Stray Dogs (Mongrels) Are Potent Reservoir of Drug-Resistant Pathogens: A Study in Peri-Urban Areas of Kolkata, India.

This study depicts the drug-resistance and phylogenomic characteristics of 365 Escherichia coli (EC) and 76 Klebsiella pneumoniae (KP) isolated from stray dogs (293) in and around Kolkata, India. Initial screening found 59 isolates, including 48 E. coli and 11 KP multidrug resistant, which included 33 extended-spectrum ß-lactamase, 41 AmpC ß-lactamase and 18 metallo-ß-lactamase producers carrying blaNDM-1 (11) and blaNDM-5 (7) genes. Majority of them had the resistant genes such as blaCTX-M (33), blaTEM (18), blaSHV (4), blaOXA (17), blaFOX (2), blaDHA (2), blaCITM (15), blaCMY-2 (13), blaGES (2) and blaVEB (2), qnrS (15), qnrB (3), aac-6'-Ib-cr (14), tetA (26), tetB (14), sul-1 (25), armA (2) and rmtB (6), in addition to adherence genes such as csgA (33), fimA (27), fliC (13), sdiA (33), rcsA (38), and rpoS (39). They also carried plasmid of diverse replicon types of which IncFIA and FIB were the most frequent. Phylogrouping categorized most of the MDR E. coli in phylogroup A (20), B1 (14), and B2 (6). Enterobacteriaceae repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) showed genetic diversity of multidrug resistant isolates irrespective of their origin, resistance, and virulence types, differentiating the EC in five clades (A-E) and KP in four clades (A-D). As these stray dogs, which had no history or scope of previous antimicrobial therapy, were found to have contracted potential antimicrobial resistance pathogens, the role of environment in spread of such pathogens and further possibility of human infections cannot be ruled out.

Prevalence of gastrointestinal parasitism in pigs of Tripura, India.

Pigs breeds are an important livestock species mostly reared by economically lower incomesection of people in India. Within North-Eastern (NE) states, pig husbandry is very much popular hence maintain the livelihood of the rural native population. Gastrointentinal (GI) parasitic infectionisone of the major constraint in profitable pig production in this area. In the present study, the GI parasitism was investigated in 388 pigs in the three districts of Tripura, NE State of India. The examination of faecal samples revealed 61.65% overall prevalence of parasitic infestation, precisely6 GI parasitic species; including 4 nematodes and 2 protozoa, while 46.91% were the mixed infections.Metastrongylus spp. (17.53%), Strongyloids spp. (19.33%),Trichuris spp. (15.98%), Coccidia spp. (12.37%), and Balantidium coli (10.82%), were detected, however, Ascaris spp. was the most prevalentrecording 32.47%. The epidemiological factors including: age, sex, season, breed, area and farming system wise when considered as markers of study showed the highest prevalence of GI parasites in grower(6-12 months) stage, female, monsoon season, non-descript breeds, Khowai district and free range farming system, recorded 71.52%, 67.27%, 65.78%, 65.71%, 64.57%, and 69.44%, respectively. Overall, our study provides a baseline data for further investigation and formulation of strategies for control of GI parasitism in pigs in Tripura.

Isolation of Reticuloendotheliosis Virus from Chorio-allantoic Membrane of SPF Chicken Eggs inoculated with Fowl Pox Virus.

Fowl Pox Viruses (FPV) infect chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth etc. The birds, affected with FPV, also show anemia and ruffled appearance which are clinical symptoms of Reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of Reticuloendotheliosis Virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the Chorio-allantoic Membrane (CAM) of 10 days old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. But the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV virus, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Ovine CD14- an Immune Response Gene Has a Role Against Gastrointestinal Nematode Haemonchus contortus-A Novel Report.

CD14 (also known as the monocyte differentiation antigen) is an important immune response gene known to be primarily responsible for innate immunity against bacterial pathogens, and as a pattern recognition receptor (PRR), binds with LPS (endotoxin), lipoproteins, and lipotechoic acid of bacteria. So far very limited work has been conducted in parasitic immunology. In the current study, we reported the role of CD14 in parasitic immunology in livestock species (sheep) for the first time. Ovine CD14 is characterized as a horse-shoe shaped bent solenoid with a hydrophobic amino-terminal pocket for CD14 along with domains. High mutation frequency was observed, out of total 41 mutations identified, 23 mutations were observed to be thermodynamically unstable and 11 mutations were deleterious in nature, causing major functional alteration of important domains of CD14, an indication of variations in individual susceptibility for sheep against Haemonchus contortus infestations. In silico studies with molecular docking reveal a role of immune response against Haemonchus contortus in sheep, which is later confirmed with experimental evidence through differential mRNA expression analysis for sheep, which revealed better expression of CD14 in Haemonchus contortus infected sheep compared to that of non-infected sheep. We confirmed the above findings with supportive evidence through haematological and biochemical analyses. Phylogenetic analysis was conducted to assess the evolutionary relationship with respect to humans and it was observed that sheep may well be used as model organisms due to better genetic closeness compared to that of mice.

