Reena Mukherjee

Selenium and vitamin E increases polymorphonuclear cell phagocytosis and antioxidant levels during acute mastitis in riverine buffaloes

Antioxidant, antiinflammatory and phagocytic activities were studied in milk polymorphonuclear cells (PMNs) isolated from healthy buffaloes (group I) and during clinical mastitis with the treatment of Enrofloxacin alone (group II) and combined treatment with Enrofloxacin and Vitamin E plus selenium (group III). On days 0,3, 8 and 15 the milk Somatic cell count (SCC) were significantly higher in mastitic milk than in milk obtained from healthy buffaloes. In group II SCC decreased significantly on day 3 and day 8, however in group III reduction in SCC was observed on day 3, day 8 and day 15 (P < 0.05). The antiinflammatory activity was evaluated by determining nitrite plus nitrate (NOx) production in the milk PMNs before treatment and on day 8. NOx activity was significantly higher in mastitic milk than from healthy controls, both before and after treatment (P < 0.05). In group II and group III the activity decreased significantly on day 8 (P < 0.05). The Glutathione peroxidase (GSH-Px) activity was estimated in the milk polymorphonuclear cell (PMNs) supernatant. GSH-Px activity was significantly lower in mastitic buffaloes than in healthy controls, both before and after treatment (P < 0.05). In group II levels did not change in response to treatment, whereas in group III levels had increased significantly on day 8 (P < 0.05). The phagocytic activity (PA) (percentage of neutrophil that had phagocytosed 1–6 bacteria) and phagocytic index (PI) (average number of bacteria/ leukocytes counted in 100 cells) of the milk PMNs was significantly lower in mastitic buffaloes (P < 0.05). In group II the PA and PI did not change in response to treatment, whereas in group III both the parameters had increased significantly on day 8 (P < 0.05). The results of the present experiment indicated enhancement of antioxidative and cellular defense and reduction of somatic cell count in the mastitic animals treated with Enrofloxacin and Vitamin E plus Selenium as compared to the Enrofloxacin treatment alone. Hence Vitamin E plus selenium therapy may be added along with the antibiotics for effective amelioration of intramammary infection in buffaloes.

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 μg 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.

Expression of cytokines and respiratory burst activity of milk cells in response to Azadirachta indica during bovine mastitis

The immuno therapeutic potential of hydro-methanolic extract of Azadirachta indica (A. indica) was studied during bovine clinical mastitis (CM). The somatic cell count (SCC), total bacterial count (TBC), milk differential leukocyte count (DLC), hydrogen peroxide (H2O2), superoxide anion (O2−) production and interleukin- 2 (IL-2) and gamma interferon (IFN-γ) cytokines expression were studied before and after intramammary infusion of A. indica extract in diseased cows. The results revealed that A. indica treatment significantly (P < 0.05) decreased the SCC, TBC, milk neutrophil percent and significantly (P < 0.05) enhanced milk lymphocyte percent, H2O2 and O2− production by milk cells. The IL-2 and IFN-γ were expressed in normal healthy cows and diseased cows after A. indica treatment, whereas both the cytokines could not be expressed in cows treated with antibiotic and in untreated diseased cows. The results of the present study indicated anti inflammatory, antibacterial and immunomodulatory potential of the herb, these activities could be due to the presence of bioactive principle in the extract. This is a preliminary trial indicated beneficial effect of the herb against bovine mastitis it can be developed as an alternative therapy where the use of antibiotics is normally restricted.

Alterations in oxidant/antioxidant balance, high-mobility group box 1 protein and acute phase response in cross-bred suckling piglets suffering from rotaviral enteritis

Rotaviral enteritis has emerged as a major cause of morbidity and mortality in piglets during their post-natal life. The present study was carried out to examine high-mobility group box 1 (HMGB1) protein, acute phase response and oxidative stress indices in the serum of suckling piglets suffering from enteritis with or without association of porcine group A rotavirus infection. The present investigation utilized 23 clinical cases with signs of acute enteritis and 12 more healthy piglets of a similar age group as control animals. Out of 23 enteritis cases, 12 cases were found to be positive for porcine group A rotavirus infection as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for group A rotavirus, and the rest were found negative. The acute enteritis cases in piglets were associated with an elevated level of HMGB1 protein and serum haptoglobin and ceruloplasmin suggestive of an acute phase response. Among the oxidative stress indices, the concentrations of malondialdehyde (MDA) and nitric oxide (NO) in serum were significantly increased. A pronounced drop of total antioxidant capacity and the activity of antioxidant enzymes such as catalase and superoxide dismutase in the serum of piglets suffering from acute enteritis compared to healthy ones were also noticed. The alterations in HMGB1 protein, acute phase response and oxidative stress indices were more pronounced in cases with the involvement of porcine rotavirus as compared to rotavirus-negative cases. It is concluded that HMGB1 protein, markers of oxidative stress and acute phase proteins might play an important role in the aetiopathogenesis of porcine diarrhoea caused by rotavirus and might be true markers in diagnosing the conditions leading to the extension of the prompt and effective therapeutic care.

