Evaluation of canine bone marrow-derived mesenchymal stem cells for experimental full-thickness cutaneous wounds in a diabetic rat model.

BACKGROUND: The wound healing potential of canine bone marrow-derived mesenchymal stem cells (BMSCs) was evaluated in the excisional wound of streptozotocin-induced diabetic rats. RESEARCH DESIGN AND METHODS: Xenogenic BMSCs were collected aseptically from the iliac crest of healthy canine donors under general anesthesia. Full-thickness experimental wounds (20 × 20 mm2) on the dorsum of forty-eight adult healthy Wistar white rats. The wounds were assigned randomly to three treatment groups: PBS (Group A) or BMSCs (Group B) injected into the wound margins on days 0, 7, and 14 or BMSCs (Group C) injected into the wound margins on days 7, 14, and 21 post-wounding. The degree of wound healing was evaluated based on macroscopical, hemato-biochemical, histopathological, and histochemical parameters. RESULTS: The results indicated granulation tissue formation with reduced exudation and peripheral swelling in the treatment groups compared to the control group A. Similarly, the degree of wound contraction was significantly higher in groups B and C animals than group A on days 14 and 21 post-wounding. The transplantation of BMSCs resulted in early drying of wounds, granulation tissue appearance, and enhanced cosmetic appearance. CONCLUSION: The histopathological, histochemical, and gross findings suggested the therapeutic potential of xenogeneic mesenchymal stem cell therapy in managing diabetic wounds. ABBREVIATIONS: BMSCs-bone marrow-derived mesenchymal stem cells, PBS-phosphate-buffered saline, MSCs-mesenchymal stem cells, FBS-fetal bovine serum, ECM-extracellular matrix.

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

A non-blinded randomized clinical trial was conducted to assess the immunomodulatory potential of ß-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.

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.

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.

Acute Phase Response and Neutrophils : Lymphocyte Ratio in Response to Astaxanthin in Staphylococcal Mice Mastitis Model.

The purpose of the study was to determine the immunotherapeutic effect of astaxanthin (AX) on total clinical score (TCS), C-reactive protein (CRP), and neutrophil : lymphocyte ratio in mice mastitis model challenged with pathogenic Staphylococcus aureus. Twenty-four lactating mice were divided in 4 equal groups: group I mice served as normal healthy control, group II, positive control, were challenged with pathogenic S. aureus, group III mice were challenged and treated with AX, and group IV were treated with amoxicillin plus sulbactum. The TCS was higher in postchallenged mice; however it was significantly higher in group II untreated mice as compared to group III and group IV mice. The neutrophil was higher and lymphocyte count was lower in group II mice at 120 hrs post challenge (PC). The CRP was positive in all the challenged group at 24 hrs PC, but it remained positive till 120 hrs PC in group II. The parameters are related to enhancement of the mammary defense and reduction of inflammation. Hence AX may be used alone or as an adjunct therapy with antibiotic for amelioration of mastitis. Development of such therapy may be useful to reduce the antibiotic burden in management of intramammary infection.

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.

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.

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 (H(2)O(2)), superoxide anion (O(2) (-)) production and interleukin- 2 (IL-2) and gamma interferon (IFN-gamma) 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, H(2)O(2) and O(2) (-) production by milk cells. The IL-2 and IFN-gamma 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.

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.

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.