Comparative analysis of tuberculin and defined antigen skin tests for detection of bovine tuberculosis in buffaloes (Bubalus bubalis) in Haryana state, India.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic disease that results from infection with any member of the Mycobacterium tuberculosis complex. Infected animals are typically diagnosed with tuberculin-based intradermal skin tests according to World Organization of Animal Health which are presently in use. However, tuberculin is not suitable for use in BCG-vaccinated animals due to a high rate of false-positive reactions. Peptide-based defined skin test (DST) antigens have been identified using antigens (ESAT-6, CFP-10 and Rv3615c) which are absent from BCG, but their performance in buffaloes remains unknown. To assess the comparative performance of DST with the tuberculin-based single intradermal test (SIT) and the single intradermal comparative cervical test (SICCT), we screened 543 female buffaloes from 49 organized dairy farms in two districts of Haryana state in India. RESULTS: We found that 37 (7%), 4 (1%) and 18 (3%) buffaloes were reactors with the SIT, SICCT and DST tests, respectively. Of the 37 SIT reactors, four were positive with SICCT and 12 were positive with the DST. The results show that none of the animals tested positive with all three tests, and 6 DST positive animals were SIT negative. Together, a total of 43 animals were reactors with SIT, DST, or both, and the two assays showed moderate agreement (Cohen's Kappa 0.41; 95% Confidence Interval (CI): 0.23, 0.59). In contrast, only slight agreement (Cohen's Kappa 0.18; 95% CI: 0.02, 0.34) was observed between SIT and SICCT. Using a Bayesian latent class model, we estimated test specificities of 96.5% (95% CI, 92-99%), 99.7% (95% CI: 98-100%) and 99.0% (95% CI: 97-100%) for SIT, SICCT and DST, respectively, but considerably lower sensitivities of 58% (95% CI: 35-87%), 9% (95% CI: 3-21%), and 34% (95% CI: 18-55%) albeit with broad and overlapping credible intervals. CONCLUSION: Taken together, our investigation suggests that DST has a test specificity comparable with SICCT, and sensitivity intermediate between SIT and SICCT for the identification of buffaloes suspected of tuberculosis. Our study highlights an urgent need for future well-powered trials with detailed necropsy, with immunological and microbiological profiling of reactor and non-reactor animals to better define the underlying factors for the large observed discrepancies in assay performance, particularly between SIT and SICCT.

Lactobacillus johnsonii CPN23 vis-à-vis Lactobacillus acidophilus NCDC15 Improves Gut Health, Intestinal Morphometry, and Histology in Weaned Wistar Rats.

The present investigation was carried out with the aim to establish the comparative efficacy of a canine-sourced probiotic meant for canine feeding and a conventional dairy-sourced probiotic. For this purpose, canine-origin Lactobacillus johnsonii CPN23 and dairy-origin Lactobacillus acidophilus NCDC15 were evaluated for potential probiotics health benefits in the rat model. Forty-eight weaned Wistar rats enrolled in this experiment of 8 weeks were fed a basal diet and divided into three dietary treatments. Rats of group I enrolled as control (CON) were given MRS placebo at 1 mL/head/day, while rats of group II (LAJ) and III (LAC) were administered with overnight MRS broth grown-culture of L. johnsonii CPN23 and L. acidophilus NCDC15, respectively, at 1 mL/head/day (108 cfu/mL). The average daily gain and net gain in body weight were significantly higher (p < 0.05) in LAJ and LAC than in CON. Fecal and digesta biochemical attributes altered (p < 0.05) positively in response to both probiotics. Total fecal and pooled digesta SCFAs were higher (p < 0.05) in both LAJ and LAC than in CON. The microbial population in cecal and colonic digesta responded (p < 0.05) positively to both probiotics. The diameter of intestinal segments was higher (p < 005) in LAJ as compared to CON. The number and height of villi in jejunum tended to be higher in LAJ as compared to CON. The humoral immune response to sheep erythrocytes as well as chicken egg-white lysozyme was higher in LAJ as compared to CON. Overall, the results of the study have demonstrated the effectiveness of the canine-sourced L. johnsonii CPN23 as a potential probiotic, with a comparatively better response than the dairy-sourced L. acidophilus NCDC15. It could thus be recommended for use in feeding dogs to help augment their health.

