Premanshu Dandapat

Potential antibacterial activity of berberine against multi drug resistant enterovirulent Escherichia coli isolated from yaks (Poephagus grunniens) with haemorrhagic diarrhoea

OBJECTIVE To evaluate the antimicrobial efficacy of berberine, a plant alkaloid. METHODS Five multi-drug resistant (MDR) STEC/EPEC and five MDR ETEC isolates from yaks with haemorrhagic diarrhoea were selected for the study. Antibacterial activity of berberine was evaluated by broth dilution and disc diffusion methods. The binding kinetics of berberine to DNA and protein was also enumerated. RESULTS For both categories of enterovirulent Escherichia coli (E. coli) isolates, berberine displayed the antibacterial effect in a dose dependent manner. The MIC(50) of berberine chloride for STEC/EPEC isolates varied from 2.07 μM to 3.6 μM with a mean of (2.95 ± 0.33) μM where as for ETEC strains it varied from 1.75 to 1.96 μM with a mean of (1.87 ± 0.03) μM. Berberine bind more tightly with double helix DNA with Bmax and Kd of (24.68±2.62) and (357.8±57.8), respectively. Berberine reacted with protein in comparatively loose manner with Bmax and Kd of (18.9±3.83) and (286.2±113.6), respectively. CONCLUSIONS The results indicate clearly that berberine may serve as a good antibacterial against multi drug resistant E. coli.

Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India.

The present study was undertaken to determine the occurrence and characterization of extended spectrum beta-lactamase (ESBL) producing Escherichia coli isolated from cattle and poultry in Odisha, India. Of 316 E. coli isolated from 305 samples (170 fecal samples from poultry and 135 milk samples from cattle), a total of 18 E. coli isolates were confirmed as ESBL producers by combination disc method and ESBL E-test. The isolates were resistant to oxyimino cephalosporins and monobactam as revealed by disc diffusion assay and determination of minimum inhibitory concentration. Resistance against other antibiotics was frequently noted as well. Further, beta-lactamase genes viz., blaSHV, blaCTXM, blaTEM and blaampC were detected in 17, 13, 9 and 2 isolates, respectively in PCR. Of the 18 ESBL strains, 16 were positive for class I integron (int1), nine of them carried sulphonamide resistance gene (sul1) and one harbored quinolone resistance gene (qnrB). Virulence markers for extraintestinal pathogenic E. coli like astA, tsh and iucD were also present in 4, 3 and 3 isolates, respectively. All the PCR amplified products were cloned and subjected to sequencing for homology analysis and data were submitted to gene bank. Sequence analysis of the amplified variable regions of class 1 integron of four representative isolates revealed the presence of aadA2 and dfrA12 gene cassettes conferring resistance to aminoglycosides and trimethoprim, respectively. Most of the ESBL producing strains emerged as single lineage through phylogenetic analysis by RAPD and ERIC PCR. This is the first ever systemic study on multidrug resistant ESBL producing E. coli in food producing animals from India.

Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus and extended spectrum β-lactamase producing Escherichia coli in bovine mastitis – three cases reported from India

Emergence of antimicrobial resistance among bovine mastitis pathogens is the major cause of frequent therapeutic failure and a cause of concern for veterinary practitioners. This study describes intra-mammary infection of methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum β-lactamase (ESBL) producing Escherichia coli in two Holstein Friesian crossbred cows with subclinical mastitis and one non-descript cow with clinical mastitis in two different districts of West Bengal, India. In total, three MRSE, one MRSA and three ESBL producing E. coli were isolated from these cases. Both the crossbreds were detected with MRSE (HFSE1 and HFSE2) and ESBL producing E. coli (HFEC1 and HFEC2), whereas, simultaneous infection of three pathogens viz. MRSA (NDSA1), MRSE (NDSE1) and ESBL producing E. coli (NDEC1) was found in the non-descript cow. The methicillin-resistant isolates possessed mecA gene and exhibited resistance to various antibiotics such as amikacin, tetracycline and glycopeptides. The ESBL producers were positive for blaCTX-M and blaTEM genes; in addition, HFEC1 and HFEC2 were positive for blaSHV and possessed the genes for class I integron (int1), sulphonamide resistance (sul1), quinolone resistance (qnrS) and other virulence factors (papC, iucD and ESTA1). All the ESBL producers exhibited resistance to a variety of antibiotics tested including third- and fourth-generation cephalosporins and were also intermediately resistant to carbapenems. This is the first ever report on simultaneous occurrence of MRSE, MRSA and ESBL producing E. coli in bovine mastitis indicating a major concern for dairy industry and public health as well.

Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei.

Blackleg, an economically important and highly fatal disease of ruminants, is caused by anaerobic bacillus, Clostridium chauvoei. Identification and differentiation of the causative agent is crucial for implementation of therapeutic and control measures in real time. Most of the diagnostic tests available for blackleg are PCR based, and only a couple of serological tests have been reported. In this study, we targeted flagellin, an important immunogenic protein of C. chauvoei, to develop a sandwich ELISA for detection of C. chauvoei. Sequence analysis of flagellin gene of related Clostridium species showed that central region of flagellin gene is unique to C. chauvoei. Hence, we cloned and expressed central region of flagellin in a prokaryotic expression system. Antiserum against recombinant flagellin was generated in rabbits and chickens. A sandwich ELISA was developed, in which rabbit anti-flagellin antibodies were used as capture antibodies and chicken anti-flagellin antibodies as detecting antibodies. The test was specific and sensitive in detection of up to 10(4) CFU/ml of C. chauvoei. This study shows that assay developed can be used for detection of C. chauvoei in suspected samples.

Intra-species sequence variability in 28s rRNA gene of Oesophagostomum venulosum isolated from goats of West Bengal, India

Objective: To identify genotypes of Oesophagostmum venulosum (O. venulosum) prevailing in West Bengal, India by comparing variation of nucleotide sequences among 28S rRNA. Methods: PCR amplification of partial segment of 28S rRNA sequence and analysis of sequence amplified product by single strand conformation polymorphism (SSCP). Results: Two distinct conformers among male and female parasites were identified by PCR-SSCP analysis. Sequence analysis among conformers revealed the presence of five single nucleotide polymorphisms (SNP) in codon 64, 66, 86, 125 and 146. Secondary RNA prediction structure showed that out of 5 SNPs, 4 occurred at interior loop of RNA which confirmed evolutionary changes among isolates prevailing in this region. Conclusions: SNPs occured in different isolates of O. venulosum might influence critical changes in rRNA folding pattern which influence evolutionary changes among isolates.

Prevalence of paratuberculosis in organized and unorganized dairy cattle herds in West Bengal, India.

Aim:: The aim of this study was to determine the prevalence pattern of Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis or Johne’s disease, in unorganized as well as organized cattle herds in West Bengal. Materials and Methods:: Four organized cattle farms with identical management practice in Nadia (n=3) and South 24 Parganas (n=1) districts and three unorganized cattle herds, one each from three districts, namely, Burdwan, North 24 Parganas, and Purba Midnapur, were selected randomly and screened for paratuberculosis by delayed-type hypersensitivity (DTH) and enzyme-linked immunosorbent assay (ELISA). Results:: Of 191 animals tested by DTH, 57 (29.8%) were found to be positive in comparison to 72 (37.7%) by ELISA. In organized farms, seropositivity varied from 13.3% to 53.1%, whereas in unorganized sector, it ranged from 5% to 6.7% with one area having exceptionally high prevalence, i.e. 53.3%. The range of positivity detected by DTH both in organized farms and backyard sectors varied from 0% to 46.7%. By employing both DTH and ELISA together, the positivity of animals in organized and unorganized herds was 19.9% and 8%, respectively. Conclusion:: The results indicate that animals in organized farms are much more prone to paratuberculosis than others. For screening the herd, both DTH and ELISA should be used simultaneously to increase the test sensitivity in order to minimize its further spread adopting control programs.

