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.

Evaluation of mammary gland immunity and therapeutic potential of Tinospora cordifolia against bovine subclinical mastitis.

Enhancement of the diseased mammary gland immunity and therapeutic potential of hydro-methanolic extract of Tinospora cordifolia (T. cordifolia; stem) in bovine subclinical mastitis was investigated. Somatic cell count (SCC), total bacterial count (TBC), phagocytic activity, and leukocyte lysosomal enzymes like myeloperoxidase and acid phosphatase activity and Interleukin-8 (IL-8) level were evaluated after intramammary infusion of hydro-methanolic extract (stem) of T.cordifolia in diseased cows. The qualitative analysis of the extract revealed the presence of polysaccharide, phenol, alkaloid, and protein. Intramammary infusion of hydro-methanolic extract of T. cordifolia treatment initially enhanced the SCC; thereafter, significant reduction in cell count (P < 0.05) was observed on day 15 of the treatment period, however, reduction in TBC was observed from day 3 onwards. The phagocytic activity of milk polymorphonuclear cells enhanced in the diseased cows treated with the T. cordifolia extract. Similarly, the lysosomal enzyme content of the milk polymorphonuclear cells enhanced significantly (P < 0.05) in diseased cows treated with T. cordifolia. The IL-8 level in milk serum also increased significantly (P < 0.05) in diseased cows treated with the herb extract. The results suggest that the hydro-methanolic extract of T.cordifolia (stem) possesses antibacterial and immunomodulatory properties. In the present study, the biological activity of the Tinospora cordifolia extract at standardized dose against bovine subclinical mastitis is reported for the first time. Development of alternative therapy with medicinal plants is an option for livestock farmers who are not allowed to use the conventional allopathic drugs under certain farming system or cannot afford to use allopathic drugs.

Immunosuppressive effect of subchronic exposure to a mixture of eight heavy metals, found as groundwater contaminants in different areas of India, through drinking water in male rats.

Immunotoxicity is an important health hazard of heavy metal exposure. Because the risk of combined exposure in the population cannot be neglected, we examined whether subchronic exposure to a mixture of metals (arsenic, cadmium, lead, mercury, chromium, nickel, manganese, and iron) via drinking water at contemporary Indian groundwater contamination levels and at concentrations equivalent to the WHO maximum permissible limit (MPL) in drinking water can induce immunotoxicity in male rats. Data on groundwater contamination with metals in India were collected from literature and metals were selected on the basis of their frequency of occurrence and contamination level above the MPL. Male albino Wistar rats were exposed to the mixture at 0, 1, 10, and 100 times the mode concentrations (the most frequently occurring concentration) of the individual metals in drinking water for 90 days. In addition, one group was exposed to the mixture at a concentration equal to the MPL of the individual metal and another group was used as positive control for immune response studies. The end points assessed were weights of organs, hematological indices, humoral and cell-mediated immune responses, and histopathology of skin and spleen. The MPL and 1x doses did not significantly affect any of the parameters and none of the doses induced any significant changes after 30 days of exposure. The mixture at 10x and 100x doses increased the relative weight of the spleen, but that of thymus, adrenals, and popliteal lymphnodes were increased with the 100x dose. After 90 days, 10x and 100x doses decreased serum protein and globulin contents and increased the albumin:globulin ratio; the albumin level was decreased only with the 100x dose. After 60 days, the total erythrocyte count (TEC), hemoglobin (Hb) level, and packed cell volume (PCV) were decreased with the 100x dose, whereas after 90 days, 10x and 100x doses reduced the TEC, total leukocyte count, Hb level, PCV, mean corpuscular volume, and mean corpuscular hemoglobin. With the 100x dose, the lymphocyte count was decreased after 60 and 90 days, but the neutrophil number was increased after 90 days. Antibody titer was decreased after 75 days with the 100x dose, but after 90 days, it was decreased with both the 10x and 100x doses. In delayed-type hypersensitivity response, these two doses decreased ear thickness after 24 and 48 h and skin biopsies showed a dose-dependent decrease in inflammatory changes. Histologically, the spleen revealed depletion of lymphoid cells and atrophic follicles with reduced follicular activity with higher doses. The findings suggest that hematopoietic and immune systems are toxicologically sensitive to the mixture, which could lead to anemia and suppression of humoral and cell-mediated immune responses in male rats at 10 and 100 times the mode concentrations of the individual components in contaminated groundwater.

Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa.

Seventy semen ejaculates were obtained from 14 Murrah buffalo bulls and were subjected to plasma separation immediately after collection by centrifugation at 2000 rpm for 20 min and stored in liquid nitrogen until analysis. In the seminal plasma the total protein concentration were estimated and the heparin and gelatin binding (HB and GB) proteins were isolated using heparin and gelatin affinity column chromatography. The molecular weight of individual isolated HB and GB protein was determined by SDS-PAGE analysis. Buffalo bull spermatozoa was collected from cauda epididymis under aseptic conditions and was used for the in vitro fertility tests (i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST)). The heparin and gelatin binding buffalo seminal plasma proteins were used in six concentrations i.e. 10, 20, 30, 40, 50 and 60 microg/ml to test their effect on in vitro fertility assessment of cauda epididymal spermatozoa. The overall mean values of total protein, HB and GB proteins were recorded as 29+/-2.7, 2.61 and 0.2mg/ml, respectively. Eighteen total protein bands were observed in the range of 12-127 kDa. Eight major HB proteins were isolated in the range of 13-71 kDa. Seven major GB proteins were isolated in the range of 13-61 kDa in the buffalo seminal plasma. The mean penetration distance (mm) travelled by the buffalo cauda spermatozoa was maximum in HB proteins (26.9+/-0.6) followed by GB proteins (25.4+/-0.6) and control (21.2+/-1.4). The difference in BCMPT values between protein treated and control group was significant (P<0.05). Almost similar trend in the effect of protein on values of HOST percentage in both HB and GB proteins treated semen samples were recorded (66.4+/-0.65 and 66.1+/-0.6, respectively). The difference in HOST values between proteins treated and control group (50.4+/-2.0) was significant (P<0.05). The present results indicate that among the isolated proteins, 4 proteins were commonly seen in both the heparin and gelatin-sepharose affinity column chromatography, and the addition of buffalo seminal plasma proteins improved the in vitro sperm functions (40 microg/ml gave best results) of buffalo cauda spermatozoa.

Immunotherapeutic potential of Ocimum sanctum (L) in bovine subclinical mastitis.

Immunotherapeutic potential of aqueous extract of Ocimum sanctum (O. sanctum) leaf in bovine sub-clinical mastitis (SCM) was investigated. Somatic cell count (SCC), total bacterial count (TBC), milk differential leukocyte count (DLC), phagocytic activity and Phagocytic index and leukocyte lysosomal enzymes like myeloperoxidase and acid phosphatase content were evaluated after intramammary infusion of aqueous leaf extract of O. sanctum. The results revealed that the aqueous extract of O. sanctum treatment reduced the TBC and increased neutrophil and lymphocyte counts with enhanced phagocytic activity and phagocytic index. Similarly, the lysosomal enzymes contents of the milk polymorphonuclear cells (PMNs) were also enhanced significantly in animals treated with the extract. The results suggest that the crude aqueous extract of O. sanctum (leaf) possesses some biologically active principles that are antibacterial and immunomodulatory in nature. As such, the present wok substantiates the therapeutic use of medicinal herb and also emphasizes on the potential of the commonly available non-toxic substances to enhance the mammary immunity.

The activity of milk leukocytes in response to a water-soluble fraction of Mycobacterium phlei in bovine subclinical mastitis.

The effect of a water-soluble fraction (WSF) of a non-pathogenic strain of Mycobacterium phlei was studied in bovine subclinical mastitis (SCM) by measuring the myeloperoxidase and acid phosphatase enzyme levels in the milk leukocytes. Forty-five cows were divided into three equal groups. Group I, consisting of 15 healthy cows, served as the control, whereas groups II and III each contained 15 cows with subclinical mastitis on the basis of a positive reaction in the California mastitis test (CMT). The cows in group II received 100 microg of WSF in 5 ml sterile phosphate-buffered saline, pH 7.4 (PBS) once only, while those in group III received 5 ml sterile PBS daily for 7 days, both treatments being given by the intramammary route. Observations were made up to 30 days after treatment (AT). The CMT of the healthy milk was negative (0), whereas it ranged between 1 and 2 points in SCM. The somatic cell count (SCC) increased significantly (p < 0.05) on day 3, then fell steeply from day 7 up to day 30 AT in the cows in group II. A steady decrease in the total bacterial count (TBC) was observed in the group treated with WSF but the bacterial counts remained high in the groups treated with PBS. The mean acid phosphatase level was enhanced by 119% on day 3 AT in group II but only by 18.7% in the cows in group III. The mean myeloperoxidase level was enhanced by 100% in the cows in group II but only by 18% in those in group III on day 3 AT. This significant reduction in the bacterial load in infected cows caused by intramammary infusion of WSF may be due to activation of the microbicidal activity of the neutrophils, but this requires confirmation.

