Comparative occurrence of ESBL/AmpC beta-lactamase-producing Escherichia coli and Salmonella in contract farm and backyard broilers.

The present study was conducted to detect the occurrence of beta-lactamase and biofilm-producing Escherchia coli and Salmonella in apparently healthy broiler birds reared in household and contract poultry farms. In total, 150 cloacal swabs were collected from apparently healthy broiler birds of various age groups reared in backyard (n = 100) and contract farms (n = 50) in West Bengal (India). The isolation rate of ESBL producers was significantly more (P < 0·05) reared in contract poultry farms than those reared in backyard. Majority of the E. coli isolates possessed blaCTX-M followed by blaSHV and blaTEM . Majority of the Salmonella strains possessed blaTEM followed by blaCTX-M , and no blaSHV was detected. The selected sequences of the PCR products were found cognate with blaCTX-M-1 , blaCTX-M-2 , blaCTX-M-9 , blaCTX-M-14 , blaCTX-M-15 , blaSHV-12 and blaTEM-52 . The study detected 46·8% of E. coli and 42·5% of Salmonella strains as biofilm producers. Beta-lactamase-producing Enterobacteriaceae strains showed resistance against tetracycline, chloramphenicol, doxycycline, co-trimoxazole and ampicillin and sensitivity to imipenem-EDTA, colistin and gentamicin. The study revealed the partial clonal relationship of ESBL sequences possessed by the poultry isolates of the present study and local clinical isolates available in the database. The study made consumer awareness about careful handling of live birds or poultry meat to avoid the zoonotic transmission of antimicrobial-resistant Enterobacteriaceae.

Multi-drug resistant, biofilm-producing high-risk clonal lineage of Klebsiella in companion and household animals.

Antimicrobial resistance is a global emergency which needs one health approach to address. The present study was conducted to detect the prevalence of beta-lactamase and biofilm-producing Klebsiella strains in rectal swabs (n = 624) collected from healthy dogs, cats, sheep and goats reared as companion or household animals in India. The dogs and cats were frequently exposed to third- or fourth-generation cephalosporins for therapy. The sheep and goats were occasionally exposed to antibiotics and had environmental exposure. Phenotypical ESBL (n = 93) and ACBL (n = 88)-producing Klebsiella were isolated significantly more (P < 0·05) from companion animals than household animals. Majority of the Klebsiella possessed blaCTX-M-15 . The sequences blaCTX-M-15.2 , blaCTX-M-197 and blaCTX-M-225 are reported first time from the companion animals. All ACBL-producing isolates possessed blaAmpC . The present study detected 65·8% of Klebsiella strains as biofilm producers possessing the studied biofilm associated genes. The isolates showed phenotypical resistance against chloramphenicol, tetracycline, doxycycline, co-trimoxazole, ampicillin, cefotaxime/clavulanic acid. The present study showed that companion and household animals (dogs, cats, sheep, goats) may act as a carrier of ESBL/biofilm-producing, multi-drug resistant, high-risk clonal lineage of Klebsiella.

Characterization of beta-lactamase and biofilm producing Enterobacteriaceae isolated from organized and backyard farm ducks.