Companion Animals Emerged as an Important Reservoir of Carbapenem-Resistant Enterobacteriaceae: A Report from India.

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) are perceived as a serious public-health threat world-wide. Despite sporadic reports, no systemic study has been carried out on CRE in companion animals in Indian subcontinent. In total, 237 canine specimens collected from five veterinary polyclinics in and around Kolkata were analyzed for isolation, antimicrobial resistance profiling and molecular characterization of carbapenem-resistant (CR) E. coli. Of the 29 CR isolates, 19 were identified as metallo-ß-lactamase producers (MP-CRE) and 10 as metallo-ß-lactamase non-producers (MNP-CRE). Eleven of them were extended spectrum ß-lactamase and/or AmpC type ß-lactamase producers and harboured fluoroquinolone-, tetracycline-, sulfonamide- and aminoglycoside-resistant genes. Beside uropathogenic virulence determinants, they carried the adhesion factors mediating biofilm production which was remarkably higher in 6 MP-CRE and one MNP-CRE isolates. Although the CRE were of diverse origin including the healthy and the diseased dogs, these were more frequently isolated from canine pyometra. The MP-CRE harboured plasmids of IncF and IncA/C types. Phylo-type B1 was observed in 38% of the CR isolates, followed by A0 in 31% and rest were attributed to A1 and D1. The Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) revealed that these isolates were genetically diverse and constituted of a heterogenous population. Detection of CRE in pet dogs despite the fact that carbapenems are not used in animals in India emphasizes the need for active surveillance to identify the transmission and dynamics of such pathogens in companion animals.

Effect of supplementing pomegranate peel infusion on body growth, feed efficiency, biochemical metabolites and antioxidant status of broiler chicken.

Pomegranate fruit peel is a great source of natural polyphenols. The objective of the present study was to evaluate efficacy of pomegranate peel infusion (PPI) on growth characteristics, feed efficiency, blood metabolites and antioxidant profile of broiler chicken. A total of 200 broiler chickens were randomly assigned to 4 treatments with 5 replicates of 10 birds. Pomegranate peel infusion was supplemented in drinking water to 3 treatment groups in a graded dose. At the end of the trial (42 days), 2 broiler chickens from each pen were sampled for serum and liver tissue. Results revealed that low-dose (50 mL/L) PPI influenced (L: P < 0.001) final body weight, daily body weight gain, and feed conversion ratio. Quadratic effect (Q: P < 0.001) was found in overall body weight, average daily gain in body weight, and average daily feed intake. It was also observed that PPI had significant (L: P < 0.05) hypo-lipidaemic effect. PPI supplementation reduced (L: P < 0.01) lipid peroxidation in all supplemented birds. Reduced glutathione and catalase in the liver tissue was also increased linearly (L: P < 0.05) by PPI supplementation, suggesting that natural polyphenols present in the PPI can stimulate antioxidant defence system. Thus, it could be concluded that low-dose supplementation of pomegranate peel infusion could be of great benefits in broiler chickens as a source of natural antioxidants.

Investigating genetic variability in Hsp70 gene-5'-fragment and its association with thermotolerance in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India.

The present study was undertaken to investigate genetic variability in a fragment comprising 5'UTR along with partial coding sequence of Hsp70 gene and its association with thermotolerance traits in Murrah buffalo at ICAR-Research Complex for Eastern Region, Patna (India). The allelic variants were identified from genomic DNA samples using SSCP technique. The PCR products were sequenced and analyzed. Data on different thermotolerance traits recorded in three seasons were analyzed by least squares ANOVA taking the SSCP genotypes as fixed effect. Two allelic variants (A and B), each of 503-bp in size, were documented with frequency of 0.59 and 0.41, respectively, and three genotypes (AA, AB and BB) with corresponding frequency of 0.30, 0.58 and 0.12. The allelic variants were due to single nucleotide substitution at 55th base position leading to a change of threonine (A) to methionine (B) in amino acid sequence. Both the allelic variants had 99.8% similarity in nucleotide sequence. In phylogenetic tree, allele A was in a cluster while allele B and Gangatiri cattle sequence formed a different cluster. The SSCP genotypes had significant effect on different thermotolerance traits in summer with thermo-humidity index of ≥ 84. Buffaloes with AA genotype had the highest (P Ë‚ 0.05) summer evening rectal temperature, respiration rate and pulse rate, inferring that the buffaloes carrying AA genotype had more stress in summer than those with AB and BB genotype. These SSCP genotypes might have differential role in heat shock protein response to induce thermotolerance of Murrah buffaloes in Gangetic plains.