Mycobacterium bovis AN5 antigens vary in their ability to induce nitric oxide production in blood monocytes of experimentally infected cattle.

Reactive nitrogen intermediates (RNI) are the principal effector molecules of activated monocyte/macrophage populations, responsible for killing and inhibiting the growth of virulent mycobacteria. In vitro nitrite production by blood monocytes of cattle inoculated with live Mycobacterium bovis AN5 was assessed from 0 day through 45 weeks post inoculation (PI). High in vitro nitrite production was observed at the 8th and 12th weeks PI in sensitized cattle but reactivity had fallen by the 20th week PI. To assess the in vitro nitrite producing ability of monocytes induced by individual polypeptides within culture filtrate antigens (CFA) of M. bovis AN5, cellular blotting was performed using peripheral blood mononuclear cells (PBMC) at the 12th week PI. It was observed that polypeptides of MW 70, 65, 60, 25, 24 and 22 kDa of CFA induced high nitrite production by blood monocytes while many polypeptides had little or no effect.

Avian reovirus induces an inhibitory effect on lymphoproliferation in chickens.

The cellular immune responses of chickens inoculated with the vaccine strain S-1133 and/or a field isolate VA-1 of avian reovirus (ARV) were studied. Both strains of virus caused inhibition of the phytohaemagglutinin (PHA)-induced lymphoproliferative response of peripheral blood mononuclear cells (PBMC) and splenic mononuclear cells (SMC) during the initial stage from day 4 up to day 10 post-inoculation (PI), with a later return to the normal value. The inhibition in the PHA-induced lymphoproliferation of SMC could be partially overcome by depletion of adherent cells. The supernatant of the PHA-stimulated SMC culture was also checked in vitro for the presence of suppressive factor(s) produced in response to ARV infection. The culture supernatant from chickens at day 5 PI caused significant inhibition of the PHA-induced lymphoproliferation of control birds, suggesting the presence of suppressive factor(s). ARV infection also significantly inhibited IL-2 production on day 5. There was a significant increase in nitric oxide production by the splenic mononuclear cells of chickens inoculated with either strain of ARV.

Optimum conditions for in vitro chicken IL-2 production and its in vivo role in Newcastle disease vaccinated chickens.

Optimum conditions for in vitro chicken interleukin-2 (IL-2) production were studied. IL-2 containing culture supernatants were generated by mitogen stimulation of splenic mononuclear cells (SMC) and the samples were tested on 72 h Concanavalin A (ConA) blasts for their proliferative ability. 3H-thymidine incorporation was used as a measurement of proliferation. Higher stimulation indices and thus maximal IL-2 production were obtained with the following culture conditions: 5 x 10(6) cells ml(-1) cultured for 24 h in the presence of 10 microg ml(-1) ConA in serum free Iscove's modified Dulbecco medium. The molecule responsible for IL-2 activity was found to have a molecular weight of 14000 as estimated by size exclusion chromatography. SMC obtained from chickens inoculated with Newcastle disease virus were used to study the immunomodulatory role of IL-2. The lymphocyte transformation test was used as an in vitro correlate of cell mediated immunity in these chickens. The mitogen responses of cells obtained from virus inoculated and control chickens were similar on the basis of stimulation indices. Antigen specific lymphocyte proliferation was demonstrated using SMC obtained from virus inoculated chickens. Uptake of exogenous IL-2 by 72 h ConA blasts was of similar magnitude in both virus inoculated and control chickens indicating that uptake of IL-2 by T lymphocytes was normal in Newcastle disease virus inoculated chickens.

Reactive nitrogen intermediates production from naive and activated monocytes by extracts of Mycobacterium bovis BCG.

Extracts of Mycobacterium bovis strain BCG were assessed for in vitro activation of monocytes to produce reactive nitrogen intermediates. The culture filtrate of M. bovis BCG was a strong inducer of nitrite production while live BCG and sonicated antigens were also potent inducers. Other extracts activated monocytes which showed an increase in nitrite production after in vitro BCG infection.

