Comparative Evaluation of Masson's Trichrome and Picrosirius Red Staining for Digital Collagen Quantification Using ImageJ in Rabbit Wound Healing Research

The therapeutic potential of Pluronic F127 (PF127) hydrogel loaded with adipose-derived stromal vascular fraction (AdSVF), mesenchymal stem cells (AdMSC), and conditioned media (AdMSC-CM) for repairing full-thickness skin wounds was evaluated using a rabbit model. The rabbits were randomly divided into eight groups with six animals each and treatment was given as per the predetermined protocol (3 doses at one-week interval): Group A (Control), Group B (AdSVF), Group C (AdMSC), Group D (AdMSC-CM), Group E (PF127), Group F (AdSVF + PF127), Group G (AdMSC + PF127), and Group H (AdMSC-CM + PF127). Skin tissue samples were collected from the healing wounds on day 28 for staining and collagen quantification. Collagen density (Area %) was quantified using tissue sections stained with Masson's Trichrome (MT) and Picrosirius Red (PSR) stain using the Colour Deconvolution plugin of ImageJ and RGB stack method, respectively. These techniques function based on separating different colour channels in the stained tissue sections to isolate the collagen fibers and then quantifying them through thresholding and image analysis. Across the treatment groups, both staining methods generally showed a trend of increased collagen density compared to the control group. For most groups, PSR staining consistently indicated slightly lower collagen densities than MT staining. However, the overall trends were similar in both staining. The comparison between PSR and MT staining methods revealed that both techniques effectively assess collagen density in healing wounds. However, there were subtle differences in the absolute values obtained, with PSR staining tending to yield slightly lower collagen density measurements than MT. These differences can be attributed to the distinct mechanisms of these staining methods. Therefore, both staining methods can digitally quantify collagen density in wound healing research

Otpornost bakterija Escherichia coli i Pseudomonas aeruginosa na karbapenem u antilope (Antilope cervicapra) i leoparda (Panthera pardus) iz zatočeništva u Indiji

The study aimed to investigate the occurrence of carbapenem resistant E. coli and P. aeruginosa in apparently healthy, captive blackbucks and leopards of India. Faecal samples of blackbucks (n = 7) and leopards (n = 7) were processed to isolate carbapenem resistant E. coli (CRE) and P. aeruginosa (CRP). Forty (leopards n = 26; blackbuck n = 14) E. coli and two P. aeruginosa (blackbuck n = 2) samples were isolated from the faecal samples (n = 14). Eleven carbapenem resistant isolates were recovered, of which 10 were CRE and one was CRP. The minimum inhibitory concentration (MIC) was determined for meropenem for carbapenem resistant isolates and was between 8 and 64 μg/mL. All the CRE and CRP were phenotypically multidrug resistant, and six CRE were extended-spectrum beta- lactamases (ESBL) producers. On genotypic screening, seven CRE and one CRP were positive for the blaNDM carbapenemase gene. Efflux pump-mediated carbapenem resistance was noticed in four CRE isolates (36.4%, 4/11). Of the six ESBL producing CRE, four isolates carried blaCTX-M-1 genes. The CRE isolates also harbored blaTEM-1, blaAmpC, qnrA, qnrB, qnrS, tetA, tetB and sul1 resistance genes. On Shiga toxin virulence screening, Stx1, Stx2 genes were detected in two and one isolates, respectively. Plasmid typing of CRE revealed that the blaNDM genes were carried on an Incl1 plasmid. The plasmid multilocus sequence typing (pMLST) of the isolates showed the Sequence Type (ST) 297. The occurrence of carbapenem resistance bacteria in captive wildlife should be a major public health priority.Cilj rada bio je istražiti slučajeve otpornosti bakterija E. coli i P. aeruginosa na karbapenem u zdravih antilopa i leoparda iz zatočeništva u Indiji. Uzorci izmeta antilopa (n = 7) i leoparda (n = 7) obrađeni su kako bi se izolirale bakterije E. coli (CRE) i P. aeruginosa (CRP) otporne na karbapenem. Iz uzoraka izmeta (n = 14) dobiveno je 40 izolata (leopard n = 26, antilopa n = 14) E. coli i 2 izolata P. aeruginosa (antilopa n = 2). Pronađeno je 11 izolata otpornih na karbapenem, od kojih je 10 E. coli i 1 P. aeruginosa. Određena je minimalna inhibicijska koncentracija (MIK) za meropenem za izolate otporne na karbapenem, od 8 za E. coli i 64 μg/mL za P. aeruginosa. Svi izolati E. coli i P. aeruginosa fenotipski su bili otporni na širok spektar lijekova, a 6 izolata E. coli proizvodilo je beta- laktamaze širokog spektra (ESBL). Genotipskim probirom 7 izolata E. coli i 1 izolat P. aeruginosa bili su pozitivni na karbapenemaza gen blaNDM. Otpornost na karbapenem putem efluks pumpe zabilježena je u 4 izolata E. coli (36,4 %, 4/11). Od 6 ESBL producirajućih CRE, 4 izolata nosila su gen blaCTX-M-1. Izolati E. coli također su sadržavali blaTEM-1, blaAmpC, qnrA, qnrB, qnrS, tetA, tetB i sul1 gene otpornosti. Pretragom na šiga-toksin, Stx1 i Stx2 geni utvrđeni su u dva odnosno jednom izolatu. Tipiziranje plazmida CRE otkrilo je prisutnost blaNDM gena na Incl1 plazmidu. Multilokusno tipiziranje sekvencija plazmida (pMLST) izolata otkrilo je sekvenciju tipa (ST) 297. Pojava otpornosti bakterija na karbapenem u divljih životinja iz zatočeništva trebala bi biti javnozdravstveni prioritet