This study was undertaken to detect the occurrence of beta-lactamase and biofilm producing Enterobacteriaceae in healthy ducks. A total 202 cloacal swabs were collected from ducks kept in organized (n = 92) and backyard (n = 110) farms in West Bengal (India). The ducks had no history of antibiotic intake. Among the 87 phenotypically beta-lactamase producing Escherichia coli, 19 (17·43%), 6 (5·05%) and 15 (13·76%) isolates possessed blaTEM , blaSHV and blaCTX-M respectively. Whereas, 5 (38·46%) Salmonella isolates were found to harbour blaCTX-M . In K. pneumoniae 10 (33·33%), 3 (13·33%), 4 (13·33%) isolates possessed blaTEM , blaSHV and blaCTX-M respectively. The sequences of selected PCR products were found 98% cognate with blaCTX-M-9, blaSHV-12 and blaTEM-1 . Beta-lactamase producing E. coli isolates belonged to 14 different serogroups such as O1, O2, O3, O5, O7, O8, O35, O83, O84, O88, O119, O128, O145 and O157. Moreover, 87 E. coli (79·82%), six Samonella (46·15%) and 13 K. pneumoniae (43·33%) isolates were detected as AmpC producers possessing blaAmpC . Majority of E. coli (46·79%), Salmonella (46·15%) and K. pneumoniae (70%) isolates were detected as biofilm producers and possessed the associated genes (csgA, sdiA, rcsA, rpoS). Significantly higher occurrence of beta-lactamase and biofilm producing Enterobacteriaceae isolates was detected in backyard ducks than organized farms. SIGNIFICANCE AND IMPACT OF THE STUDY: Consumption of antibiotic through feed or during therapy is considered as potential reason for generation of antimicrobial resistant bacteria in birds. This study provides valuable evidence that exposure to contaminated environment may be an additional source for generation of antimicrobial resistant bacteria in backyard ducks. The backyard ducks are reared by marginal farmers in India who cannot offer antibiotics to them either through feed or during therapy due to high cost. The study also reveals a significant correlation between biofilm formation and possession of antimicrobial resistance genes in the bacterial isolates from the ducks.

Isolation, molecular characterization and antibiotic resistance of Shiga Toxin-Producing Escherichia coli (STEC) from buffalo in India.

In total, 363 Escherichia coli were isolated from 165 faecal samples of healthy buffaloes in West Bengal, India. Twenty-four of these isolates (6·61%) were found to carry at least one gene characteristic for Shiga toxin-producing Escherichia coli (STEC). These STEC strains belonged to 13 different O-serogroups. The stx1 gene was present in 23 (95·8%) of total STEC isolates, whereas 20 (83·3%) STEC isolates carried the gene stx2. Twelve strains of E. coli (50% of total STEC isolates) possessed enterohaemolysin (ehxA) gene in combination with others. Fourteen (58·33%) isolates found to possess saa gene. However, no E. coli was detected harbouring gene for intimin protein (eaeA). Of 23 stx1 -positive isolates, seven (30·43%) were positive for genes of the stx1C subtype. Of the 20 isolates with the stx2 gene, 25% (5/20) possessed stx2C and 10% (2/20) possessed stx2d gene. The phylogenetic analysis after RAPD of STEC strains revealed six major clusters. The isolated STEC strains were resistant most frequently to erythromycin (95·83%), cephalothin (62·5%), amikacin (54·17%), kanamycin (45·83%) and gentamicin (41·67%) group of antibiotics. No ESBL-producing (blaCTXM , blaTEM , blaSHV ) or quinolone resistance gene (qnrA) was detected in the STEC isolates.

Occurrence of canine parvovirus type 2c in diarrhoeic faeces of dogs in Kolkata, India.

Canine parvovirus-2(CPV-2) causes a highly contagious disease of dogs characterised by acute hemorrhagic gastroenteritis, lethargy, vomiting, fever and usually bloody or mucoid diarrhoea. In the present study, 41 faecal samples collected from dogs exhibiting the signs of fever, vomition, bloody or mucoid diarrhoea in Kolkata, India were screened by haemagglutination test and PCR for detection of capsid protein coding VP2 gene. The viral genotype was detected by multiplex PCR and analysis of partial VP2 gene nucleotide sequences of selected PCR products with bioinformatics tool. Thirteen (31.71%) samples were found positive with HA titre ≥ 32 whereas 28 (68.29%) samples were positive by PCR of VP2 gene indicating higher sensitivity of PCR. Highest occurrence of CPV-2 was observed in the age group of 1-6 months (80.65%) and non-descript breeds with no history of vaccination (85%). Three samples were antigenic type CPV-2a, rest were CPV-2b/CPV 2c. Six CPV sequences were found to be highly similar to published CPV 2c sequences in BLAST analysis revealing a maximum identity of 99-100% with other CPV-2c strains and clustered together with CPV-2c strains of India and other countries in phylogenetic analysis. The present study highlights the need for continuous monitoring of samples to detect gradual changes in circulating CPV-2 genotypes in India.

