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.

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.

Nuclear lipid peroxidation induced in rat liver by T-2 mycotoxin.

Oral administration to rats of T-2 mycotoxin (1.25 mg/kg) for five days causes an increase in lipid peroxidation (ascorbate-induced as well as NADPH-dependent) in hepatic nuclei, while the activity of liver glutathione-S-transferase (EC. 2.5.1.18) is decreased. The hepatotoxicity could be due to lipid peroxidation induced by depletion of hepatic reduced glutathione and/or production of free radicals.

T-2 toxin induced changes in liver and serum enzymes of rats.

The effects on liver and serum enzymes of feeding a single dose (2 mg/kg) and daily doses (0.75 mg/kg) of T-2 toxin were studied in young male rats. Sample times were 8, 16 and 24 hr for single dose administration and 7, 14 and 21 days for daily dose administration. T-2 toxin in single and daily doses significantly reduced activities of hepatic glutamate pyruvate transaminase (GPT) and alkaline and acid phosphatases at all the sampling periods. In both feeding trials, levels of serum GPT increased, while that of acid and alkaline phosphatases significantly decreased at all the sampling times. This study indicates that the liver is affected by feeding T-2 toxin to rats.

Effects of T-2 toxin on glucose and tryptophan uptake and intestinal mucosal enzymes.

We studied the uptake of D-glucose and L-tryptophan by the small intestine and estimated the activities of the intestinal brush border enzymes (sucrase, lactase, NA+-K+-ATPase and alkaline phosphatase) and lysosomal enzymes in rats receiving T-2 toxin orally. considerable decrease occurred in glucose and tryptophan uptake and in brush border sucrase, lactase and (Na+-K+)-ATPase. Alkaline phosphatase activity and release of lysosomal enzymes (acid phosphatase and acid ribonuclease) was unchanged.

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.

Effect of T-2 toxin administration to rats on lipid metabolism in liver.

The effect of oral dosing of rats with 1.5 mg T-2 toxin/kg body weight daily for 4 days on metabolism of liver lipids was studied. T-2 toxin significantly elevated total liver lipids, triglycerides, free cholesterol, total phospholipids and phosphatidyl choline, whereas the level of sphingomyelin + lysophosphatidyl ethanolamine was reduced. No change in the esterified cholesterol and phosphatidyl ethanolamine contents was observed. Incorporation of [1-14C]acetate into liver lipids, esterified cholesterol, triglycerides, free cholesterol and phosphatidyl ethanolamine was reduced in T-2 toxin-treated animals, implying reduced lipogenesis. Increased lipids in liver in T-2 toxin-treated rats are possibly due to an impaired secretion of lipids from the liver.

Effects of feeding T-2 toxin on RNA, DNA and protein contents of liver and intestinal mucosa of rats.

Young male albino rats were fed 1.5 mg T-2 toxin/kg body weight daily for 4 days and the effects on body weight, liver weight and protein, RNA, and DNA contents of liver and intestinal mucosa were studied. A significant decrease in body weight and an increase in liver weight were observed. Liver protein and DNA and intestinal mucosal protein contents were significantly decreased, whereas intestinal mucosal RNA content was increased. Decreased liver and intestinal mucosal protein synthesis in T-2 toxin-fed rats was inferred from the [14C]leucine incorporation studies.

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.

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.

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.

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.

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.

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.

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.

Effects of T-2 toxin gavage on the synthesis and contents of rat-liver macromolecules.

Effects of oral administration of T-2 toxin (0.75 mg/kg body weight/day) for 7, 14 or 21 days on the liver and plasma of young male rats were studied. A significant decrease in body weight and an increase in liver weight were observed in rats treated with T-2 toxin. Liver protein and glycogen levels were significantly lower than in controls after 21 days of treatment, but no significant differences were observed after 7 or 14 days. Levels of RNA in liver were significantly increased after 7, 14 and 21 days of treatment whereas liver DNA levels were significantly lower than in controls at each time interval. Liver microsomal protein was significantly decreased after 14 and 21 days, but microsomal RNA contents were significantly increased at 7 days and significantly decreased at 21 days. The levels of serum protein at 7, 14 and 21 days and of blood glucose at 14 and 21 days were significantly lower in T-2 toxin-treated rats. The levels of incorporation of [14C]leucine and [3H]uridine into liver protein and RNA, and into liver microsomal protein and RNA, were higher than in controls at 7 days, but then decreased. The incorporation of [3H]thymidine into liver DNA was not significantly altered in animals treated with the toxin.

RNA synthesis in liver nuclei of young rats fed varying amounts of vitamin A.

Twenty five days old male rats were feed 5000, 20000 and 40000 I.U. of vitamin A for two days and its effects studied on RNA contents and its synthesis from orotic acid-6(-14)C in liver and its nuclear and mitochondrial fractions. RNA contents and its synthesis from orotic acid-6(-14)C in liver, its nuclear and mitochondrial fractions was not affected in rats 5000 I.U. of vitamin A, but these were reduced in rats fed 20000 and 40000 I.U. of vitamin A. The turnover rate of nuclear RNA was significantly greater in rats fed 5000 I.U. of vitamin A as compared to the controls.

Studies on mode of action of vitamin A.

Effect of feeding 5000,10 000, 20 000, 30 000 and 40 000 I.U. of vitamin A for two days to 25 days old rats has been studied on liver protein, RNA and DNA, liver and plasma lipids and adrenal cholesterol. The incorporation of palmitate- I-14-C into liver and plasma lipids has been studied in vitamin A fed rats. Two patterns of effects of vitamin A were observed in rats on their liver protein, RNA and DNA and liver and plasma lipids. At lower amounts of vitamin A feeding either the above did not change or were increased, but at higher amounts of vitamin A feeding the above were decreased as compared to the controls. Plasma nonesterified fatty acids were increased and adrenal cholesterol decreased in rats fed 5000 I.U. of vitamin A, but were not affected in rats fed higher amounts of vitamin A. The utilization of palmitate-I-14-C for the synthesis of liver triglycerides and phosphoglycerides was reduced in rats fed higher amounts of vitamin A, but was not significantly different from the controls in rats fed liver amounts of vitamin A. The effects of vitamin A are influenced by the age of the animal as well as the amounts of vitamin A fed. The results show that the effects of vitamin A are exerted by the stimulation of adrenal activity (increased corticoid synthesis).