Studies on immunopathological changes induced by commercial IBD live vaccines in poultry birds.

Intermediate plus live strain infectious bursal disease virus (IBDV) vaccines are used to control IBDV endemic infections in India. In the present study, immunopathological changes induced by commercial infectious bursal disease live vaccines with different immunization regimes were compared. A total of days old 108 Cobb broiler chicks were randomly divided into five groups with 24 chicks each in groups I, II, III and 18 chicks each in group IV and V. Group I served as control I (no immunization) and group II and III chicks were immunized with a single dose of vaccines 1 and 2 on 17th day of age (DOA), respectively. The group IV and V chicks were immunized with vaccines 1 and 2, respectively with primary dose on 17th DOA followed by booster dose on 24th DOA. Both intermediate plus live vaccines produced gross and histopathological lesions in lymphoid organs (bursa of Fabricius, thymus, spleen and caecal tonsils). Increased CD4 + , CD8 + T cells in affected bursa of Fabricius was evidenced by immunohistochemistry. Further, up-regulation in relative mRNA expression of IFN-γ, IL-1ß and IL-6 were observed in bursa of Fabricius of treated birds, with maximum alteration particularly on 14th day post single immunization and 7th day post booster immunization. The findings suggest that single immunization regime on the 17th day of age showed immunization equivalent to booster immunization with lesser lesions, therefore, may be practiced and promoted in the field conditions for the better economic returns and animal welfare.

Epidemiology, risk factors and molecular characterization of small ruminant morbillivirus in Haryana, India.

Peste des petitis ruminants is an economically important transboundary and notifiable viral disease of sheep and goats. In this study, 14 PPR suspected outbreaks among sheep and goats were investigated in four districts of Haryana, India, during July 2020 to October, 2021. The causative agent of the disease; small ruminant morbillivirus was detected by Reverse Transcription Polymerase Chain Reaction targeting full gene sequences of fusion protein gene and confirmed by sequencing. The overall morbidity and cumulative mortality in these outbreaks were 37.56% and 12.09%, respectively. Risk factor analysis identified significant difference in mortality based on age with higher mortality in young ones; 21% as compared to adults; 7.55%. Analysis of the vaccination status revealed significant difference in morbidity and mortality with higher morbidity and mortality in un-vaccinated animals as compared to vaccinated ones. The sequencing and phylogenetic analysis of representative samples revealed that the strains of the present study fall in lineage IV (96.6-99.1%) along with other Indian isolates but made a separate cluster (sub-lineage). The comparison of deduced amino acid (aa) sequence analysis of fusion protein of circulating field strains with reference vaccine strain and other lineage IV strains revealed four N-linked glycosylation sites instead of three. The findings of the present study revealed changes in fusion protein of some of the circulating field strains of SRMV in Haryana, India. Further detailed studies are warranted to delineate the molecular details of these circulating field strains and to evaluate the effectiveness of currently used vaccine against these mutated strains.

Acceleration of wound healing by quercetin in diabetic rats requires mitigation of oxidative stress and stimulation of the proliferative phase.

Increased oxidative stress in diabetic wound areas impairs wound healing. Quercetin exhibits significant antioxidant properties. We investigated the effects of topical quercetin on antioxidant status in diabetic wound areas and its effect on wound healing in rats. A 2 cm2 cutaneous wound was produced on the back of streptozotocin induced diabetic and normal rats. Rats were divided into three groups of 20: normal healthy control group, diabetic group and quercetin treated diabetic group. The control and diabetic groups were treated topically with ointment base once daily for 21 days. The quercetin treated diabetic rats were treated similarly with ointment containing quercetin. The quercetin treated diabetic group exhibited increased levels of catalase, glutathione peroxidase, superoxide dismutase and total thiols compared to the diabetic group. Nitrite levels in the diabetic group were decreased significantly on day 3 compared to the healthy control group. Malondialdehyde levels were decreased in the quercetin treated diabetic group compared to the diabetic group. The expression of proliferating cell nuclear antigen) (PCNA) was greater in the quercetin treated diabetic group on day 7 compared to healthy control and diabetic groups. Formation of granulation tissue and the quality of healed tissue was improved in the quercetin treated diabetic group compared to the diabetic group. Quercetin improves antioxidant status in wounds of diabetic rats and stimulates the proliferation phase, which accelerates wound healing.