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Not AvailableA study on the occurrence of Burkholderia cepacia complex in ultrasound gels used in different veterinary clinical settings in IndiaNot Availabl

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Not AvailableBackground: Semen cryopreservation results in deleterious effects on spermatozoa, including lipid peroxidation and a reduction in the total antioxidant components of seminal plasma. The ultimate outcome of these changes is a reduction in post-thaw semen quality. A mitochondrial derived peptide, humanin, a potent cytoprotective and antioxidant agent was used in the present study. Objective: To evaluate the efficacy of a mitochondrial-derived peptide, humanin to improve the post-thaw quality of buffalo spermatozoa. Materials and methods: A total of 18 ejaculates from three Murrah buffalo bulls (n=6 each) were collected. Each ejaculate was divided into four aliquots. The first aliquot was diluted with standard EYTG dilutor (Group I, control), whereas the other three aliquots were diluted with EYTG supplemented with 2 µM (Group II), 5 µM (Group III) and 10 µM humanin (Group IV), respectively. Semen was evaluated for physico-morphological and functional attributes such as progressive motility, viability, abnormality, acrosome integrity, plasmamembrane integrity of fresh samples, pre-freeze and post-thaw stages. Oxidative stress parameters [lipid peroxidation (LPO) and total antioxidant capacity (TAC)] were also measured at the pre-freeze and post-thaw stages. Results: Humanin supplementation resulted in significantly higher (p < 0.05) post-thaw motility in all treatment groups and, higher (p < 0.05) viability in Groups III and IV in comparison to the control at the post-thaw stage. Spermatozoa with intact acrosome and plasma membrane were higher (p < 0.05) in Groups III and IV as compared to Groups I and II. The LPO levels at the post-thaw stage were found to be lower (p < 0.05) in all treatment groups versus the control group, whereas, higher (p≤0.05) TAC values were recorded in Groups III and IV in comparison to the control and Group II. Conclusion: Humanin supplementation in the extender improved the freezabilty of buffalo spermatozoa.Not Availabl

Experimental pathology of two highly pathogenic H5N1 viruses isolated from crows in BALB/c mice

