A comparative genomics approach for identifying genetic factors in Escherichia coli isolates associated with bovine diseases.

AIMS: E. coli are ubiquitously present bacterial pathogens that cause septicaemia, diarrhoea and other clinical illness in farm animals. Many pathogen factors can be associated with disease conditions. Currently, studies inferring E. coli genetic factors associated with infection in bovines are limited. Hence, the present study envisaged to determine the pathogen genetic factors associated with bovine disease conditions. METHOD AND RESULTS: The comparative genomic analysis involved genome sequence data of 135 diseased and 145 healthy bovine origin E. coli strains. Phylogroups A and C, as well as pathotypes ExPEC and EPEC, were found to have a strong connection with bovine disease strains. STEC strains, including EHEC, seem to play a less important role in bovine disease. Sequence types (STs) predominant among strains from diarrhoeal origin were ST 301 (CC 165) and ST 342. Correlation of core genome phylogeny with accessory gene-based clustering, phylogroups and pathotypes indicated lineage-specific virulence factors mostly associated with disease conditions. CONCLUSIONS: Comparative genomic analysis was applied to infer genetic factors significant in bovine disease origin E. coli strains. Isolates from bovine disease origin were enriched for the phylogroups A and C, and for the pathotypes ExPEC and EPEC. However, there was minimal evidence of STEC involvement. The study also indicated predominant genetic lineages and virulence genes (pap, sfa and afa) associated with disease origin strains. SIGNIFICANCE AND IMPACT OF STUDY: The study revealed significant pathotypes, phylogroups, serotypes and sequence types associated with bovine disease conditions. These identified genetic factors can be applied for disease diagnosis, implementing vaccines and therapeutic measures. In addition, E. coli isolates from the bovine species revealed a complex pattern of disease epidemiology.

Development of novel iron-regulated Pasteurella multocida B: 2 bacterin and refinement of vaccine quality in terms of minimum variation in particle size and distribution vis-a-vis critical level of iron in media.

To enhance the qualitative bacterial biomass per unit of media and to overcome the limitations of the existing haemorrhagic septicaemia (HS) vaccines, a comprehensive study was undertaken encompassing the role of iron on the bacterial biomass of Pasteurella multocida B: 2 to vaccine development. Trypsin digested hydrochloric acid-treated sheep blood (THSB) as a novel iron rich supplement had been devised for the first time for augmenting the qualitative bacterial biomass per unit of media which was evident with growth kinetic study. The higher recovery of iron from THSB became evident via atomic absorbance spectrophotometry. The critical level of iron in the media as well as mode of iron supplementation showed a major impact on the outer membrane protein profile of P. multocida B:2 and variation in droplet size and particle-size distribution of formulated vaccine. Immune response study against iron-regulated bacterin adjuvanted with aluminum hydroxide gel in mouse model showed that 3% THSB supplementation of casein sucrose yeast (CSY) not only augmented the growth of P. multocida B:2 significantly but conferred highest pre-challenged ELISA IgG titer and protection against pasteurellosis. Thus, THSB supplementation of CSY can resolve existing up-scaling and immunogenic potential problems of HS vaccine production.

Effect of alum co-adjuvantation of oil adjuvant vaccine on emulsion stability and immune responses against haemorhagic septicaemia in mice.

BACKGROUND AND OBJECTIVES: Haemorrhagic septicaemia (HS), caused by Pasteurella multocida, is the most important bacterial disease of cattle and buffaloes in India. Oil adjuvant vaccine (OAV) is the most potent vaccine available for the control of HS. The study aims to evaluate the effect of alum co-adjuvantation of OAV on emulsion stability and immune response. MATERIALS AND METHODS: Two different oil adjuvant vaccines viz., standard oil adjuvant vaccine (OAV) and alum precipitated oil adjuvant vaccine (A-OAV) were prepared with Pasteurella multocida antigen. Emulsion stability was tested by centrifugation, storage at 37 °C for 3 months and microscopy. Immune responses were evaluated by ELISA antibody titer, CD4, CD8 T cell populations and survival post challenge by P. multocida in mice. RESULTS: The separation of aqueous and oil phase of emulsion by centrifugation and storage test were 0 and 6.76% in A-OAV as compared to 11.00 and 26.39% in OAV, respectively. The mean droplet size was significantly smaller (p<0.01) in A-OAV as compared to OAV. The A-OAV recorded higher ELISA antibody titer (p<0.05) up to 21st days post vaccination, and higher CD4 (p>0.05) and CD8 T cell (p<0.05) populations compared to OAV. The A-OAV group conferred 100% protection after challenge with both 100 LD50 and 1000 LD50 as compared to 100 and 60% respective protection by OAV group. CONCLUSION: The results indicates that A-OAV had better emulsion stability, produces higher level of CD4, CD8 T cells and antibody titer with better protection compared to oil adjuvant vaccine.

Prokaryotic expression of ovis aries conglutinin encoding neck and carbohydrate recognition domain and its functional characterization.