Temporal Effects of Different Vehicles on Wound Healing Potentials of Quercetin: Biochemical, Molecular, and Histopathological Approaches.

Development of novel drugs or formulations to accelerate the wound healing process is the need of current era. Quercetin (Q), a bioflavonoid, at 0.3% concentration has showed some wound healing potential in our preliminary studies. The present study was aimed to explore the wound healing potential of 0.3% quercetin formulated in 3 different vehicles, that is, dimethyl sulfoxide (DMSO; 10%), ointment base, and corn oil. Ninety experimentally wounded rats were grouped in 6 groups. The 0.3% quercetin mixed with DMSO, ointment base, and corn oil was topically applied once daily for 21 days on the wounds of groups 2, 4, and 6, respectively. DMSO, ointment base, and corn oil alone was applied similarly in groups 1, 3, and 5, respectively. Gross evaluation and wound contraction results revealed accelerated wound closure in all quercetin-treated groups. The mRNA expressions of vascular endothelial growth factor, transforming growth factor-ß1, and interluekin-10 were markedly upregulated in healing tissues of quercetin-treated groups. Tumor necrosis factor-α mRNA expression and protein levels were lowered by quercetin treatment. Quercetin-treated groups also showed increased activities of SOD (superoxide dismutase) and catalase, and levels of total thiols in wound tissues on day 7. Levels of superoxide anion radicals and malondialdehyde were markedly lower in quercetin-treated groups. Histologically, wound sections of quercetin-treated groups showed early dominance of fibroblasts, increased blood vessels, marked collagen deposition, and regenerated epithelial layer. The significant effects were more pronounced in ointment + Q group among all the quercetin-treated groups. In conclusion, 0.3% quercetin mixed in ointment base produced the fastest and better wound healing in rats.

A Defined Antigen Skin Test for Diagnosis of Bovine Tuberculosis in Domestic Water Buffaloes (Bubalus bubalis).

Bovine tuberculosis (bTB) remains endemic in domestic water buffaloes (Bubalus bubalis) in India and elsewhere, with limited options for control other than testing and slaughter. The prescribed tuberculin skin tests with purified protein derivative (PPD) for diagnosis of bTB preclude the use of Bacille Calmette-Guérin (BCG)-based vaccination because of the antigenic cross-reactivity of vaccine strains with Mycobacterium bovis and related pathogenic members of the M. tuberculosis complex (MTBC). For the diagnosis of bTB in domestic water buffaloes, we here assessed a recently described defined-antigen skin test (DST) that comprises overlapping peptides representing the ESAT-6, CFP-10 and Rv3615c antigens, present in disease-causing members of the MTBC but missing in BCG strains. The performance characteristics of three doses (5, 10 or 20 µg/peptide) of the DST were assessed in natural tuberculin skin test reactor (n = 11) and non-reactor (n = 35) water buffaloes at an organized dairy farm in Hisar, India, and results were compared with the single intradermal skin test (SIT) using standard bovine tuberculin (PPD-B). The results showed a dose-dependent response of DST in natural reactor water buffaloes, although the SIT induced a significantly greater (P < 0.001) skin test response than the highest dose of DST used. However, using a cut-off of 2 mm or greater, the 5, 10, and 20 µg DST cocktail correctly classified eight, 10 and all 11 of the SIT-positive reactors, respectively, suggesting that the 20 µg DST cocktail has a diagnostic sensitivity (Se) of 1.0 (95% CI: 0.72-1.0) identical to that of the SIT. Importantly, none of the tested DST doses induced any measurable skin induration responses in the 35 SIT-negative animals, suggesting a specificity point estimate of 1.0 (95% CI: 0.9-1.0), also identical to that of the SIT and compares favorably with that of the comparative cervical test (Se = 0.85; 95% CI: 0.55-0.98). Overall, the results suggest that similar to tuberculin, the DST enables sensitive and specific diagnosis of bTB in water buffaloes. Future field trials to explore the utility of DST as a defined antigen replacement for tuberculin in routine surveillance programs and to enable BCG vaccination of water buffaloes are warranted.