In this study, we assessed the pathogenicity of two H5N1 viruses isolated from crows in mice. Eighteen 6–8 weeks BALB/c mice each were intranasally inoculated with 106 EID50/ml of H5N1 viruses A/crow/India/03CA04/2015 (H9N2-PB2 reassortant H5N1) and A/crow/India/02CA01/2012 (Non-reassortant H5N1). The infected mice showed dullness, weight loss and ruffled fur coat. Histopathological examination of lungs showed severe congestion, haemorrhage, thrombus, fibrinous exudate in perivascular area, interstitial septal thickening, bronchiolitis and alveolitis leading to severe pneumonic changes and these lesions were less pronounced in reassortant virus infected mice. Viral replication was demonstrated in nasal mucosa, lungs, trachea and brain in both the groups. Brain, lung, nasal mucosa and trachea showed significantly higher viral RNA copies and presence of antigen in immunohistochemistry in both the groups. This study concludes that both the crow viruses caused morbidity and mortality in mice and the viruses were phenotypically highly virulent in mice. The H5N1 viruses isolated from synanthropes pose a serious public health concern and should be monitored continuously for their human spill-over. •H5N1 highly pathogenic avian influenza viruses isolated from crows in India caused significant pathology in BALB/c mice.•Non-reassortant H5N1 virus caused more severe lesions compared to H9N2-PB2 reassortant H5N1 virus in mice.•The H5N1 viruses in crows pose a significant public health concern as crows harbour near human dwellings

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Not AvailableBuffalo spermatozoa are vulnerable to cryo-injuries due to inherent deficiency of endogenous antioxidants, high polyunsaturated fatty acids (PUFA) content in plasma membrane and low cholesterol/phospholipid (C/P) ratio. Humanin is a potent cytoprotective agent that protects the cells against oxidative stress and apoptosis. The present study was designed to establish the presence of Humanin in buffalo and effect of Humanin supplementation on freezability of buffalo spermatozoa. Indirect immunofluorescence test revealed presence of Humanin in ejaculated and epididymal spermatozoa, and, elongated spermatids and interstitial space in the testicular tissue section. Humanin levels in seminal plasma were significantly and positively correlated with sperm concentration and individual progressive motility (IPM) in good (n = 22; IPM >70%) and poor (n = 10; IPM <50%) quality ejaculates. For supplementation studies, a total of 24 ejaculates (IPM ≥70%) were collected and each ejaculate was then divided into four aliquots. First aliquot was diluted with egg yolk-tris-glycerol (EYTG) extender without Humanin and served as control group (Group I). Rest three aliquots were diluted with extender containing 2 (Group II), 5 (Group III) and 10 μM Humanin (Group IV), respectively. Semen was cryopreserved using standard protocol and evaluated at pre-freeze for lipid peroxidation (LPO) and post-thaw stages for spermatozoa kinematics, LPO, mitochondrial membrane potential (MMP), capacitation, apoptotic status and DNA integrity. The treatment group that showed best results (5 μM) was compared with control group for in vitro fertility assessment by homologous zona binding assay. The LPO levels were lower (p < 0.05) in 5 and 10 μM Humanin supplemented group. The MMP and DNA integrity were higher (p < 0.05) in 5 μM group than other groups. F-pattern was higher (p < 0.05) and B-pattern was lower (p < 0.05) in 5 and 10 μM Humanin supplemented groups. Lower apoptotic and higher viable spermatozoa (p < 0.05) were observed in 5 μM Humanin group. The mean number of spermatozoa bound to zona pellucida was higher (p < 0.05) in 5 μM Humanin treated group than the control group. The study established the presence of Humanin in buffalo spermatozoa and seminal plasma for very first time and concluded that Humanin supplementation at 5 μM concentration improves the freezability and in vitro fertility of buffalo spermatozoa.Not Availabl

Metabolism of aceclofenac to diclofenac in the domestic water buffalo Bubalus bubalis confirms it as a threat to Critically Endangered Gyps vultures in South Asia

