Tapas Kumar Goswami

Escheriosomes entrapped DNA vaccine co-expressing Cu–Zn superoxide dismutase and IL-18 confers protection against Brucella abortus

In the present study, we evaluated prophylactic prospective of liposome based DNA vaccine co-expressing Cu-Zn superoxide dismutase (SOD) along with interleukin-18 (IL-18) against experimental murine brucellosis. The immunization schedule involves liposome-mediated delivery of pVsod (encoding SOD of Brucella abortus) and pVIL18-sod (encoding IL-18 of mouse and SOD of B. abortus) DNA constructs. The data highlight potential of Escherichia coli lipid liposome (escheriosome) based DNA delivery vehicle to induce SOD specific humoral and cellular immune responses in the immunized mice. The co-expression of SOD along with IL-18 ensued in higher lymphoproliferative response and IFN-gamma production in comparison to the group of animals that were immunized with free form of SOD-DNA. Antibody response developed upon immunization with both DNA vaccines was of IgG2a type mainly. The results of the present study show that co-expression of IL-18 along with SOD polarized the antigen specific immune responses toward Th-1 direction, a desirable feature to control intracellular pathogens.

Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice

Brucella abortus S19 is a smooth strain used as live vaccine against bovine brucellosis. Smooth lipopolysaccharide (LPS) is responsible for its residual virulence and serological interference. Rough mutants defective of LPS are more attenuated but confers lower level of protection. We describe a modified B. abortus S19 strain, named as S19Δper, which exhibits intermediate rough phenotype with residual O-polysaccharide (OPS). Deletion of perosamine synthetase gene resulted in substantial attenuation of S19Δper mutant without affecting immunogenic properties. It mounted strong immune response in Swiss albino mice and conferred protection similar to S19 vaccine. Immunized mice produced higher levels of IFN-γ, IgG2a and thus has immune response inclined towards Th1 cell mediated immunity. Sera from immunized animals did not show agglutination reaction with RBPT antigen and thus could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccine. S19Δper mutant displayed more susceptibility to serum complement mediated killing and sensitivity to polymyxin B. Pregnant guinea pigs injected with S19Δper mutant completed full term of pregnancy and did not cause abortion, still birth or birth of weak offspring. S19Δper mutant with intermediate rough phenotype displayed remarkable resemblance to S19 vaccine strain with improved properties of safety, immunogenicity and DIVA capability for control of bovine brucellosis.

Experimental iron-inactivated Pasteurella multocida A: 1 vaccine adjuvanted with bacterial DNA is safe and protects chickens from fowl cholera.

Fowl cholera is a serious problem in large and small scale poultry production. The present study describes the development and testing of an inactivated whole-cell, low-cost, safe, and effective vaccine for fowl cholera based on a previous work (Vaccine 23:5590-5598). Pasteurella multocida A: 1 grown in the presence of low FeCl(3) concentrations, inactivated with higher concentrations of FeCl(3), and adjuvanted with bacterial DNA from P. multocida B: 2 containing immunostimulatory CpG motifs protect chickens with a lethal P. multocida A: 1 challenge. Chickens were immunized with two whole-cell inactivated vaccine doses at 4 weeks apart and challenged 4 weeks after booster immunization. Experimental vaccines were pure, easy injectable, and caused very little distress in chickens due to their aqueous consistency. Vaccines and bacterial DNA (bDNA) posed no safety problems when chickens were injected subcutaneously (s.c.) with a single, double, and overdose of these preparations. Immunized chickens produced systemic IgY antibodies (Ab) responses and vaccine adjuvanted with bDNA protected 100% chickens from lethal intrapertoneal (i.p.) P. multocida A: 1 challenge. This work suggests that use of bDNA as an adjuvant can improve the cost-effectiveness of inactivated veterinary vaccines for their use in developing countries. Our future studies will focus on safety and potency evaluation of experimental and current vaccines using bDNA as an adjuvant.

Methionine sulfoxide reductase A (MsrA) contributes to Salmonella Typhimurium survival against oxidative attack of neutrophils.