Genetic profiling of Hsp70 gene in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India.

This study was aimed to genetic profiling of heat shock protein 70 (Hsp70) gene in Murrah buffalo investigating 50 unrelated adult animals at ICAR-Research Complex for Eastern Region, Patna (India) in winter, spring, and summer. PCR ready genomic DNA samples and season-wise total RNA samples were prepared. The PCR products of Hsp70 eluted from agarose gel were sequenced and analyzed. The first-strand cDNA was synthesized and concentration was equalized to 25 ng/µl. Expression kinetics of mRNA transcripts in different seasons was studied using Brilliant SYBR Green QPCR technique and the data retrieved was analyzed by least-squares ANOVA. DNA sequencing by primer walking revealed four allelic variants of Hsp70 gene. Alignment study revealed one substitution in 5'UTR, six substitutions in coding region, and one addition in 3'UTR. The highest percent identity and negligible phylogenetic distance were found among the alleles and reference bovine sequences. The relative mRNA expression was significantly higher in summer when THI ≥ 84 than the spring and winter; fold change increased by 4.5 times in summer than the spring whereas found nearly half in winter. These findings can be useful for heat stress management in buffaloes and help in understanding the mechanism of thermo-regulation well.

Novel decellularized animal conchal cartilage graft for application in human patient.

Restoration of the external ear and nose in human patients, in either congenital deformity or acquired defects, is a challenge in reconstructive surgery. Optimization of the currently available materials is necessary for rhinoplasty and microtia correction to avoid intraoperative manoeuvring and early rejection. The aim of this study was to develop cross-linked decellularized caprine conchal cartilages as biocompatible, robust, and non-toxic matrix template. The characterization of the decellularized tissue encompasses in vitro lymphoproliferation assay, cytotoxicity test, agar gel precipitation test, in vivo immunocompatibility study, histology, and determination of pro-inflammatory cytokines in animal model. Decellularized cartilage was implanted in human volunteer at R. G. Kar Medical College and Hospital, Kolkata, India, and samples were assessed histologically by retrieving those after 4 months. The processed cartilages were implanted in rhinoplasty (nine) and microtia patients (six) keeping autogenous cartilage graft as control up to 18 months after surgery. Primary outcomes were viability and safety of the material, both in animal model and human pre-application in actual site. Secondary outcomes included self-assessed clinical findings on gross examination. This study is under the ethical approval no. RKC/14 dated January 27, 2012. The in vitro cellular reactivity was less in processed cartilage protein than control. Histology of retrieved tissues in animal model and human volunteer showed no adverse reactions. Production of IL-2, IL-6, and TNF-α cytokines was lower at 4 weeks. The rhinoplasty and microtia operation in clinical patients utilizing the processed cartilage showed satisfactory recovery with improved facial look. These low cost, easily available, biocompatible, safe xenocartilage biomatrices of caprine conchal cartilage origin are very flexible in shape and size, enabling them as potential bioimplant for repair of nasal and auricular structure without any rejection or diverse biomedical applications.

Prevalence of CTX-M-Producing Klebsiella spp. in Broiler, Kuroiler, and Indigenous Poultry in West Bengal State, India.

This study was undertaken to detect the prevalence of CTX-M-producing Klebsiella spp. in healthy broiler, indigenous, and kuroiler birds reared in West Bengal (India) during November 2014-February 2015. In addition to CTX-M gene, the study was also conducted to reveal the occurrence of other ß-lactamase and class I integron genes in Klebsiella spp. isolates along with their clonal relationship. A total of 321 cloacal swabs from healthy broiler, indigenous, and kuroiler birds were collected from different places of West Bengal, India. Klebsiella spp. isolation rate varies among different types of poultry birds (43.8-72.3%). In total, 33 (10.7%) Klebsiella spp. isolates were detected phenotypically as CTX-M producers and all the isolates possessed blaCTX-M in polymerase chain reaction. Whereas 17 (51.5%) and 16 (48.5%) Klebsiella spp. isolates possessed blaSHV, and blaTEM with blaCTX-M, respectively. None of the CTX-M-producing Klebsiella spp. isolates in this study possessed class I integron gene. Randomly amplified polymorphic DNA-based phylogenetic tree revealed the presence of clonal relationship among the CTX-M-producing Klebsiella spp. isolates, recovered from broilers and indigenous birds. This study identified broilers and indigenous game birds as a potential reservoir of CTX-M-producing Klebsiella spp., which could be transmitted to the human food chain directly or indirectly.