Effects of dietary supplementation of Pseudomonas aeruginosa FARP72 on the immunomodulation and resistance to Edwardsiella tarda in Pangasius pangasius.

Edwardsiella tarda is one of the serious bacterial pathogens infecting both cultured and wild catfish urging an immediate need for effective protection strategies. This study assessed the effects of dietary supplementation of Pseudomonas aeruginosa FARP72 at 108 cells/g feed (PA diet) for 30 days on the innate immunity parameters, viz., respiratory oxidative burst (ROB) activity, lysozyme, ceruloplasmin, myeloperoxidase, in-vitro nitric oxide (NO) production in addition to the expression of immune genes encoding interleukin-1ß, C3 and transferrin in yellowtail catfish Pangasius pangasius and their resistance to Edwardsiella tarda challenge at a sub-lethal dose of 1.50 × 107 cells/fish. A significant increase in the innate immunity parameters was noted in PA diet-fed catfish on 30 dpf compared to the control. Post E. tarda challenge, the levels of immune parameters increased significantly and peaked at 5 dpi irrespective of feeding to confer protection against E. tarda. Their levels, however, decreased on and from 10 dpi. The results on the expression of immune genes encoding interleukin-1ß, C3 and transferrin in the kidney and liver tissue samples of PA diet-fed P. pangasius upon challenge with E. tarda further confirmed the ability of P. aeruginosa to stimulate primary immune organs at the gene level. The effects of feeding P. aeruginosa FARP72 on the immune functions of catfish as examined by the functional immune assays, thus, demonstrating the innate immune responses of catfish that are differentially stimulated by the PA diet. The findings of our study would help evolve management strategies to confer protection against E. tarda infection in commercial catfish aquaculture.

Detection and characterization of pathogenic Pseudomonas aeruginosa from bovine subclinical mastitis in West Bengal, India.

AIM: Subclinical mastitis in bovines is mainly responsible for the huge economic loss of the dairy farmers, of which Pseudomonas aeruginosa is one of the causative agents. The study was aimed at a screening of suspected milk samples from different cattle farms of West Bengal for detection and confirmation of P. aeruginosa strains followed by their characterization. MATERIALS AND METHODS: Around 422 milk samples were screened from different dairy farms primarily by on-spot bromothymol blue (BTB) test and then in the lab by somatic cell counts (SCC) to finally consider 352 samples for detection of P. aeruginosa. Selective isolation and confirmation of the isolates were done using selective media, viz., cetrimide and Pseudomonas agar followed by confirmation by fluorescent technique. Molecular characterization of the strains was done by polymerase chain reaction for the presence of toxA (enterotoxin A, 352 bp) and exoS (exoenzyme S, 504 bp) genes. RESULTS: Approximately, 371 (87.9%) samples were positive in on-spot BTB test among which 352 (94.8%) samples revealed high SCC values (more than 3 lakh cells/ml) showing infection when screened. Among these, 23 (6.5%) samples yielded typical Pseudomonas sp. isolates out of which only 19 (5.4%) isolates were confirmed to be P. aeruginosa which showed characteristic blue-green fluorescence due to the presence of pigment pyoverdin under ultraviolet light. Out of these 19 isolates, 11 isolates were positive for toxA, 6 isolates for exoS, and 2 for both these pathogenic genes. CONCLUSION: Approximately, 5.4% cases of bovine subclinical mastitis infections in South Bengal were associated with P. aeruginosa which possess pathogenic genes such as toxA (63.2%) and exoS (36.8%).

An annotated checklist of Culicoides Latreille, 1809 (Insecta: Ceratopogonidae: Diptera) with incorporation of a vector species list from India.