Topical application of quercetin improves wound repair and regeneration in diabetic rats.

Purpose: There is an urgent need of effective drug/formulation to speed up the healing process in diabetic wounds. In our earlier studies, quercetin has accelerated the healing of nondiabetic wounds. So, we investigated the wound-healing potentials of quercetin in diabetic rats.Materials and methods: A square-shaped cutaneous wound (≈400 mm2) was created on the back of nondiabetic and diabetic rats. They were divided into three groups, viz. healthy control (nondiabetic), diabetic control and diabetic-treated group. Ointment base was topically applied for 21 days in healthy and diabetic control groups. Quercetin (0.3%) ointment was similarly applied in third group. Effects of quercetin on repair and regenerations of diabetic wounds in terms of wound closure, inflammation, angiogenesis, fibroblast proliferation, collagen synthesis, epithelialization, axonal regeneration etc was studied.Results: Quercetin accelerated the wound closure and increased the expressions of IL-10, VEGF and TGF-ß1 in granulation/healing tissue of diabetic wound. However, quercetin decreased the expression of TNF-α, IL-1ß, and MMP-9. Histopathological evaluation revealed amelioration of persistence of inflammatory cells by quercetin in diabetic wounds. There was good quality of granulation tissue, marked fibroblast proliferation, well organized collagen deposition, early regeneration of epithelial layer etc. in the quercetin treated diabetic wounds in comparison to diabetic control group. Results of immunohistochemistry showed more angiogenesis, faster phenotypic switching of fibroblast to myofibroblasts and increased GAP-43 positive nerve fibers in quercetin-treated diabetic wounds.Conclusion: Quercetin ointment at 0.3% w/w concentration modulates cytokines, growth factors and protease, thereby improved repair and regenerations of cutaneous diabetic wounds in rats.

Quercetin accelerated cutaneous wound healing in rats by modulation of different cytokines and growth factors.

Quercetin on wounds could be favorable for healing based on its variety of biological effects. Eighty wounded rats were divided into four groups i.e. dimethyl sulfoxide, 0.03% quercetin, 0.1% quercetin, and 0.3% quercetin-treated. Different treatments were topically applied for 20 days. Quercetin (0.3%) caused the fastest wound closure and markedly improved the oxidative stress. Quercetin treatment increased the expressions of IL-10, VEGF, TGF-ß1, CD31, α-SMA, PCNA, and GAP-43, and decreased the expressions of TNF-α. Early infiltration of inflammatory cells and formation of good quality granulation tissue dominated by fibroblast proliferation, angiogenesis, and collagen deposition in quercetin treated groups was also evident. All these effects were more pronounced at 0.3% quercetin concentration. The earliest regeneration of epithelial layer was also observed in 0.3% quercetin-treated wounds. In conclusion, 0.3% quercetin accelerates wound healing efficiently by modulating antioxidant system of wound, cytokines, growth factors, other proteins and cells involved in healing.

Quercetin loaded chitosan tripolyphosphate nanoparticles accelerated cutaneous wound healing in Wistar rats.