Salmonella Typhimurium (ST) must evade neutrophil assault for infection establishment in the host. Myeloperoxidase generated HOCl is the key antimicrobial agent produced by the neutrophils; and methionine (Met) residues are the primary targets of this oxidant. Oxidation of Mets leads to methionine sulfoxide (Met-SO) formation and consequently compromises the protein function(s). Methionine sulfoxide reductase A (MsrA) reductively repairs Met-SO to Mets. In this manner, MsrA maintains the function(s) of key proteins which are important for virulence of ST and enhance the survival of this bacterium under oxidative stress. We constructed msrA gene deletion strain (ΔmsrA). The primers located in the flanking regions to ΔmsrA gene amplified 850 and 300 bp amplicons in ST and ΔmsrA strains, respectively. The ΔmsrA strain grew normally in in vitro broth culture. However, ΔmsrA strain showed high susceptibility (p<0.001) to very low concentrations of HOCl which was restored (at least in part) by plasmid based complementation. ΔmsrA strain was hypersensitive (than ST) to the granules isolated from neutrophils. Further, the ΔmsrA strain was significantly (p<0.05) more susceptible to neutrophil mediated killing.

Influence of iodine on nutritional, metabolic and immunological response of goats fed Leucaena leucocephala leaf meal diet

Fifteen indigenous nondescript kids (8·2 kg; 8 months initial age), randomly allotted into three equal groups, were used to study the effects of supplementation of extra iodine on their performance when fed a leucaena (Leucaena leucocephala) leaf meal containing diet. Group I (CON) was fed a control concentrate supplement consisting of a conventional protein source whereas the other two groups (LL and LLI) were fed a concentrate containing leucaena leaf meal so as to supply 0·5 of the net crude protein (CP) requirements. Additionally, animals in group LLI were given supplemental iodine (as potassium iodide solution) at 0·25 mg/head/day. Wheat straw was provided ad libitum as the sole source of roughage during the 120 days of the experimental period. A metabolism trial, conducted at the end of the feeding trial, revealed no variation in the dry matter intake (DMI) among the groups. A significant (P<0·01) decline was evident in digestibility of CP in both the leucaena-fed groups (0·463 and 0·482 versus 0·586) whilst that of the other organic components remained unaffected. Animals on the LL diet exhibited lower (P<0·01) nitrogen retention and average daily gain (ADG) in live weight (LW). Blood collected periodically was analysed for the thyroid hormones triiodothyronine (T 3 ) and thyroxine (T 4 ) as well as other biochemical parameters. At the end of the experimental feeding, the cell-mediated immune (CMI) response of the goats was assessed by intra-dermal inoculation of phytohaemagglutinin-P and measuring the change in skin thickness at various postinoculation hours. The results revealed that the serum concentration of glucose was significantly (P<0·05) higher in the LLI group of animals fed leucaena with iodine. The concentration of cholesterol in serum of LL animals increased significantly (P<0·05) compared to the CON and LLI groups. No variation due to dietary interventions was evident in other indices of metabolic profile. While the concentration of circulating T 3 remained unaffected due to dietary intervention, that of T 4 reduced significantly (P<0·05) in the LL group. Moreover, the T 4 concentration in the LLI group remained similar to that of control indicative of positive impact of iodine supplementation. The immune response revealed that the skin thickness of animals in the LL group was lower (P<0·05) as compared to the control, indicating a compromise of CMI response due to feeding of leucaena leaf meal. Supplementation of iodine appeared to be partially effective in potentiating the response. In conclusion, iodine supplementation could be adopted as a strategic management strategy to ameliorate the negative impacts of feeding leucaena leaf meal in growing kids.

Protein-L-Isoaspartyl Methyltransferase (PIMT) Is Required for Survival of Salmonella Typhimurium at 42°C and Contributes to the Virulence in Poultry

Poultry birds are asymptomatic reservoir of Salmonella Typhimurium (S. Typhimurium) but act as source of human infection for this bacterium. Inside the poultry, S. Typhimurium experiences several stresses, 42°C body temperature of birds is one of them. Proteins are highly susceptible to temperature mediated damage. Conversion of protein bound aspartate (Asp) residues to iso-aspartate (iso-Asp) is one of such modifications that occur at elevated temperature. Iso-Asp formation has been linked to protein inactivation and compromised cellular survival. Protein-L-isoaspartyl methyltransferase (PIMT) can repair iso-Asp back to Asp, thus enhances the cellular survival at elevated temperature. Here, we show that the pimt gene deletion strain of S. Typhimurium (Δpimt mutant strain) is hypersensitive to 42°C in vitro. The hypersusceptibility of Δpimt strain is partially reversed by plasmid based complementation (trans-complementation) of Δpimt strain. Following oral inoculation, Δpimt strain showed defective colonization in poultry caecum, and compromised dissemination to spleen and liver. Interestingly, we have observed three and half folds induction of the PIMT protein following exposure of S. Typhimurium to 42°C. Our data suggest a novel role of pimt gene in the survival of S. Typhimurium at elevated temperature and virulence.

