔznuA strain for development as a live vaccine capable of inducing reliable cross-serovar protection following intratracheal immunisation.

Outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) have led to large economic losses in China. The attenuated vaccine (HP-PRRSV JXA1-R) was used to control HP-PRRSV. However, in recent years, co-infection with classical PRRSV (C-PRRSV), HP-PRRSV, and/or HP-PRRSV JXA1-R has been increasing in China, resulting in a significant impact on PRRSV diagnostics and management. To facilitate rapid discrimination of HP-PRRSV JXA1-R from HP-PRRSV and C-PRRSV, a multiplex RT-PCR assay for the visual detection of HP-PRRSV JXA1-R, HP-PRRSV, and C-PRRSV was established and evaluated with reference PRRSV strains and clinical samples. Primer specificities were evaluated with RNA/DNA extracted from 10 viral strains, and our results revealed that the primers had a high specificity for PRRSV. The assay sensitivity was 24 copies/μL for PRRSVs. A total of 516 serum samples were identified, of which 12.21% (63/516) were HP-PRRSV-positive, 2.33% (12/516) were HP-PRRSV JXA1-R-positive, and 1.16% (6/516) were C-PRRSV-positive, respectively, which was completely consistent with the sequencing method. The high specificity, sensitivity, and reliability of the multiplex RT-PCR assay described in this study indicate that it is useful for the rapid and differential diagnosis of HP-PRRSV JXA1-R, HP-PRRSV, and C-PRRSV.

In a previous study, we performed serial brain-to-brain passages of swine Japanese encephalitis virus in mice and sequenced the complete genomes of the F5 and F20 passaged mouse-adapted variants. In the current study, we analyzed the differences between their genome sequences and found 12 amino acid substitutions in the nonstructural proteins. We also assessed the growth characteristics of these two variants in mammalian cells in vitro and in vivo. Our investigations revealed that the F20 variant had enhanced growth characteristics and modified virulence compared with the F5 variant. We therefore conclude that multiple amino acid substitutions in the nonstructural proteins of swine Japanese encephalitis virus alter its virulence in mice.