Conglutinin, a soluble pattern recognition receptor of innate immune system in bovines is known for its potential defensive activity against microorganisms either by direct agglutination in the presence of calcium or by acting as opsonin. In the present study, sheep (Ovis aries) conglutinin encoding neck and carbohydrate recognition domain (rSCGN) was expressed in the E coli BL21 expression host. The recombinant conglutinin revealed molecular weight of 27 kDa in SDS PAGE and also in western blotting using antibuffalo conglutinin polyclonal serum. The protein was characterized further for its functional activity in various assays. In ELISA based sugar and LPS binding assay, the rSCGN revealed its high binding activity toward N-acetyl glucosamine and E. coli LPS in the presence and the absence of calcium ions, respectively. Hemagglutination of chicken red blood cells caused by Newcastle disease virus was not inhibited in the presence of rSCGN as it lacked complete collagenous region present in the native protein. In virus neutralization test, the recombinant protein was found to reduce multiplication of bovine herpes virus-1 propagated in MDBK cells. This prokaryotically expressed 27 kDa recombinant sheep conglutinin can serve as antigen in future studies to develop sandwich ELISA for assessing the level of native conglutinin in sheep serum.

Effect of different levels of selenium supplementation on growth rate, nutrient utilization, blood metabolic profile, and immune response in lambs.

Eighteen male lambs (8-9 months of age, 25.00 +/- 0.90 kg body weight) were divided into three groups of six animals in each and fed a total mixed ration (TMR) containing concentrate mixture (30% maize grain, 27% soybean meal, 40% wheat bran, 2% mineral mixture, and 1% common salt) and wheat straw in 65:35 ratio and supplemented with selenium (Se) as sodium selenite at 0 (T1, control), 0.15 (T2), and 0.30 ppm (T3) levels. Experimental feeding was done for a period of 90 days including a 6-day metabolism trial. To assess the growth performance, lambs were weighed every 15 days throughout the experimental period. All the lambs were intramuscularly inoculated with a single dose (2 ml) of haemorrhagic septicaemia oil adjuvant vaccine on 0 day to evaluate the humoral immune response. Blood samples were collected on 0 day and thereafter at 30 days interval. Results revealed that supplementation of Se both at 0.15 and 0.30 ppm levels had no significant (P > 0.05) effect on intake and digestibility of dry matter, organic matter, crude protein (CP), ether extract, neutral detergent fiber, acid detergent fiber, and hemicellulose; balances of calcium and phosphorus; and level and intake of digestible CP and total digestible nutrients. Se supplementation also had no significant (P > 0.05) effect on the levels of serum total cholesterol, total protein, albumin, globulin, albumin/globulin ratio, tri-iodothyronine (T(3)), thyroxine (T(4)), and T(4)/T(3) ratio; and serum glutamate oxaloacetate transaminase and serum glutamate pyruvate transaminase enzyme activity in the lambs. However, there was a significant (P < 0.05) increase in the plasma Se levels, red blood cell glutathione peroxidase enzyme activity, and humoral immune response in both the Se-supplemented groups. Feed (TMR) required per kilogram gain was less by 11.1% and 16.5% in groups T2 and T3, respectively, as compared to control (T1) group. Average daily gain was highest (108.5 g) in group T3, followed by group T2 (98.2 g), and lowest (89.06 g) in the control group (T1). These results indicated that supplementation of 0.15 and 0.3 ppm Se in the diet (having 0.19 ppm Se) of lambs significantly improves their immune response and antioxidant status.

Effect of zinc supplementation from different sources on growth, nutrient digestibility, blood metabolic profile, and immune response of male Guinea pigs.

Forty weaned male guinea pigs of 208.20 +/- 6.62 g mean body weight were divided into 4 groups of 10 animals in a randomized block design. All of the guinea pigs were fed a basal diet [25% ground maize hay, 30% ground maize grain, 22% ground chickpea (Cicer arietinum L.), 9.5% deoiled rice bran, 6% soybean meal, 6% fish meal, 1.45% mineral supplement (without Zn) and 0.05% ascorbic acid] and available green fodder. Group I served as the control (no Zn supplementation), whereas 20 ppm Zn was added in the diet in groups II, III, and IV either as zinc sulfate (ZnSO(4)), zinc amino acid complex (ZAAC), and ZnSO4 + ZAAC in equal parts, respectively. Experimental feeding lasted for 70 d, including a 3-d digestibility trial. Blood was collected through cardiac puncture from four animals in each group at d 0 and subsequently at the end of experimental feeding. After 40 d of experimental feeding, four animals from each group were injected with 0.4 mL of Brucella abortus cotton strain-19 vaccine to assess the humoral immune response of the animals. After 10 wk of study, four animals from each group were sacrificed to study the concentration of Zn, Cu, Co, Fe, and Mn in the liver, pancreas and spleen. Results revealed no significant difference in the feed intake, body weight gain, and digestibility of the nutrients, except for crude protein (CP) digestibility, which was significantly (p < 0.05) lower in group IV. Although concentrations of serum glucose, Ca, and P and the albumin:globulin (A:G) ratio were similar in the different groups, the total protein, albumin, and serum alkaline phosphatase activity were higher in all of the Zn-supplemented groups on d 70. The serum Zn levels at the end of experimental feeding were significantly higher in groups II and III, whereas serum Mn levels were found to be significantly (p < 0.05) higher in groups III and IV. The organ weights (as percentage of body weights) did not show any differences among the treatment groups. Although the Mn concentration was significantly (p < 0.05) higher in the pancreas, the Cu concentration was significantly (p < 0.05) reduced in the spleen in all of the Zn-supplemented groups. The humoral immune response (antibody titer values) on d 14 of vaccination was significantly (p < 0.05) higher in all of the Zn-supplemented groups. It was concluded that the 20-ppm level of Zn in the diet might be adequate for growth and nutrient utilization in guinea pigs, but supplementation of 20-ppm zinc significantly improved the immune response and impact was more prominent with the ZAAC (organic source) compared to ZnSO(4) (inorganic source).