Pramod Kumar Nanda

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.

A comparative study on the expression profile of MCTs and HSPs in Ghungroo and Large White Yorkshire breeds of pigs during different seasons.

Thermal stress has a significant adverse effect on commercial swine production but it is not easy to measure. Animals may adapt to stress conditions by an alteration in the expression of stress-related genes such as heat shock proteins (HSPs) and monocarboxylate transporters (MCTs). The present study presents a comparative analysis of seasonally varied effects on the expression profiles of HSPs (27, 70, and 90) and MCTs (1, 2, and 4) transcripts in thigh muscle and colon tissue of Ghungroo and Large White Yorkshire (LWY) breeds of pig. By real-time polymerase chain reaction, the mRNA expression of HSP27 and HSP90 genes was found to be higher in both thigh muscle and colon tissue in Ghungroo compared to Large White Yorkshire pigs during the summer. However, the relative expression of HSP70 was significantly higher (P < 0.01) in Ghungroo compared to Large White Yorkshire pigs during both seasons in both thigh muscle and colon tissue. The expression of HSP90 was higher in Ghungroo when compared to LWY though the variation was non-significant (P > 0.05) in the colon during different seasons. However, in Ghungroo, the mRNA expression of MCT1 was found to be significantly (P < 0.05) higher in thigh muscle and colon regions during the summer compared to LWY, whereas MCT2 was expressed more in the colon in LWY compared to Ghungroo during the summer. The relative expression of mRNA of MCT4 was found to be significantly (P < 0.05) higher in thigh region in both summer and winter in Ghungroo compared with LWY. Thus, the study demonstrated that both HSPs and MCTs gene expression during thermal stress suggests the possible involvement of these genes in reducing the deleterious effect of thermal stress, thus maintaining cellular integrity and homeostasis in pigs. These genes could be used as suitable markers for the assessment of stress in pigs.

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.

Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India

The present study reports on 23 extended spectrum β-lactamase producing Klebsiella pneumoniae (KP), isolated from milk samples (n=340) of healthy cows (n=129) and cows with subclinical (n=159) and clinical (n=52) mastitis, from three different states of India viz. West Bengal, Jharkhand and Mizoram. Seven of them were AmpC type β-lactamase producers, as well. The ESBL producing KP were significantly (P=0.006, χ2=10.04, df=2) and more frequently detected in milk samples of mastitic cows than healthy ones. The β-lactamase genes - blaCTX-M, blaTEM and blaSHV were detected in 19, 8 and 3 isolates, respectively. In all but one CTX-M positive isolates, the genetic platform - ISEcp1-blaCTX-M-orf477 was detected. Ten of the isolates carried plasmid mediated quinolone resistance gene - qnrS and 1 isolate possessed qnrB. Again 11 of them were found to have sulfonamide resistance gene - sul1 and 12 possessed class I integron. Sequencing of the class 1 integron revealed the presence of dfrA12/dfrA17 and aadA2/aadA5 gene cassettes conferring resistance to trimethoprim and aminoglycosides, respectively. All the isolates, characterized by enterobacterial repetitive intergenic consensus (ERIC) PCR, yielded distinct fingerprinting profile. However, most of the isolates from Jharkhand were clustered along with two isolates each from West Bengal and Mizoram indicating their clonal relatedness even though isolated from geographically different areas. Isolation of ESBL producing KP from bovine milk samples implies its public health significance; as such pathogens may enter the human food chain causing severe health hazards.

Antioxidant Efficacy of Litchi (Litchi chinensis Sonn.) Pericarp Extract in Sheep Meat Nuggets

In the present study, the efficacy of litchi fruit pericarp (LFP) extract (0.5%, 1.0% and 1.5% concentration) in retarding lipid oxidation of cooked sheep meat nuggets was evaluated and compared to butylated hydroxyl toluene (BHT, 100 ppm). The total phenolic content and antioxidant potential of LFP extracts were determined. The thiobarbituric acid reactive substance (TBARS) values were evaluated to assess the potential of LFP extracts as natural antioxidants for oxidative stability of cooked nuggets during 12 days of refrigerated storage. Results show that total phenolics content in 10 mg LFP powder was comparable to 100 ppm BHT, but 15 mg LFP powder had significantly higher (p < 0.05) total phenolics content and reducing power than the synthetic antioxidant. LFP extract did not affect pH, cooking yield and the sensory attributes of cooked nuggets. Non-treated control and nuggets with 1.0% LFP extract had significantly lower total phenolics than nuggets with 1.5% extract and BHT. TBARS values were significantly lower (p < 0.05) throughout the storage period in cooked meat nuggets containing either LFP extract or BHT than in non-treated control. Results indicate that LFP extracts are promising sources of natural antioxidants and can potentially be used as functional food additives in meat products at 1.5% without affecting products’ acceptability.

