Pathology, virulence-associated gene profiling, antimicrobial susceptibility, and pathogenicity of untypeable capsular serotypes of Pasteurella multocida isolated from slaughtered pigs of India.

Pasteurella multocida is widely distributed in all pig-rearing countries, affecting the economic viability and profitability of pig production. The present research highlights the molecular characterization and pathology of untypeable capsular serotypes of P. multocida in slaughtered pigs from prominent pig-rearing states of India. The prevalence of Pasteurellosis was 27.17% by Pasteurella multocida specific Pasteurella multocida specific PCR (PM-PCR). assay, while isolation rate was 7.62%. The microscopic lesions of bronchopneumonia, tonsillitis, and the presence of bacterial antigens in immunohistochemistry confirmed P. multocida with pathologies. In capsular typing, the majority of the isolates were untypeable with prevalence of 52.15% and 43.58% in molecular and microbiological methods, respectively. All the isolates showed the uniform distribution of virulence genes such as exbB, nanB, sodC, plpB, and oma87 (100%), while the variations were observed in ptfA, hasR, ptfA, pfhA, hsf-1, and plpE genes. The untypeable isolates showed higher prevalence of hsf-1 gene as compared to others. The untypeable serotypes showed a higher degree of resistance to ampicillin, amoxicillin, and penicillin antibiotics. The mouse pathogenicity testing of untypeable capsular isolates confirmed its pathogenic potential. The higher frequency of pathogenic untypeable isolates with antibiotic resistance profile might pose a serious threat to the pigs, and therefore, preventive measures should be adopted for effective control.

Delineating virulence of Vibrio campbellii: a predominant luminescent bacterial pathogen in Indian shrimp hatcheries.

Luminescent vibriosis is a major bacterial disease in shrimp hatcheries and causes up to 100% mortality in larval stages of penaeid shrimps. We investigated the virulence factors and genetic identity of 29 luminescent Vibrio isolates from Indian shrimp hatcheries and farms, which were earlier presumed as Vibrio harveyi. Haemolysin gene-based species-specific multiplex PCR and phylogenetic analysis of rpoD and toxR identified all the isolates as V. campbellii. The gene-specific PCR revealed the presence of virulence markers involved in quorum sensing (luxM, luxS, cqsA), motility (flaA, lafA), toxin (hly, chiA, serine protease, metalloprotease), and virulence regulators (toxR, luxR) in all the isolates. The deduced amino acid sequence analysis of virulence regulator ToxR suggested four variants, namely A123Q150 (AQ; 18.9%), P123Q150 (PQ; 54.1%), A123P150 (AP; 21.6%), and P123P150 (PP; 5.4% isolates) based on amino acid at 123rd (proline or alanine) and 150th (glutamine or proline) positions. A significantly higher level of the quorum-sensing signal, autoinducer-2 (AI-2, p = 2.2e-12), and significantly reduced protease activity (p = 1.6e-07) were recorded in AP variant, whereas an inverse trend was noticed in the Q150 variants AQ and PQ. The pathogenicity study in Penaeus (Litopenaeus) vannamei juveniles revealed that all the isolates of AQ were highly pathogenic with Cox proportional hazard ratio 15.1 to 32.4 compared to P150 variants; PP (5.4 to 6.3) or AP (7.3 to 14). The correlation matrix suggested that protease, a metalloprotease, was positively correlated with pathogenicity (p > 0.05) and negatively correlated (p < 0.05) with AI-2 and AI-1. The syntenic organization of toxS-toxR-htpG operon in V. campbellii was found to be similar to pathogenic V. cholerae suggesting a similar regulatory role. The present study emphasizes that V. campbellii is a predominant pathogen in Indian shrimp hatcheries, and ToxR plays a significant role as a virulence regulator in the quorum sensing-protease pathway. Further, the study suggests that the presence of glutamine at 150th position (Q150) in ToxR is crucial for the pathogenicity of V. campbellii.

Immunogenicity assessment of Clostridium perfringens type D epsilon toxin epitope-based chimeric construct in mice and rabbit.

Epsilon toxin (Etx) belongs to family of pore-forming toxin and is produced by Clostridium perfringens type D. The Etx toxin is responsible for the pathogenesis of enterotoxaemia in sheep and goats, and occasionally in other livestock animals. The present study aimed to develop a Clostridium perfringens epsilon toxin-based chimeric epitope construct having immunodominant B-cell epitope and universal T-cell epitope and its immunogenicity was evaluated in mice and rabbit. An artificial chimeric epitope construct (CEC) was prepared by joining tandem repeats of a peptide containing amino acids (aa) 134-145 of epsilon toxin B-cell epitope and universal T-cell epitopes. The CEC was expressed in the Escherichia coli following codon optimization for efficient translational efficiency and purified by affinity chromatography. The antigenic reactivity of r-CEC proteins was confirmed by western blot with rabbit anti-r-Etox hyperimmune sera. The immunogenicity of the recombinant single CEC was examined in mice and rabbit by indirect ELISA. It was found that r-CEC yielded high titers of neutralizing antibodies (≥ 1.035 IU/ml) in immunized mice and rabbit. The potency of chimeric protein immunized serum was observed to be higher than the recommended level (0.1-0.3 IU/ml) for protection in sheep and goats. This indicated the potential ability of the chimeric protein as a vaccine candidate. This further requires studying the immune response in targeted host species (sheep and goat).

Translational control of NKT cell cytokine production by p38 MAPK.

NKT cells are known to rapidly produce a large amount of cytokines upon activation. Although a number of signaling pathways that regulate the development of NKT cells have been identified, the signaling pathways involved in the regulation of NKT cell cytokine production remain unclear. In this study, we show that the p38 MAPK pathway is dispensable for the development of NKT cells. However, NKT cell cytokine production and NKT-mediated liver damage are highly dependent on activation of this pathway. p38 MAPK does not substantially affect cytokine gene expression in NKT cells, but it regulates the synthesis of cytokines through the Mnk-eIF4E pathway. Thus, in addition to gene expression, translational regulation by p38 MAPK could be a novel mechanism that contributes to the overall production of cytokine by NKT cells.

IP3 receptor-mediated Ca2+ release in naive CD4 T cells dictates their cytokine program.

IP(3) (inositol 1,4,5-trisphosphate) receptors (IP(3)Rs) regulate the release of Ca(2+) from intracellular stores in response to IP(3). Little is known about regulation of the expression of IP(3)Rs and their role during the activation of CD4 T cells. In this study we show that mouse naive CD4 T cells express IP(3)R1, IP(3)R2, and IP(3)R3, but that gene expression of IP(3)R3 primarily is down-regulated upon activation due to loss of the Ets-1 transcription factor. Down-regulation of IP(3)R expression in activated CD4 T cells is associated with the failure of TCR ligation to trigger Ca(2+) release in these cells. We also show that down-regulation of specific IP(3)Rs in activated CD4 T cells correlates with the requirement of IP(3)R-mediated Ca(2+) release only for the induction of, but not for the maintenance of, IL-2 and IFN-gamma expression. Interestingly, while inhibition of IP(3)R function early during activation blocks IL-2 and IFN-gamma production, it promotes the production of IL-17 by CD4 T cells. Thus, IP(3)Rs play a key role in the activation and differentiation of CD4 T cells. The immunosuppressive effect of pharmacological blockers of these receptors may be complicated by promoting the development of inflammatory CD4 T cells.

Novel Rath peptide for intracellular delivery of protein and nucleic acids.

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant beta structure with a small alpha helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.