Culicoides Latreille, 1809 (Insecta : Diptera : Ceratopogonidae) are small nematocerous biological vectors of a wide range of pathogens of veterinary and medical importance. They are distributed worldwide but prefer warm, damp, and muddy areas. Female midges require blood for egg maturation. Studies on taxonomy, proper identification keys, and distribution patterns of these flies across different geographical regions of India of these flies are limited. This article provides an updated checklist of Culicoides spp. from India collected from various scattered publications, along with their synonyms and details on their subgenera, geographical distribution, and type locality. A compiled list of different Culicoides vectors from India has also been included separately in this article, along with the type of the diseases spread.

Molecular characterization and antibiotic susceptibility pattern of caprine Shiga toxin producing-Escherichia coli (STEC) isolates from India.

The present study was conducted to detect the occurrence, serotype, genotype, phylogenetic relationship and antimicrobial resistance pattern of STEC from healthy goats of West Bengal, India. From the 125 faecal samples collected from healthy goats, 245 isolates were identified as Escherichia coli. The E. coli harbouring any gene for Shiga toxins (stx 1/stx 2) was detected in 36 (14.7%) of the 245 E. coli isolates. These STEC strains belonged to 22 different serogroups (O2, O5, O20, O21, O22, O25, O41, O44, O45, O60, O71, O76, O84, O85, O87, O91, O103, O112, O113, O120, O156, and O158) and three were untypeable. The stx 1 and stx 2 was detected in 26 (72.2%) and 21 (58.3%) of Shiga toxin producing-E. coli (STEC) isolates, respectively. Further, E. coli harbouring eaeA only (Enteropathogenic E. coli) and ehxA was detected in 22 (61.1%) and 28 (77.7%) isolates, respectively. Whereas the saa was present in 8 (22.2%) E. coli isolates. The subtyping of the 26 E. coli strains possessing stx 1 showed that 73.% (19/26) of these isolates were positive for stx 1C subtype. Of the 21 isolates with the stx 2 gene, 42.8% (9/21) were positive for stx 2C, and 38.1% (8/21) were positive for stx 2d gene. The phylogenetic analysis of STEC strains after RAPD reveals eight major clusters. However, no serogroup specific cluster was observed. Resistance was observed most frequently to erythromycin (80.5%), amikacin (52.7%), cephalothin (50%), kanamycin (41.6%), neomycin (36.1%) and gentamycin (36.1%) and less frequently to norfloxacin (2.7%), enrofloxacin (2.7%), and ciprofloxacin (2.7%). Multidrug resistance was observed in eleven STEC isolates.

Comparative possession of Shiga toxin, intimin, enterohaemolysin and major extended spectrum beta lactamase (ESBL) genes in Escherichia coli isolated from backyard and farmed poultry.

The present work was conducted to compare the occurrence of Escherichia coli possessing virulence and ESBL genes in backyard and farmed poultry. Three hundred and sixty samples from the poultry kept in backyard system and 120 samples from the farmed birds were collected from West Bengal, India. Among the E. coli isolates of backyard poultry (O2, O10, O25, O55, O60, O106, UT), none of them possessed any of the Shiga toxin genes and eight E. coli isolates (8/272; 2.9%) harboured eaeA gene alone. Whereas among the E. coli isolated from the farmed poultry (O17, O20, O22, O102, O114, O119, rough, UT), four isolates (4/78, 5.1%) harboured stx 1/stx 2 gene and 11 isolates (11/78, 14.1%) possessed eaeA gene. None of the E. coli isolates from the backyard poultry harboured any studied ESBL gene. Whereas 29.4% of E. coli isolates from the farmed poultry were found to possess the ESBL genes.

Evaluation of egg production after adoption of biosecurity strategies by backyard poultry farmers in West Bengal.