Immunogenic activity of viral polypeptides of an Indian isolate of infectious bursal disease virus.

An Indian isolate of infectious bursal disease virus, i.e. IBDV-P/AD/81, was analysed for immunogenic activity of its structural polypeptides. Virus was purified from infected bursal homogenate by sucrose density gradient centrifugation. It showed five different structural polypeptides of 75.8, 45, 40.7, 33.1 and 27 kDa molecular weights in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Anti infectious bursal disease virus (IBDV) antibodies were tested using enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT). Polypeptide 40.7 kDa (VP2) was known to have neutralizing epitopes. However, polyclonal anti VP2 failed to neutralize the virus. It was interpreted that VP2 had labile neutralizing epitopes which get altered confirmationally by SDS. Surprisingly, polyclonal anti 33.1 kDa (VP3) had mild neutralizing activity.

Antigenic characterization of Mycobacterium bovis BCG culture filtrate.

Mycobacterium bovis BCG culture filtrate, on gelfiltration chromatography revealed four prominent regions. Of these 4, region II possessed the highest antigenic reactivity by enzyme-linked immunosorbent assay (ELISA) and delayed type hypersensitivity (DTH) skin testing in guinea pigs. On anion-exchange chromatography, region II was resolved into 5 prominent fractions of which fraction II was found to have the highest antigenic reactivity. On sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), M. bovis BCG culture filtrate revealed 11 structural polypeptides. Fraction II yielded a homogenous polypeptide of 15.3 kDa molecular weight. All fractions cross-reacted with various mycobacteria used in this study except a 15.3 kDa polypeptide which was specific to M. bovis BCG.

Antigenic characterization of Mycobacterium bovis BCG soluble antigens.

The proteins in Mycobacterium bovis BCG sonicated antigens were separated by gel filtration chromatography. A total of 6 regions were observed. All the regions were analyzed separately for their antigenic reactivity using enzyme linked-immunosorbent assay (ELISA), delayed type hypersensitivity (DTH) skin testing and an in vitro lymphocyte transformation test (LTT). Region I of high molecular weight was found to have more reactivity in comparison to regions of lower molecular weight. When region I was subjected to anion exchange chromatography, it resolved into 5 further regions. Of these regions only region III was found to have highest reactivity when tested in ELISA, DTH and LTT. This was interpreted to indicate that region III of anion exchange chromatography has immunodominant epitopes. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, this region was found to be a homogeneous polypeptide of 71 kDA molecular weight. All regions were found to crossreact with various mycobacteria used in this study.

Effect of costimulants on interleukin-2-like activities from goat peripheral blood mononuclear cells.

Culture supernatants containing interleukin-2-like activities (CS-IL2) were prepared from goat peripheral blood cells (mononuclear cells 75% and polymorphonuclear cells 25%). These were stimulated with three costimulants, (tetradecanoyl phorbol acetate, indomethacin and calcium ionophore A23187), either alone or in different combinations, in RPMI-1640 medium (containing 0.5% bovine serum albumin (BSA)) with or without serum. After 18 h of incubation with costimulants, concanavalin A (Con A) was added and the incubation was continued for next 48 h. Higher interleukin-2 (IL-2)-like activities were generated in the culture supernatants prepared in RPMI-1640 growth medium containing 0.5% BSA without serum. Further, IL-2-like activities were much higher in culture supernatants obtained by stimulation with all the three costimulants, as well as Con A, than the two costimulants with Con A or any of the costimulants with Con A.

Determination of optimal in vitro conditions for caprine alternative complement pathway assay.

Optimal in vitro testing conditions for caprine alternative complement pathway assay were determined. Effects of the following variables were tested: heterologous erythrocytes; pH, ionic strength and Mg2+ ion concentration of the complement diluent; incubation time and temperature. Rabbit erythrocytes were the optimal target cells. The optimal buffer conditions were: pH 8.0, ionic strength 0.06 mmol NaCl and 5mmol Mg2+ ion. Optimal incubation time and temperature were 75 min and 30 degrees C, respectively.

Bovine interleukin 2: production kinetics in normal and in vivo antigen-primed cattle.