Previous studies have shown that quercetin on topical application improved cutaneous wound healing in rats, but hydrophobic nature and less skin penetration limits its potential as topical healing agent. Therefore, present study was planned to investigate wound healing potential of chitosan based quercetin nanoparticles. Quercetin loaded nanoparticles were synthesized by ionic gelation method and characterized by various standard techniques. A 2 × 2 cm2 square shaped wound was created on the thoraco-lumbar part of rats. Wounded rats were divided into 6 groups namely, gel (20%), blank nanoparticle (0.16%), bulk quercetin (0.3%), quercetin nanoparticles (0.03%), quercetin nanoparticles (0.1%) and quercetin nanoparticles (0.3%) treated groups. Different formulations of quercetin nanoparticles were applied on the wounds for the duration of study and healing tissues were collected on 7th, 14th and 21st day to study various parameters. Quercetin loaded nanoparticles were 361.16 ± 9.72 nm size and spherical in shape. We observed quercetin nanoparticles (0.03%) treatment caused marked reduction in the tumor necrosis factor-alpha, whereas expressions of interleukin 10, vascular endothelial growth factor and transforming growth factor beta1 was increased significantly with treatment. The granulation tissue of quercetin nanoparticles (0.03%) treated group showed better quality healing and maturity as supported by the increased blood vessels density, decreased inflammatory cells, increased number of myofibroblasts, deposition and arrangement of collagen fibers and re-epithelialization. In conclusion, quercetin nanoparticles (0.03%) treatment significantly improved wound healing by modulation of cytokines and growth factors involved in inflammatory and proliferative phases of wound healing.

Clinico-haematological alterations and therapeutic management of tick borne fever in cross bred cow.

A 5 years old crossbred cow was brought to the Veterinary Clinical Complex of Lala Lajpat Rai University of Veterinary and Animal Sciences Hisar with history of progressive weakness, pale mucous membrane, anorexia, high fever (105 °F), tachycardia, laboured breathing and coffee coloured urine. Analysis of haematological parameters revealed severe anaemia, leucocytopenia, marked poikilocytosis of erythrocytes. Blood smear examination showed presence of signet ring shaped Theileria organisms, Babesia piroplasms and condensed dot forms of Anaplasma marginale in the stained erythrocytes. Further animal was treated with buparvaquone @ 2.5 mg/kg b.wt deep I/M in neck region and long acting oxytetracycline at 25 mg/kg b. W. slow I/V daily in normal saline solution for 5 days. Berenil (Diminazene aceturate 5%) injection was also administered @ 1 ml/20 kg b.wt. I/M along with supportive therapy. Clinical signs started to subside 3 days post treatment. Complete recovery was achieved by 4 weeks post treatment however animal succumbed to death due to immunosuppression.

Neurobehavioral alterations and histopathological changes in brain and spinal cord of rats intoxicated with acrylamide.

The aim of this project was to study the clinical manifestations, neurobehavioral, hematobiochemical, oxidative stress, genotoxicity, and histopathological changes during acrylamide toxicity in rats. A total of 30 adult male Wistar rats were divided in 5 equal groups and received 0, 10, 15, and 20 mg/kg body weight acrylamide as oral gavage, while group 5 was micronucleus (MN) control. Functional observational battery (FOB) parameters were studied at the 28th day of post treatment. Toxicological manifestations were evident in acrylamide-treated rats from 14th day onward. FOB revealed a significant change in central nervous system, neuromuscular, and autonomic domains. The hematological changes include significant decrease in concentration of hemoglobin, total erythrocyte count, packed cell volume, and mean corpuscular volume. The biochemical parameters aspartate aminotransferases, alkaline phosphatase, and albumin showed significant increase, while the levels of serum globulin and glucose were found to decrease significantly. The MN assay revealed the significant increase in frequencies of micronuclei and number of polychromatic erythrocytes. The oxidative stress parameters revealed no significant difference as compared to control rats. Histopathological changes observed in brain include neuronal degeneration, edema, and congestion, while spinal cord revealed demyelination in low-dose group and bilateral necrosis with malacia, liquefaction of white matter, and loss of myelin from gray matter in high-dose groups. The result indicates pathological alterations in brain and spinal cord and is responsible for neurobehavioral changes in rats. The FOB changes and histopathological alterations in spinal cord are in dose dependent to acrylamide intoxication. Various toxicological effects observed in experiment direct us to focus on a deep study and evaluate the possible causes pertaining to toxicity of this chemical. It would furnish the scientists with better options that would help them to search for a median path regarding the use of this chemical and take preventive measures to save the living beings from the hidden disasters of this chemical.