Contribution of protein isoaspartate methyl transferase (PIMT) in the survival of Salmonella Typhimurium under oxidative stress and virulence.

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p<0.001) more susceptible to H2O2 and hypochlorite (HOCl). Further, the Δpimt mutant strain shows hypersusceptibility (p<0.001) to INF-γ stimulated macrophages. This susceptibility is reversed by pharmacological inhibition of reactive oxygen species (ROS) but not reactive nitrogen species (RNS) production. Further, plasmid based complementation enhances the survival of Δpimt mutant strain against oxidants in vitro and also inside the macrophages. In mice model, the LD50 for wild type ST and mutant Δpimt has been 1.73×10(4) and 1.38×10(5), respectively. Further, the mutant strain shows reduced dissemination to spleen and liver in mice. Following infection with a mixture of wild type ST and the Δpimt mutant (co-infection experiment), we recover significantly (p<0.001) less numbers of mutant bacteria from the spleen and liver of mice.

Iodine supplementation to a diet containing Leucaena leucocephala leaf meal: consequences on nutrient metabolism, clinical chemistry and immunity of goats

The present study was aimed at investigating the metabolic effects of iodine supplementation in goats when fed on a diet containing Leucaena leucocephala, intended to create mild iodine deficiency. Two groups of indigenous adult goats were fed on a leucaena leaf meal-based diet without (CON) or with 0.1 mg iodine per day per animal (EXP) for a period of 180 days. Dry matter intake and dry matter digestibility were higher (P < 0.05) in the EXP group. The retention of nitrogen and phosphorus was higher (P < 0.05) in the EXP group. Retention of calcium was also higher (P = 0.082) in the EXP group. The net loss of bodyweight in the CON group (-1.21 kg) was reversed in EXP goats, which showed a net gain (+1.79 kg). The serum metabolic profile reflected a positive influence of iodine supplementation, which also induced greater (P < 0.001) circulating thyroxine concentration. The cell-mediated immune response was higher (P < 0.05) in the EXP group, whereas humoral immunity remained unaltered. The results suggested that supplementation with extra iodine could be used as an effective strategy to counteract functional disorders of the thyroid and associated adverse effects induced in goats by feeding of leucaena leaf meal.

Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide.

ABSTRACT Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.[1] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26 kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.

Effect of Varying the Energy Density of Protein-adequate Diets on Nutrient Metabolism, Clinical Chemistry, Immune Response and Growth of Muzaffarnagari Lambs

Effects of varied dietary energy densities on immune response and performance of Muzzafarnagari lambs were ascertained in a 180-d study. Animals (n = 24), in three groups, were fed diets providing 100% (100E), 80% (80E) or 70% (70E) of their metabolizable energy requirement. Mean nutrient digestibilities varied significantly among treatments. Nitrogen intake was lower (p<0.01) in the 70E. Nitrogen retention, was reduced (p<0.001) in 80E and 70E vs 100E. The average daily gain (p<0.001) was 47.01±4.23, 13.54±1.72 and -16.67±8.24 g for 100E, 80E and 70E, respectively. Hemoglobin concentration, haematocrit, total and differential leukocyte counts were lower (p<0.001) for 80E and 70E than for 100E with a similar trend (p<0.05) for serum glucose and total protein. Serum cortisol was reduced (p<0.001) with decreased energy availability. Antibody titre to Brucella abortus S19 showed an initial reduction in 80E and 70E vs 100E. Delayed-type hypersensitivity response was lower (p<0.001) in 80E and 70E vs 100E, accompanying a lower (p<0.001) nitric oxide production by the peripheral lymphocytes. It is concluded that the reduced dietary energy density significantly affects the growth performance and immune response of lambs.