Conditions influencing production kinetics of bovine interleukin 2 (IL-2), viz. cell concentration, mitogen and its concentration, length of incubation, nutrient medium and in vivo antigen-priming were investigated. Peripheral blood mononuclear cells (PBL) of outbred cattle of different age groups showed considerable variation in their ability to secrete IL-2 which possibly reflects their immune competence. Of the cultures initiated with PBL, 5 x 10(6) cells/ml cultured in serum free Iscove's medium and stimulated with 5 micrograms Con A/ml for 24 hr produced maximal amount of IL-2 activity. In vivo antigen-priming of bovine lymphocytes with the live attenuated rinderpest virus revealed that IL-2 production was not affected by rinderpest virus but the in vivo antigen-priming possibly resulted in concomitant production of suppressor factor(s) which suppressed the already produced IL-2. The implications of this factor(s) in relation to regulation of immune responses in the disease process are discussed.

Bovine interleukin 2: biochemical and biological characterization.

Interleukin 2 (IL-2), secreted by bovine peripheral blood mononuclear cells (PBL) on stimulation with concanavalin A (Con A), was purified and characterized by different chromatographic and electrophoretic techniques. The ability of IL-2 to support proliferation of Con A-stimulated bovine lymphoblasts was used to assay and quantitate IL-2 activity. Bovine IL-2 having an apparent MW of 27,000 eluted from a gel-filtration column; from an anion exchange column peak activity was detected at 190 mM NaCl. Binding of bovine IL-2 to phenyl-Sepharose gel and elution with 35-60% ethanediol indicated its hydrophobic nature. Studies on cross-species reactivity revealed that both buffalo and goat lymphocytes respond to cattle IL-2 and detected 35% of activity from a standard cattle IL-2 preparation. Sheep lymphocyte response to cattle IL-2 was negligible.

Effect of oral administration of T-2 toxin on glutathione shuttle enzymes, microsomal reductases and lipid peroxidation in rat liver.

Effects of T-2 toxin on liver lipid peroxidation, glutathione shuttle enzymes and microsomal reductases have been studied in rats at 8, 16 and 24 hr after feeding a single dose of toxin (2.0 mg/kg) and at 7, 14 and 21 days after feeding of toxin (0.75 mg/kg) daily. Feeding of a single dose of T-2 toxin caused significant increase in liver lipid peroxidation in rats at 8, 16 and 24 hr post treatment. The liver lipid peroxidation was also significantly increased at 14 and 21 days after feeding of 0.75 mg/kg of T-2 toxin daily to rats. The activities of liver GSH-shuttle enzymes, i.e. glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase, were significantly higher in rats after both feeding schedules of T-2 toxin. NADPH-cytochrome c reductase activity was significantly lower at 8, 16 and 24 hr in liver of rats fed a single dose of T-2 toxin, whereas NADH-cytochrome b5 reductase was significantly higher until 16 hr and then declined below normal at 24 hr post treatment. In rats fed multiple doses of T-2 toxin, both liver microsomal reductases were significantly reduced. These results suggest that T-2 toxin/or its metabolites in the liver may be involved in the generation of free radicals which cause the observed increase in lipid peroxidation.

Liver lipid metabolism in T-2 toxicosis. I. Effects of a single dose feeding of T-2 toxin to rats.

The acute effects of oral administration of a single dose of T-2 toxin (2.0 mg/kg body wt) to rats on whole liver lipid metabolism were studied at 8, 16 and 24 h post-treatment. Administration of T-2 toxin significantly increased liver and microsomal total lipids, free cholesterol, esterified cholesterol and triglycerides initially at 8 h, which subsequently returned to control values at 24 h. However, no significant alterations were observed in the contents of whole liver and liver microsomal total phospholipids and phosphatidyl choline, except that phosphatidyl ethanolamine and sphingomyelin + lysophosphatidyl ethanolamine contents in liver at 16 and 24 h and sphingomyelin + lysophosphatidyl ethanolamine content in liver microsomes at all three periods were significantly lower. The incorporation of 1-14C-acetate into whole liver and liver microsomal total lipids was reduced at 16 and 24 h post feeding. However, the incorporation of 1-14C-acetate into liver and microsomal free cholesterol, esterified cholesterol and triglycerides was significantly higher at 8 h, subsequently returning to the control value at 24 h; incorporation was significantly lower even into microsomal triglycerides. The incorporation of 1-14C-acetate into liver and its microsomal total phospholipids, phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin + lysophosphatidyl ethanolamine, was significantly decreased at all three periods post toxin treatment. The results suggested that T-2 toxin inhibited the incorporation of 14C-acetate mainly into liver and its microsomal phospholipids and their subfractions in rats.