Prevalence of Deg Nala disease in eastern India and its reproduction in buffaloes by feeding Fusarium oxysporum infested rice straw

OBJECTIVE To undertake a study on prevalence of Deg Nala disease in eastern states of India and to reproduce the disease in buffaloes by the Fusarium spp., isolated from the affected region. METHODS During this investigation, a survey was conducted covering four states of eastern region to identify the Deg Nala cases as well as to isolate and characterize the causative agent(s). An experimental study was carried out to reproduce the disease in healthy male buffaloes (2-3 years age) by randomly dividing them into five groups (four in each group). Each individual group was fed with rice straw artificially infested with either of the two representative isolates of Fusarium oxysporum (F. oxysporum) (F01, F02) or representative reference strains of Fusarium equiseti (F. equiseti) (ITCCF-2470) and Fusarium moniliforme (F. moniliforme) (ITCCF-4821) for 30 days, whereas the control group was fed with normal rice straw only. RESULTS A total of 658 Deg Nala cases were recorded and 12 Fusarium isolates were identified from the mouldy rice straw collected from these affected areas. The characterization of the isolates revealed three species viz., F. oxysporum, F. equiseti and F. moniliforme, among which F. oxysporum was predominant. The disease was artificially reproduced in three buffaloes in F01 group and one in F02 group within 20-23 days by feeding F. oxysporum infested rice straw which resembled the clinical symptoms and gross lesions of natural Deg Nala cases. CONCLUSIONS The field investigation and laboratory studies, including experimental production of Deg Nala disease suggest the possible involvement of mycotoxins. However, further investigations needs to be done to understand nature of the toxic factors involved in production of the Deg Nala disease.

A cross-sectional study on prevalence of bovine tuberculosis in Indian and crossbred cattle in Gangetic delta region of West Bengal, India

Aim : The aim of this study was to investigate the incidence of bovine tuberculosis (BTB), an old chronic disease having zoonotic potential, covering four districts in Gangetic delta region of West Bengal, India, and to find the prevalence in organized as well as backyard herds and variation in relation to their age, sex, and breeds. Methods : The incidence of BTB in exotic and indigenous breeds of cattle (n=173) of various age groups was investigated employing tuberculin (single intradermal tuberculin and comparative cervical tuberculin) tests and gamma interferon assay. Further, milk samples (n=96) from milching animals and antemortem (n=519) samples (nasal swab, buccal swab, and aspirates from pre-scapular lymph nodes) were also screened employing bacteriological and molecular techniques. Results : In total, 36 (25.4%) animals from organized and one (3.2%) from backyard farming sector were found positive to BTB. Polymerase chain reaction (PCR) of milk samples based on 16S rRNA amplified the 1030 bp band in four samples indicating them belonging to genus Mycobacterium. Species-specific primers used to differentiate between Mycobacterium bovis and M. tuberculosis confirmed the presence of M. bovis. Prevalence of BTB in exotic crossbred animals (34.6%) was significantly higher (p<0.001) compared to indigenous cattle (10.5%). Further, gender-wise analysis of data with respect to BTB revealed higher positivity (p<0.05) among cows/heifers (25.8%) compared to bulls/bullocks (7.3%). Although BTBpositive cattle were detected in all the age groups, no statistical difference (p=0.779) was found among them. Conclusion : The findings indicate a higher prevalence of BTB in exotic crossbred animals in Gangetic delta and variation in breed susceptibility, thereby suggesting an urgent review of the present policy on adopting national crossbreeding program and implementation of “One Health” approach.

Epidemiological and laboratory investigation of a zoonotic anthrax outbreak in West Bengal, India

Premanshu Dandapat, Arijit Chakrabarty, Sonjoy Dey, Pramod Kumar Nanda, Suresh Chandra Das, Suman Dey, Arun Kurien, Apurba Chakraborty, Samiran Bandyopadhyay, Subhasish Bandyopadhyay, Raj Kumar Singh Eastern Regional Station, ICAR-Indian Veterinary Research Institute, 37, Belgachia Road, Kolkata, PIN 700 037, West Bengal, India Department of Paediatrics, Bankura Sammilani Medical College & Hospital, P.O.: Kenduadihi, Dist.: Bankura, PIN – 722 102, West Bengal, India Animal Resources Development (Veterinary Research & Investigation), Toxicology Laboratory, Jhargram, Dist: West Midnapur, PIN 721 507, West Bengal, India