Pmorf0222, a Virulence Factor in Pasteurella multocida, Activates Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase via Toll-Like Receptor 1/2.

Pasteurella multocida primarily causes hemorrhagic septicemia and pneumonia in poultry and livestock. Identification of the relevant virulence factors is therefore essential for understanding its pathogenicity. Pmorf0222, encoding the PM0222 protein, is located on a specific prophage island of the pathogenic strain C48-1 of P. multocida. Its role in the pathogenesis of P. multocida infection is still unknown. The proinflammatory cytokine plays an important role in P. multocida infection; therefore, murine peritoneal exudate macrophages were treated with the purified recombinant PM0222, which induced the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) via the Toll-like receptor 1/2 (TLR1/2)-nuclear factor kappa B (NF-κB)/mitogen-activated protein kinase (MAPK) signaling and inflammasome activation. Additionally, the mutant strain and complemented strain were evaluated in the mouse model with P. multocida infection, and PM0222 was identified as a virulence factor, which was secreted by outer membrane vesicles of P. multocida. Further results revealed that Pmorf0222 affected the synthesis of the capsule, adhesion, serum sensitivity, and biofilm formation. Thus, we identified Pmorf0222 as a novel virulence factor in the C48-1 strain of P. multocida, explaining the high pathogenicity of this pathogenic strain.

A functional mutation associated with piglet diarrhea partially by regulating the transcription of porcine STAT3.

The present study aimed to search for functional mutations within the promoter of porcine STAT3 and to provide causative genetic variants associated with piglet diarrhea. We firstly confirmed that STAT3 expressed higher in the small intestine than in the spleen, stomach and large intestine of SPF piglets, respectively (P < 0.05). Then, 10 genetic variations in the porcine STAT3 promoter region was identified by direct sequencing. Among them, three mutations SNP1: g.-870 G>A, SNP2: g.-584 A>C and a 6-bp Indel in the promoter region that displayed significant differential transcriptional activities were identified. Association analyses showed that SNP1: g.-870 G>A was significantly associated with piglet diarrhea (P < 0.05) and the GG animals had lower diarrhea score than AA piglets (P < 0.01) in both Min and Landrace population. Further functional analysis revealed that E2F6 repressed the transcriptional efficiency of STAT3 in vitro, by binding the G allele of SNP1. The present study suggested that SNP1: g.-870 G>A was a piglet diarrhea-associated variant that directly affected binding with E2F6, leading to changes in STAT3 transcription which might partially contribute to piglet diarrhea susceptibility or resistance.

A 12-bp indel in the 3'UTR of porcine CISH gene associated with Landrace piglet diarrhea score.

Variations in Cytokine inducible SH2-containing protein (CISH) gene influence human susceptibility to common infectious diseases, but little is known about CISH in swine. The objectives of this study were to 1) determine porcine CISH (pCISH) mRNA expression level in different tissues of piglets, 2) predict putative functional genetic variations within pCISH, 3) investigate the association between a identified variation in the 3'UTR and piglets phenotype traits in Min (n = 226) and Landrace (n = 186) population, and explore the function of this variation. Results of quantitative PCR showed pCISH mRNA expressed in all the collected tissues with higher level in lung and ileum than colon (p < 0.05). In-silico analysis indicated none of the functional ns-SNPs existed in pCISH coding region. Results from the characterizing of 3'UTR presented a novel 12-bp insertion/deletion (indel) mutation. Statistical analysis demonstrated that this 12-bp indel associated with piglets diarrhea score in the Landrace population, and animals with AA genotype (12-bp insertion) presented lower diarrhea score when compared with BB (p < 0.05) or AB (p < 0.01) carriers. The in vitro study indicated that the luciferase activity of reconstruct plasmid psiCHECK-2-CISH-AA or psiCHECK-2-CISH-BB was significantly lower than the negative control (p < 0.05), and luciferase activity of psiCHECK-2-CISH-AA was higher than that of the psiCHECK-2-CISH-BB (p < 0.05). Although results herein suggested the 12-bp indel might affect Landrace piglet susceptibility to diarrhea, further association studies in more populations are needed before this preliminary finding could be used for pig breeding.