Evaluation of protective effect of vitamin e on acrylamide induced testicular toxicity in wister rats.

Male wistar rats (weighting 160-180 g) were divided in six groups of 6 animals per group. Group A and F served as control. Groups B, C, D and E received acrylamide at 20 mg/kg body weight for 28 days and groups C and E received additionally vitamin E (50 IU/kg body weight) for 1 to 28 days and 29 - 42(nd) days of experiment, respectively. The animals from groups A, B, and C were sacrificed on day 28(th) of experiment and from groups D, E, and F on 42(nd) day of experiment, respectively. There was significant decrease in the total sperm count and significant increase in the dead sperm count on day 28(th) of study due to acrylamide toxicity. At recovery period, there was significant increase in the total sperm count of vitamin-E-treated group of animals as compared to untreated toxicated rats. But, values were significantly lower than control animals. Microscopically, the lesions in the testes of acrylamide intoxicated rats at 28(th) day revealed destruction of seminiferous tubules at periphery. No spermatid and spermatocytes were seen in the seminiferous tubules. Detachment of spermatogonial cells started at periphery of seminiferous tubules. Atrophy of seminiferous tubules was a constant finding. Some tubules showed vacuolar degenerative changes in germinal epithelium. During the recovery period, destruction of seminiferous tubules, detachment of spermatogonial cells, and atrophy of seminiferous tubules were observed in group D and E. Few sections revealed only spermatogonial cells. At recovery period vitamin-E-treated rats revealed somewhat better architecture of the seminiferous tubules. Late spermatids were seen in few seminiferous tubules and other revealed starting of spermatogenesis. Thus, it appears that Vitamin E is not able to protect testes from acrylamide toxicity during active feeding, but after cessation of acrylamide feeding treatment with vitamin E revealed faster recovery as compare to not treated group.

Ameliorative effect of ocimum sanctum on meloxicam induced toxicity in wistar rats.

An ameliorating effect of Ocimum sanctum on the toxic effect of meloxicam, a new non-steroidal anti-inflammatory drug was studied by evaluating haemato-biochemical parameters, oxidative stress, gross and histopathological changes in various organs of Wistar rats. A total of thirty-six male rats were divided in six experimental groups each comprising of six rats and numbered from G(1) to G(6). Meloxicam toxicity was induced by oral feeding of meloxicam at 1.2 mg/kg and 2.4 mg/kg body weight in G(2) and G(3) respectively for 28 days. Group G(4) and G(5) were fed with 1.2-mg/kg body weight and 2.4-mg/kg body weight of meloxicam along with 200 mg/kg body weight of aqueous extract of Ocimum sanctum. Group G(1) serve as control while group G(6) was kept as treatment control and fed only aqueous extract of Ocimum sanctum at 200 mg/kg body weight. Clinical finding showed mild diarrhea from 23(rd) day onwards in-group treated with 2.4-mg/kg body of meloxicam. Significant reduction of hemoglobin and packed cell volume (PCV) was observed in both the group treated with 1.2 mg/kg and 2.4-mg/kg body wt. of meloxicam. Ocimum sanctum could restore the hemoglobin and PCV value in-group treated with meloxicam at low dose level. Serum alkaline phosphatase, serum glutamic pyruvic transaminase, Serum glutamic oxaloacetic transaminase and total bilirubin were found elevated in meloxicam treated groups and indicated hepatotoxic activity of meloxicam. Ocimum sanctum could reduce hepatotoxic activity of meloxicam in group G4 receiving meloxicam at lower dose rate along with Ocimum sanctum failed to regulate creatinine level in meloxicam treated groups. In meloxicam toxicity elevated Lipid peroxidation values was noticed in liver and kidneys, while superoxide dismutase and glutathione did not revealed any change. Stomach and intestine revealed hemorrhagic gastroenteritis and ulcers. Perivascular necrosis with infiltration with inflammatory cells was evident in liver. Interstitial nephritis, myocardial necrosis and spongiform encephalopathy were important lesions. The Ocimum sanctum could only counteract the toxic effect of meloxicam in liver and gastrointestinal tract.