Dynamics of milk leukocytes in response to a biological response modifier during bovine subclinical mastitis.

The objective of this study was to evaluate the dynamics of milk leukocyte in response to intramammary infusion of a biological response modifier (BRM) prepared from Nocardia globerula during bovine subclinical mastitis (SCM). The somatic cell count (SCC), total bacterial count (TBC) in milk, cyclooxygenase (COX) activity, phagocytic activity, production of hydrogen peroxide (H2O2) and myeloperoxidase (MPO) activity in milk leukocytes were evaluated after intramammary infusion of BRM in quarters inflicted with SCM. Intramammary infusion of BRM significantly enhanced the SCC in earlier phase with subsequent reduction on day 7 after initiation of treatment. Similarly, the COX activity in milk cell lysate increased on day 3 and reduced on day 5. However reduction in TBC could be observed from day 3 onwards. The phagocytic activity of milk leukocytes was lower in mastitic cows whereas, significant enhancement in phagocytic activity was recorded in post treated cows with BRM. Similarly, the H2O2 production and MPO activities in milk leukocytes were enhanced significantly in diseased cows in response to BRM infusion. Significant enhancement of phagocytic activity, H2O2 and MPO activities indicate the priming of resident milk leukocytes in response to BRM infusion. Initial influx of SCC and raised COX activity also indicate the immunomodulatory activity of BRM. Reduction of TBC could be due to increased leukocytosis or direct microbicidal activity of activated milk cells. In the present study, the biological activity of BRM at standardized dose against bovine SCM is reported for the first time. Development of such therapy is warranted to reduce the drug resistance of microorganisms and contamination of milk with antibiotic residue.

Activity of cyclooxygenase-2 and nitric oxide in milk leucocytes following intramammary inoculation of a bio-response modifier during bovine Staphylococcus aureus subclinical mastitis.

The objective of the study was to evaluate the effects of intramammary infusion of a bio-response modifier (BRM) prepared from Nocardia globerula on certain inflammatory markers and percentage of neutrophil/lymphocyte in mammary secretions during bovine Staphylococcus aureus subclinical mastitis (SCM). The somatic cell count (SCC), total bacterial count (TBC) in milk, cyclooxygenase-2 (COX-2) activity, production of nitrite and nitrate (NOx) in milk leukocytes and neutrophil % and lymphocyte % in milk were evaluated before and after intramammary infusion of BRM in healthy and quarters inflicted with S. aureus SCM. Intramammary infusion of BRM significantly enhanced the SCC in earlier phase with subsequent reduction on day 7 after initiation of treatment. Whereas, the reduction of TBC was observed from day 3 onwards. The COX-2 activity and NOx production in milk cell increased initially on day 3 of post treatment but reduced on day 5 in SCM infected quarters following BRM infusion. The neutrophil % and lymphocyte % in milk also enhanced significantly on day 3 but reduced on day 5 in SCM infected quarters in response to BRM infusion. Initial influx of SCC, increased neutrophil%, lymphocyte % and enhanced COX-2 and NOx activity indicate the immunomodulatory potential of BRM in S. aureus SCM. Reduction of TBC could be due to increased leukocytosis or direct microbicidal activity of the activated milk cells. The beneficial effect of the BRM could be used as alternative therapy in the control of S. aureus SCM in cows, either alone or in conjunction with antibiotic therapy.

Assay of alterations in oxidative stress markers in pigs naturally infested with Sarcoptes scabiei var. suis.

The present study aimed to examine the status of antioxidant systems of the pigs naturally suffering from sarcoptic mange. Fifty nine pigs were divided into three groups, healthy control (group I, n=15), subclinical sarcoptic mange (group II, n=22) and clinical sarcoptic mange (group III, n=22). To assess the status of antioxidant systems; lipid peroxides (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), ascorbic acid, zinc and copper concentrations in the blood samples as well as LPO, SOD, CAT and glutathione-s-transferase (GST) activities in the skin were measured. The GSH, SOD, GPx, ascorbic acid, zinc, copper concentrations in blood were significantly lower in the pigs suffering from clinical and subclinical sarcoptic mange, when compared with the healthy control. However, LPO content of these infested pigs was significantly higher. The CAT, SOD and GST activities in the skin of the diseased pigs were significantly lower, whereas LPO was significantly higher as compared to the healthy control. From the present study, it may be concluded that sarcoptic mange bestows remarkable alterations in the oxidative stress markers and imposes compromisation of the antioxidant status of the infested pigs.