Characterization, mRNA expression profile, subcellular distribution and association analysis with piglet diarrhea of porcine matrix metallopeptidase 7 (pMMP7).

Matrix metalloproteinase 7 (MMP7) is involved in the degradation of extracellular matrix in disease processes and therefore plays an important role in host disease resistance/susceptibility. To better understanding the effects of porcine MMP7 (pMMP7) on piglets diarrhea trait, we characterized pMMP7 gene, identified genetic variations in pMMP7 and explored the relationship between pMMP7 polymorphisms and piglets diarrhea in Min pig and Landrace populations. The complete coding sequence of pMMP7 is 804 bp encoding a protein of 267 amino acids. Sequence alignment showed that the identity between pMMP7 and human MMP7 was approximately 80%. The expression of pMMP7 in the gut of healthy piglets were weak and the distribution of the pMMP7-EGFP fusion protein was observed mainly in the cytoplasm. After the identification of 21 genetic variations in 5' flanking region and exons, Hae III and Eco72 Ⅰ PCR-RFLP were established to genotype SNP rs327380117 and rs329429922, respectively. Statistical analysis indicated that Landrace piglets with a TT genotype at rs327380117 had a lower diarrhea score and day-14 wt than TC piglets (p < 0.05); the diarrhea score of AA Landrace animals with rs329429922 was lower than that of GG individuals (p < 0.05). The findings presented here contribute to the understanding of the biological function of pMMP7 and may provide new molecular markers for pig breeding.

Expression pattern and association analysis of porcine matrix metallopeptidase 9 (MMP9) with diarrhea and performance traits in piglets.

Matrix metalloproteinase 9 (MMP9) plays critical roles in multiple biological processes, such as reproduction, cell proliferation and differentiation, and host defenses. The aim of this study was to evaluate whether MMP9 is a candidate gene for resistance to diarrhea in piglets. In this study, quantitative real-time PCR was used to analyze the expression of MMP9 mRNA in different tissues of specific pathogen-free piglets. MMP9 was expressed in all the tissues (heart, liver, spleen, lung, kidney, stomach, duodenum, jejunum, ileum, and colon) analyzed. An association analysis between MMP9 polymorphisms and piglet diarrhea score and performance traits were performed in Min (Chinese indigenous breed) and Landrace populations. In the statistical analysis, at the g.48178429 G>A locus, AA piglets had a lower diarrhea score than that of GA in the Min population (P < .05), whereas GG had higher day-35 body weight and average daily gain (ADG) than AA in the Landrace breed (P < .05). At the rs336583561 locus, Min piglets with the GG genotype have a lower diarrhea score than AG piglets (P < .05). At g.48184777C>T, CC animals have higher body weight than TC Landrace piglets (P < .05 or P < .01). A 5' flanking deletion assay indicated that g.48178429 G>A was not located in the MMP9 promoter region. Our results suggest that the A allele at the g.48178429 G>A locus and the G allele at rs336583561 are resistance alleles in Min pigs. Before these markers are used in pig breeding programs, more studies in larger populations are needed.

Identification of porcine CTLA4 gene polymorphism and their association with piglet diarrhea and performance traits.

The objective of this study was to evaluate the association between the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) gene and piglet diarrhea. In this study, the mRNA expression of the CTLA4 gene increased significantly in IPEC-J2 cells after Escherichia coli K88 infection. Single nucleotide polymorphisms (SNPs) located in the 5' flanking region (SNPs g.107281989C>T) and 3'-untranslated region (3'-UTR; SNPs g.107288753C>A) were identified, and they were in linkage disequilibrium in both Min pigs and the Landrace population. Association analysis showed that Landrace piglets with a TT or AA genotype had a lower diarrhea index, and AA animals had higher average daily gain when compared to CC pigs, respectively (p < 0.05). However, the relationship between SNPs and diarrhea and performance traits in the Min population was not significant. Haplotype analysis indicated that the TC haplotype had the lowest diarrhea index. The 5' flanking deletion assay suggested that SNP g.107281989C>T was a molecular marker instead of the functional marker. This research demonstrated that genetic variances in the CTLA4 gene had significant effects on Landrace piglet diarrhea resistance.