Plasma mineral profiles and hormonal activities of normal cycling and repeat breeding crossbred cows: A comparative study.

AIM: The present study was carried out to compare the associated role of micro minerals and hormones in repeat breeding animals with the normal crossbred cows. MATERIALS AND METHODS: Blood samples were collected from 10 normal cycling and 10 repeat breeding crossbred cows of Ramakrishna Mission Ashram, Narendrapur to study the plasma mineral profile and hormonal activities. RESULTS: Zn was found to be highly significant (p<0.01) between the two groups. Follicle stimulating hormone (FSH) and progesterone showed significant (p<0.05) difference in repeat breeding animal from the normal cyclic animal, whereas no significant differences were observed in Ca, P, Cu, Se, Co, luteinizing hormone and estradiol level. CONCLUSION: It may conclude that repeat breeding condition of crossbred cows in farm condition is mainly due to the low level of progesterone, FSH and zinc.

Isolation and purification of beta-lactoglobulin from cow milk.

AIM: The present study was undertaken to standardize a convenient method for isolation and purification of ß-lactoglobulin (ß-lg) from cow milk keeping its antigenicity intact, so that the purified ß-lg can be used for detection of cow milk protein intolerance (CMPI). MATERIALS AND METHODS: Raw milk was collected from Gir breed of cattle reared in Haringhata Farm, West Bengal. Milk was then converted to skimmed milk by removing fat globules and casein protein was removed by acidification to pH 4.6 by adding 3 M HCl. ß-lg was isolated by gel filtration chromatography using Sephacryl S-200 from the supernatant whey protein fraction. Further, ß-lg was purified by anion-exchange chromatography in diethylaminoethyl-sepharose. Molecular weight of the purified cattle ß-lg was determined by 15 percent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was analyzed by gel documentation system using standard molecular weight marker. RESULTS: The molecular weight of the purified cattle ß-lg was detected as 17.44 kDa. The isolated ß-lg was almost in pure form as the molecular weight of purified ß-lg monomer is 18kDa. CONCLUSION: The study revealed a simple and suitable method for isolation of ß-lg from whey protein in pure form which may be used for detection of CMPI.

Anti-inflammatory activity of the ethanol extract of Dictamnus dasycarpus leaf in lipopolysaccharide-activated macrophages.

BACKGROUND: Dictamnus dasycarpus is widely used as a traditional remedy for the treatment of eczema, rheumatism, and other inflammatory diseases in Asia. The current study investigates the molecular mechanism of anti-inflammatory action of the ethanol extract of Dictamnus dasycarpus leaf (DE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. METHODS: Nitric oxide (NO) production was assessed by Griess reaction and the mRNA and protein expressions of pro inflammatory cytokines, transcription factor, and enzymes were determined by real-time RT-PCR and immunoblotting analysis. RESULTS: DE (0.5 and 1 mg/mL) suppressed the NO production by 10 and 33%, respectively, compared to the untreated group in LPS-stimulated RAW 264.7 cells. DE (0.5 and 1 mg/mL) reduced the mRNA expression of key transcription factor nuclear factor-κB by 7 and 24%, respectively compared to the untreated group in LPS activated macrophage. The pro inflammatory cytokines such as tumor necrosis factor α and interleukin 1ß were also decreased by DE treatment. Moreover, the protein expression of pro inflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase 2 were also dramatically attenuated by DE in a dose dependent manner. CONCLUSIONS: These results suggest that Dictamnus dasycarpus leaf has a potent anti-inflammatory activity and can be used for the development of new anti-inflammatory agents.

Virulence repertoire, characterization, and antibiotic resistance pattern analysis of Escherichia coli isolated from backyard layers and their environment in India.