Immunomodulatory potential of β-glucan as supportive treatment in porcine rotavirus enteritis

A non-blinded randomized clinical trial was conducted to assess the immunomodulatory potential of β-glucan (BG) in piglet diarrhoea associated with type A rotavirus infection. A total of 12 rotavirus-infected diarrheic piglets were randomly divided into two groups: wherein six rotavirus-infected piglets were treated with supportive treatment (ST) and other six rotavirus-infected piglets were treated with BG along with ST (ST-BG). Simultaneously, six healthy piglets were also included in the study which served as control. In rotavirus-infected piglets, marked increase of Intestinal Fatty Acid Binding Protein-2 (I-FABP2), nitric oxide (NOx), Interferon-γ (IFN-γ) concentrations and decrease of immunoglobulin G (IgG) were noticed compared to healthy piglets. The faecal consistency and dehydration scores were significantly higher in rotavirus-infected piglets than healthy piglets. The ST-BG treatment progressively reduced the I-FABP2 and increased the IgG concentrations over the time in rotavirus-infected piglets compared to piglets received only ST. A pronounced enhancement of NOx and IFN-γ concentrations was observed initially on day 3 and thereafter the values reduced on day 5 in ST-BG treated piglets in comparison to piglets which received only ST. Additionally, ST-BG treatment significantly reduced faecal consistency and dehydration scores on day 3 compared to ST in rotavirus-infected piglets. These findings point that BG represents a potential additional therapeutic option to improve the health condition and reduce the piglet mortality from rotavirus associated diarrhoea where porcine rotavirus vaccine is not available.

Adding a bio-response modifier and zinc oxide to piglet weaner diets influences immunological responses to weaning

The effect of zinc oxide (ZnO) and a Mycobacterium smegmatis-derived bio-response modifier (BRM) supplementation on blood neutrophil functions, high-mobility group box 1 (HMGB1) protein and pro-inflammatory cytokine responses was studied in early weanling piglets. In total, 45 piglets were placed in the following five groups: basal diet only (I), supplemented with ZnO (II), supplemented with BRM (III), supplemented with ZnO plus BRM (IV) in basal diet and basal diet without weaning from dam (V). The phagocytic activity, superoxide anion and myeloperoxidase production in blood neutrophils and the concentrations of HMGB1, TNF-α, IFN-γ and IL-1β in blood plasma were measured before and after weaning. The neutrophil functions were impaired and the concentrations of HMGB1, inflammatory cytokines, were elevated in piglets during the post-weaning period. The neutrophil functions were not improved until Day 7 of weaning (P > 0.05) and pronounced elevation (P < 0.05) in the concentration of pro-inflammatory cytokines and HMGB1 was observed until Days 14 and 21 respectively, in Groups II and III. The addition of BRM plus ZnO in basal diet improved superoxide anion and myeloperoxidase production on Day 2 (P < 0.05) and decreased the TNF-α and IFN-γ concentrations on Day 7 (P < 0.05), with no significant change in the level of IL-1β and HMGB1 in Group IV. Finally, it is concluded that addition of ZnO plus BRM in the diet induced the neutrophil functions and reduced the inflammatory cytokine response much earlier to stimulate innate immunity than did ZnO or BRM alone.

Activity of milk leukocytes in response to a biological response modifier during bovine subclinical mastitis

