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).

Marker-less deletion of cctA gene of Clostridium chauvoei.

Clostridium chauvoei causes blackleg disease in domestic animals, especially cattle and sheep. The pathogen produces several toxins including CctA - a hemolysin and protective antigen. Molecular pathogenesis of the disease is poorly understood, possibly due to lack of genetic manipulation tools for C. chauvoei. In the present study, we report the marker-less deletion of cctA gene using the CRISPR-Cas9 system. The C. chauvoei cctA deletion mutant had negligible hemolytic and significantly reduced cytotoxic activities. To the best of our knowledge, this is the first report of genetic manipulation of C. chauvoei. The method we used in this study can be applied for genetic manipulation of C. chauvoei to better understand the pathogenesis and genetics of the pathogen.

Cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of Clostridium chauvoei.

AIM: Blackleg disease is caused by Clostridium chauvoei in ruminants. Although virulence factors such as C. chauvoei toxin A, sialidase, and flagellin are well characterized, hyaluronidases of C. chauvoei are not characterized. The present study was aimed at cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of C. chauvoei. MATERIALS AND METHODS: C. chauvoei strain ATCC 10092 was grown in ATCC 2107 media and confirmed by polymerase chain reaction (PCR) using the primers specific for 16-23S rDNA spacer region. nagH gene of C. chauvoei was amplified and cloned into pRham-SUMO vector and transformed into Escherichia cloni 10G cells. The construct was then transformed into E. cloni cells. Colony PCR was carried out to screen the colonies followed by sequencing of nagH gene in the construct. RESULTS: PCR amplification yielded nagH gene of 1143 bp product, which was cloned in prokaryotic expression system. Colony PCR, as well as sequencing of nagH gene, confirmed the presence of insert. Sequence was then subjected to BLAST analysis of NCBI, which confirmed that the sequence was indeed of nagH gene of C. chauvoei. Phylogenetic analysis of the sequence showed that it is closely related to Clostridium perfringens and Clostridium paraputrificum. CONCLUSIONS: The gene for virulence factor nagH was cloned into a prokaryotic expression vector and confirmed by sequencing.

Modulatory effect of betaine on expression dynamics of HSPs during heat stress acclimation in goat (Capra hircus).

Changing climatic scenario with expected global rise in surface temperature compelled more focus of research over decoding heat stress response mechanism of animals and mitigation of heat stress. Recently betaine, a trimethyl form of glycine has been found to ameliorate heat stress in some species of animals. To overcome deleterious effect of heat stress, an attempt was taken to investigate the effect of betaine supplementation on heat stress mitigation in goats. Eighteen female Barbari goats were taken and randomly divided into 3 groups (n=6) such as control, HS (Heat stressed), HS+B (Heat stressed administered with betaine). Except for the control group, other groups were exposed to repeated heat stress (42 °C) for 6 h for sixteen consecutive days. Blood samples were collected at the end of heat exposure on day 1 (Initial heat stress acclimation - IHSA), day 6 (Short term heat stress acclimation - STHSA) and day 16 (Long term heat stress acclimation - LTHSA). When the groups were compared between different heat stress acclimatory phases, expression of all HSPs (HSP60, HSP70, HSP90 and HSP105/110) showed a similar pattern with a first peak on IHSA, reaching a basal level on STHSA followed by second peak on LTHSA. The messenger RNA (mRNA) and protein expression of HSPs was observed to be higher (P<0.05) in HS group than HS+B group except HSP90 on IHSA and HSP60 on STHSA. HSP105/110 expression was highest (P<0.05) on LTHSA. Immunocytochemical analysis revealed that HSPs were mainly localized both in nucleus and cytoplasm of PBMCs. In conclusion, heat stress increases HSPs expression and betaine administration was shown to have a dwindling effect on expression of HSPs, suggesting a possible role of this chemical chaperone on heat stress amelioration.

Expression analysis of NOS family and HSP genes during thermal stress in goat (Capra hircus).

Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher (P < 0.05) during peak summer, and iNOS and eNOS expressions were also observed to be significantly higher (P < 0.05) during peak winter season as compared with moderate season. The iNOS, eNOS, cNOS, HSP70, and HSP90 were mainly localized in plasma membrane and cytoplasm of PBMCs. To conclude, data generated in the present study indicate the possible involvement of the NOS family genes in amelioration of thermal stress so as to maintain cellular integrity and homeostasis in goats.

Expression of HSPs: an adaptive mechanism during long-term heat stress in goats (Capra hircus).

Menacing global rise in surface temperature compelled more focus of research over understanding heat stress response mechanism of animals and mitigation of heat stress. Twenty-four goats divided into four groups (n = 6) such as NHS (non-heat-stressed), HS (heat-stressed), HS + VC (heat-stressed administered with vitamin C), and HS + VE + Se (heat-stressed administered with vitamin E and selenium). Except NHS group, other groups were exposed to repeated heat stress (42 °C) for 6 h on 16 consecutive days. Blood samples were collected at the end of heat exposure on days 1, 6, 11, and 16. When groups compared between days, expression of all heat shock proteins (HSPs) showed a similar pattern as first peak on day 1, reached to basal level on the sixth day, and followed by second peak on day 16. The relative messenger RNA (mRNA) and protein expression of HSP 60, HSP70, and HSP90 was observed highest (P < 0.05) in HS group, followed by antioxidant-administered group on days 1 and 16, which signifies that antioxidants have dampening effect on HSP expression. HSP105/110 expression was highest (P < 0.05) on day 16. We conclude that HSP expression pattern is at least two-peak phenomenon, i.e., primary window of HSP protection on the first day followed by second window of protection on day 16. HSP60, HSP70, and HSP90 play an important role during the initial phase of heat stress acclimation whereas HSP105/110 joins this cascade at later phase. Antioxidants may possibly attenuate the HSP expression by reducing the oxidative stress.

Impact of short-term heat stress on physiological responses and expression profile of HSPs in Barbari goats.

Six, nonpregnant, Barbari goats aged 4-5 years were selected for the study. For the first 6 days, the animals were kept in psychrometric chamber at thermoneutral temperature for 6 h each day to make them acclimated to climatic chamber. On the 7th day, the animals were exposed to 41 °C temperature for 3 h and then to 45 °C for the next 3 h. Cardinal physiological responses were measured, and blood samples (3 ml) were collected at 1-h interval during the heat exposure period and then once after 6 h of the heat exposure. The rectal temperature (RT) and respiratory rate (RR) increased significantly (P < 0.05) during the heat exposure compared to pre- and postexposure. The relative messenger RNA (mRNA) expression of heat shock protein (HSP)60, HSP70, and HSP90 increased significantly (P < 0.05) within 1 h after exposure to heat stress at 41 and 45 °C and decreased significantly (P < 0.05) in next 2 h but remain significantly (P < 0.05) elevated from preexposure. HSP105/110 relative mRNA expression level remained unchanged during the first 4 h, and thereafter, it increased significantly (P < 0.05) and reached the peak at 6 h. Relative protein expression pattern of HSPs during exposure to heat stress showed similar trend as observed for the relative mRNA expression. Given the response sensitivity and intensity of HSP genes to environmental stresses, HSP70 was found to be the most sensitive to temperature fluctuation, and it could be used as an important molecular biomarker to heat stress in animals.