AIM: On the basis of identified source of major bacterial infections at four agro-climatic zones in West Bengal the cost-effective biosecurity strategy was formulated for backyard poultry farmers. The aim of the present study was to assess the adoption. So, the study was aimed to detect the adoption level of the formulated biosecurity strategy to mitigate the Salmonella and Escherichia coliweek post-hatch period chicks were contamination level in the sources and its correlation with egg production in West Bengal. MATERIALS AND METHODS: A questionnaire was prepared querying regarding the biosecurity measures presently followed by the farmers, if any and egg production of their birds. Subsequent to the interview the formulated biosecurity strategy was conveyed. After 3 months, the interview with the same questionnaire was conducted to the same farmers to detect their adoption level. RESULTS: The change in practices were noted in certain parameters which differs significantly (p<0.01 or p<0.05). As a consequence, the average egg production/flock was increased in 3 months after adoption of the strategy (618.2±37.77/flock) in comparison to last 3 months average before adoption of the strategy (495.3±30.00/flock) which also differs significantly (p<0.01). CONCLUSION: The present study detected the implementation of the biosecurity strategy in backyard poultry farming in West Bengal can substantially benefit the farmers in terms of increased egg production.

Mycobacterium bovis AN5 antigens vary in their ability to induce nitric oxide production in blood monocytes of experimentally infected cattle.

Reactive nitrogen intermediates (RNI) are the principal effector molecules of activated monocyte/macrophage populations, responsible for killing and inhibiting the growth of virulent mycobacteria. In vitro nitrite production by blood monocytes of cattle inoculated with live Mycobacterium bovis AN5 was assessed from 0 day through 45 weeks post inoculation (PI). High in vitro nitrite production was observed at the 8th and 12th weeks PI in sensitized cattle but reactivity had fallen by the 20th week PI. To assess the in vitro nitrite producing ability of monocytes induced by individual polypeptides within culture filtrate antigens (CFA) of M. bovis AN5, cellular blotting was performed using peripheral blood mononuclear cells (PBMC) at the 12th week PI. It was observed that polypeptides of MW 70, 65, 60, 25, 24 and 22 kDa of CFA induced high nitrite production by blood monocytes while many polypeptides had little or no effect.

Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.).

Aeromonas hydrophila causes disease under stress conditions or in concert with infection by other pathogens in goldfish. Sero-diagnostic and/or immunoprophylactic tools against Aeromonas infection in goldfish are not available so far. The present study was undertaken to fractionate and characterise the outer membrane proteins (OMP) of A. hydrophila and to identify suitable immunoreactive components. A total of 10 fractions were generated from crude OMP antigens upon gel permeation and subsequent ion-exchange chromatography. One of the fractionated antigens (GPID2), primarily a 57-kDa polypeptide, showed maximum sero-reactivity, even higher than the crude OMP. Suitability of GPID2 antigen for use in diagnostic preparations was assessed by dip-stick ELISA. In vitro goldfish lymphoproliferative ability of fractionated antigen, GPIID2 (primarily a 23-kDa polypeptide) was observed to be higher than all the fractionated antigens as well as crude OMP. It can be concluded that the 57 kDa and 23 kDa polypeptides of the OMP of A. hydrophila, possessing high immunoreactivity, should be given due attention while preparing immunodiagnostic and immunoprophylatic tools against Aeromonas infections in goldfish.

Adjuvant effect of mushroom glucan and bovine lactoferrin upon Aeromonas hydrophila vaccination in catla, Catla catla (Hamilton).

Mushroom glucan and bovine lactoferrin (Lf), known for their immunostimulatory potential, were used as adjuvant in conjunction with a formalin-killed Aeromonas hydrophila vaccine in catla, Catla catla. In vitro antigen-specific responsiveness of catla leucocytes and protective responses against experimental challenge with homologous antigen were monitored following immunization. Antigen-specific proliferation, 'macrophage activating factor' (MAF) production and antibody production were significantly higher in fish injected with glucan adjuvanted vaccine. Lf adjuvanted preparations showed a weak proliferative response and MAF production, although the antibody production was significantly higher than the controls. A good degree of protection was achieved with the glucan adjuvanted vaccine. However, in spite of producing significant anti-A. hydrophila antibody, Lf adjuvanted vaccine did not confer any protection following challenge with A. hydrophila. The potential of adjuvanticity of mushroom glucan and bovine Lf in intraperitoneal vaccination is discussed.