Conception rates in dairy cattle in the United States have decreased to 25 – 35%, potentially a result of luteal insufficiency. Different methods have been tried to increase circulating progesterone (P4) concentrations to improve pregnancy rates. We developed a method that increases the size and P4 production of the primary corpus luteum (CL). Chronic administration of a gonadotropin releasing hormone (GnRH) agonist increases luteinizing hormone (LH) secretion, subsequent P4 production by the CL, and ovarian follicular dynamics. Chronic administration of a GnRHagonist during the early luteal phase increases luteal size, P4 circulating concentrations, and conception rates using a small number of lactating dairy cows of various parity. Ovarian follicular dynamics are different during the second ovarian follicular wave in first lactation heifers as compared to multiparous cattle. In all cattle, co-dominance of antral follicles occurs during the first ovarian follicular wave. Codominance of follicles is more prevalent on the ovary containing the CL than the contralateral ovary in cows administered treatment. It is unclear the difference between ovarian follicular dynamics between the ipsilateral and contralateral ovary, but is speculated that intraovarian communication is occurring that has yet to be determined. Furthermore, if the cows were not bred the GnRH treatment had no effect on the size of the ovulatory follicle or length of the estrous cycle following termination of treatment. The current model of chronic GnRH agonist treatment controlsluteal size, circulating P4 production, ovarian follicular dynamics, length of the estrous cycle, and conception rates in lactating dairy cows.I was reported that adult somatic cells could be reprogrammed in enucleated oocytes and developed to a normal sheep after nuclear transfer in 1997. But, it is also clarified that the successful rate of animal cloning is still very limited. One of the possible reasons for the low success is the failure of reprogramming of somatic cells in ooplasm after nuclear transfer. Failure of the reprogramming of somatic cell nuclei in ooplasm causes variedabnormalities to SCNT embryos in every developmental stage, including both preimplantation and postimplantation stages, and resulted in the low successful rate at birth. But the mechanism of reprogramming or reprogramming factors in the enucleated oocytes are still unclear.I will review the recent progress of animal cloning in this talk. Especially, whether goodembryos can be selected before embryo transfer, and how to raise the viability of clonedembryos after implantation will be discussed.Twelve (12) matured lactating ewes weighting between 16 and 25kg live weights were used to investigate the effect of udder stimulation, stage of lactation, and parity on milk yield in West African Dwarf (WAD) sheep in a semi intensive system of management. For udder stimulation, the animals were divided into 2; 6 animals were stimulated by massaging, cleaning and drying the udder for about 2 minutes before milking and while the other 6 were not stimulated, the udder were just milked. Stage of lactation was divided into 4; early, mid, late, very late respectively with 3weeks interval. The animals were on first and second parity. The animals were allowed to graze in an established paddock consisting of Stylosanthas hamata, Panicum maximum,Pennisetum purpureum etc. for about 5 hours and then supplemented with concentrate (17% CP at 5% body weight). Each quarter of the udder was hand milked separately once daily at 8.00am for 12 weeks. Their lambs were removed 5 pm each day until after milking the following day when they were released to suckle the dam. The 15 hour milk yield was divided by 15 and multiplied by 24 to obtain the 24 hour milk yield. The milk yield obtained was recorded in mass (g) and volume (cm) respectively. The result of the study showed that udder stimulation, stage of lactation and parity significantly affected milk yield in WAD sheep.S aureus is a major human and animal pathogen that can cause a variety of diseases. The exported α-toxin (hla) is an important virulence factor in many S. aureus infections. Although the complicated regulation of hla expression has been well studied in human S. aureus isolates, the mechanisms of hla regulation in bovine S. aureus isolates remain poorly defined. We found that many bovine S. aureus isolates generate tremendous amounts of α-toxin in vitro culture compared to human clinical S. aureus isolates. To explore potential regulatory mechanisms, we identified predominant single nucleotide polymorphisms (SNPs) at the hla promoter regions. Using bioinformatics, site-directed mutagenesis and hla promoter-lux reporter approaches, we identified and demonstrated that the SNPs contribute to the differential control of hla expression among bovine and human S. aureus isolates. Using a DNA affinity assay, gel-shift assays and a null mutant, we identified and revealed that an hla positive regulator, SarZ, mainly contributes to the involvement of the SNPs in mediating hla expression. In addition, we found that the hyper-production of α-toxin in these bovine S. aureus isolates may be partially attributable to higher transcription levels of hla positive regulators, including agrA, saeR, and arlR, but a lower expression level of hla repressor rot compared to the human S. aureus isolate. These findings provide new insights into the regulatory mechanisms of hla expression in S. aureus. Moreover, the identification of predominant SNPs in the hla promoter region may provide a novel method for genotyping the S. aureus isolates.The objective of the present investigation was to evaluate the activity of milk leukocyte in response to intramammary infusion of a Nocardia globerula derived biological response modifier (BRM) in bovine subclinical mastitis (SCM). The somatic cell count (SCC), total bacterial count (TBC) in milk, milk differential leukocyte count (DLC), cyclooxygenase (COX) activity, phagocytic activity, production of hydrogen peroxide (H2O2), nitric oxide (NOx) and myeloperoxidase (MPO) activity in milk leukocytes were evaluated before as well as after intramammary infusion of BRM in cows inflicted with SCM. Intramammary infusion of BRM significantly enhanced the SCC in earlier phase with subsequent reduction on day 7 onwards after initiation of treatment. Similarly, the COX activity in milk cell lysate increased on day 3 and reduced on day 5. However reduction in TBC could be observed from day 3 onwards. The significant enhancement in milk lymphocyte%, phagocytic activity, H2O2, NOx production and MPO activities was recorded in post treated quarters with BRM. Significant enhancement of phagocytic activity, H2O2, NOx and MPO activities indicate the priming of resident milk leukocytes in response to BRM infusion. Initial influx of SCC and raised COX activity also indicate the immunomodulatory activity of BRM. Reduction of TBC could be due to increased leukocytosis or direct microbicidal activity of activated milk cells. In the present study, the biological activity of BRM at standardized dose against bovine SCM is reported for the first time. Development of such therapy is warranted to reduce the drug resistance microorganisms and contamination of milk with antibiotic residue.