Vulture declines in South Asia were caused by accidental poisoning by the veterinary non-steroidal anti-inflammatory drug (NSAID) diclofenac. Although veterinary use of diclofenac has been banned, other vulture-toxic NSAIDs are legally available, including aceclofenac, which has been shown to metabolise into diclofenac in domestic cattle. We gave nine domestic water buffalo the recommended dose of aceclofenac (2 mg kg−1 body weight), collected blood at intervals up to 48 h, and carried out a pharmacokinetic analysis of aceclofenac and its metabolite diclofenac in plasma. Aceclofenac was rapidly converted to diclofenac, and was barely detectable in plasma at any sampling time. Diclofenac was present within 20 min, and peaked 4–8 h after dosing. Aceclofenac is a prodrug of diclofenac, and behaves similarly in domestic water buffalo as it did in domestic cattle, posing the same risk to vultures. We recommend an immediate ban on the veterinary use of aceclofenac across vulture-range countries.The Haryana Forest Development Corporation.https://www.elsevier.com/locate/etap2023-09-30hj2023Paraclinical Science

Metabolism of aceclofenac to diclofenac in the domestic water buffalo Bubalus bubalis confirms it as a threat to Critically Endangered Gyps vultures in South Asia.

Vulture declines in South Asia were caused by accidental poisoning by the veterinary non-steroidal anti-inflammatory drug (NSAID) diclofenac. Although veterinary use of diclofenac has been banned, other vulture-toxic NSAIDs are legally available, including aceclofenac, which has been shown to metabolise into diclofenac in domestic cattle. We gave nine domestic water buffalo the recommended dose of aceclofenac (2 mg kg-1 body weight), collected blood at intervals up to 48 h, and carried out a pharmacokinetic analysis of aceclofenac and its metabolite diclofenac in plasma. Aceclofenac was rapidly converted to diclofenac, and was barely detectable in plasma at any sampling time. Diclofenac was present within 20 min, and peaked 4-8 h after dosing. Aceclofenac is a prodrug of diclofenac, and behaves similarly in domestic water buffalo as it did in domestic cattle, posing the same risk to vultures. We recommend an immediate ban on the veterinary use of aceclofenac across vulture-range countries.Funding was provided by the Haryana Forest Development Corporation

Experimental safety testing shows that the NSAID tolfenamic acid is not toxic to Gyps vultures in India at concentrations likely to be encountered in cattle carcasses.

Population declines of Gyps vultures across the Indian subcontinent were caused by unintentional poisoning by the non-steroidal anti-inflammatory drug (NSAID) diclofenac. Subsequently, a number of other NSAIDs have been identified as toxic to vultures, while one, meloxicam, is safe at concentrations likely to be encountered by vultures in the wild. Other vulture-safe drugs need to be identified to reduce the use of those toxic to vultures. We report on safety-testing experiments on the NSAID tolfenamic acid on captive vultures of three Gyps species, all of which are susceptible to diclofenac poisoning. Firstly, we estimated the maximum level of exposure (MLE) of wild vultures and gave this dose to 40 Near Threatened Himalayan Griffons G. himalayensis by oral gavage, with 15 control birds dosed with benzyl alcohol (the carrier solution for tolfenamic acid). Two birds given tolfenamic acid died with elevated uric acid levels and severe visceral gout, while the remainder showed no adverse clinical or biochemical signs. Secondly, four G. himalayensis were fed tissues from water buffaloes which had been treated with double the recommended veterinary dose of tolfenamic acid prior to death and compared to two birds fed uncontaminated tissue; none suffered any clinical effects. Finally, two captive Critically Endangered vultures, one G. bengalensis and one G. indicus, were given the MLE dose by gavage and compared to two control birds; again, none suffered any clinical effects. The death of two G. himalayensis may have been an anomaly due to i) the high dose level used and ii) the high ambient temperatures at the time of the experiment. Tolfenamic acid is likely to be safe to Gyps vultures at concentrations encountered by wild birds and could therefore be promoted as a safe alternative to toxic NSAIDs. It is manufactured in the region, and is increasingly being used to treat livestock