This study was undertaken to observe the prevalence, serogroup, avian pathogenic Escherichia coli (APEC)-associated virulence gene, randomly amplified polymorphic DNA (RAPD) pattern, and antibiotic resistance genes of E. coli in backyard layers and their environment in India. From the 360 samples of healthy layers and their environment, 272 (75.5%) E. coli were isolated. The majority (28.67%) of them were untypeable. Among the studied virulence genes (papC, tsh, iucC, astA), 52 (14.32%) isolates were found to possess astA, including the isolates from the drinking water of the birds (4/272, 1.47%). These strains belonged to 18 different serogroups. Most of the isolates were typeable by RAPD and they produced different patterns. Phenotypic resistance of the isolates was most frequently observed to erythromycin (95.83%), chloramphenicol (87.52%), and cotrimoxazole (78.26%). None of the isolates was found to possess extended-spectrum beta-lactamases (bla(TEM), bla(SHV), bla(CTX-M) or quinolone resistance (qnrA) genes by PCR. The present study was the first attempt in India to assess APEC distribution in backyard poultry production.

Polymorphism of fecundity genes (FecB, FecX, and FecG) in the Indian Bonpala sheep.

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.

Polymorphism of BMPR1B, BMP15 and GDF9 fecundity genes in prolific Garole sheep.

Mutation studies in different prolific sheep breeds have shown that the transforming growth factor beta super family ligands viz. the growth differentiation factor 9 (GDF9/FecG), bone morphogenetic protein 15 (BMP15/FecX) and associated type I receptors, bone morphogenetic protein receptor (BMPR1B/FecB), are major determinant of ovulation rate and consequent increase in litter size. The Garole sheep is a highly prolific sheep breed of India. Characterization of fecundity genes in these animals could substantially improvise the breeding programme in these animals as well as other sheep breeds of the region. The present study was therefore designed with the objective of polymorphism study of fecundity genes in these prolific microsheep. A total of 11 point mutations were detected by polymerase chain reaction (PCR)-based method. A competitive technique called tetra-primer amplification refractory mutation system-PCR was adapted to type a total of ten points of two ovine fecundity genes (GDF9 and BMP15). The FecB locus of the BMPR1B gene and G1 locus of GDF9 gene were found to be polymorphic. In FecB locus, two genotypes, wild type (FecB(+)) and mutant (FecBB), were detected with allele frequencies of 0.39 and 0.61, respectively. At G1 locus, two genotypes, mutant (A) and wild types (G) were detected with allele frequencies of 0.18 and 0.82, respectively. This study reports Garole sheep as the fourth sheep breed after Belclare/Cambridge, Lacaune and Small-tailed Han sheep, where coexisting polymorphism has been found in two different fecundity genes (BMPRIB and GDF9 genes).

Surveillance of bluetongue virus antibody in goats using a recombinant VP7-based indirect ELISA in the coastal saline area of West Bengal, India.

The authors describe the serological surveillance of bluetongue virus (BTV) group-specific antibody in goats of the coastal saline (Sunderban) area of West Bengal, India. A recombinant viral protein 7 (rVP7)-based indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the antibody in sera. The bacterially expressed rVP7 was purified by affinity chromatography. The diagnostic performance of the assay was assessed by comparing it to the commercially available previously validated competitive ELISA. Using the control and 1 202 test sera, the cut-off value, sensitivity and specificity as well as other performance characteristics e.g. the Youden index, efficiency, positive and negative predictive value and prevalence were estimated. Field-collected goat sera (n = 1 202) were tested and a serological prevalence rate of 47% was observed in the study area.

Observations on the epidemiology of bovine cryptosporidiosis in India.

The significance of Cryptosporidium as a causative agent of diarrhea has been assessed in bovine for a period of 2 years. A total of 940 faecal samples (470 samples in each year) both from diarrhoeic and non-diarrhoeic bovine (0-12 months age) were examined during three different seasons (rainy season, summer and winter). Overall Cryptosporidium was detected in 17.46% and 18.04% cases in first and second year, respectively. Out of 50.21% diarrhoeic and 49.79% non-diarrhoeic cases Cryptosporidium was detected in 26.79% and 8.13% in first year and 27.49% and 8.59% in second year. Year did not have any significant effect on the occurrence of cryptosporidiosis in bovine during this study period. The prevalence of cryptosporidiosis, both in diarrhoeic (61.64%) and non-diarrhoeic (47.22%) cases was highest in 0-1-month age group (P<0.01). Such a high percentage of cryptosporidiosis in clinically asymptomatic animals indicated that the particular age group of animals might be reservoir for the parasite. During this study period highest prevalence was recorded in rainy season (27.55%) followed by summer (16.99%) and winter (8.71%) (P<0.01). A total of 166 positive cases were genotyped. Molecular characterization of bovine cryptosporidiosis has been carried out by PCR-RFLP analysis of SSU rRNA gene and results indicated that Cryptosporidium parvum mainly responsible for diarrhea in bovine in India.