Changes in expression of monocarboxylate transporters, heat shock proteins and meat quality of Large White Yorkshire and Ghungroo pigs during hot summer period

Objective Present study explores the effect of hot summer period on the glycolytic rate of early post-mortem meat quality of Ghungroo and Large White Yorkshire (LWY) pig and comparative adaptability to high temperature between above breeds by shifting the expression of stress related genes like mono-carboxylate transporters (MCTs) and heat shock proteins (HSPs). Methods Healthy pigs of two different breeds, viz., LYW and Ghungroo (20 from each) were maintained during hot summer period (May to June) with a mean temperature of about 38°C. The pigs were slaughtered and meat samples from the longissimus dorsi (LD) muscles were analyzed for pH, glycogen and lactate content and mRNA expression. Following 24 h of chilling, LD muscle was also taken from the carcasses to evaluate protein solubility and different meat quality measurements. Results LWY exhibited significantly (p<0.01) higher plasma cortisol and lactate dehydrogenase concentration than Ghungroo indicating their higher sensitivity to high temperature. LD muscle from LWY pigs revealed lower initial and ultimate pH values and higher drip loss compared to Ghungroo, indicating a faster rate of pH fall. LD muscle of Ghungroo had significantly lower lactate content at 45 min postmortem indicating normal postmortem glycolysis and much slower glycolytic rate at early postmortem. LD muscle of LWY showed rapid postmortem glycolysis, higher drip loss and higher degrees of protein denaturation. Ghungroo exhibited slightly better water holding capacity, lower cooking loss and higher protein solubility. All HSPs (HSP27, HSP70, and HSP90) and MCTs (MCT1, MCT2, and MCT4) in the LD muscle of pigs inclined to increase more in Ghungroo than LWY when exposed to high temperature. Conclusion Effect of high temperature on the variation of HSPs and MCTs may play a crucial role in thermal tolerance and adaptation to different climatic conditions, pH regulation, muscle acidification, drip loss, protein denaturation and also in postmortem meat quality development.

Effect of Nigella sativa seed extract on lipid and protein oxidation in raw ground pork during refrigerated storage

Purpose Black cumin (Nigella sativa L.) is well known for its strong, hot, peppery taste and has many nutritional, pharmaceutical and traditional therapeutic uses. The aim of this study was to investigate the antioxidant effect of different solvent extracts of black cumin seed to retard lipid and protein oxidation in raw ground pork meat during refrigerated storage (4 ± 1°C) for nine days. Design/methodology/approach Black cumin extracts (BCEs) were prepared using different solvents, namely, ethanol, water, ethanol:water (60:40) and methanol:hot water (60:40). Extracts were analysed for total phenolic content (TPC), 1,1 diphenyl-2-picrylhydrazil (DPPH) radical scavenging activity and reducing power. Based on the results, water extract (WE) and ethanol–water extract (EHWE) of black cumin were selected and incorporated at 1.5 per cent into freshly minced pork meat and compared with a synthetic antioxidant, butylated hydroxytoluene (BHT; 100 ppm), in retarding lipid and protein oxidation. Treated and control samples were aerobically packed in low-density polyethylene bags for analysis of various parameters (pH, colour and odour score, peroxide, lipid and protein oxidation) during nine-day refrigerated storage study. Findings Results showed that BCEs had a good amount of TPC (4.4-7.4 mg gallic acid equivalents/g) and also DPPH scavenging activities (33.96-44.23 per cent), with WE and EHWE extracts showing highest reducing power and promising antioxidant capacity. Hence, BCEs (WE and EHWE) incorporated at 1.5 per cent into freshly minced pork meat was tested, compared to BHT (100 ppm) and control samples, in retarding lipid and protein oxidation during storage. In BCE-treated samples, thiobarbituric acid reacting substances, free fatty acids, peroxide, formation of protein carbonyls and off-odour or rancid odour development were lower than control and values were comparable with BHT. Incorporation of BCE did not negatively affect the colour of ground pork. Originality/value BCEs (WE and EHWE) at 1.5 per cent inhibited protein and lipid oxidation and it could be exploited commercially as an effective alternative in retarding oxidative deterioration of meat products.

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.

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.

Inhibition of lipid and protein oxidation in raw ground pork by Terminalia arjuna fruit extract during refrigerated storage

Objective Terminalia arjuna plant, specially its leaves, bark, and roots, are widely used in traditional herbal medicine due to presence of bioactive components and being a rich source of natural antioxidants. But its fruit has not been used for any such purposes despite its potential to retard oxidation. Hence, the antioxidant potential of Arjuna fruit extract (AFE) in retarding lipid and protein oxidation of raw ground pork was evaluated during refrigerated storage for 9 days. Methods The AFEs were prepared using different solvents viz. ethanol (EH), water, ethanol: water (60:40) and methanol:hot water (60:40). The AFEs were analysed for total phenolic content (TPC), 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power. Water extract (WE) and ethanol-water extract (EH-WE) were selected and incorporated at 1.0% into freshly minced pork meat and compared with a synthetic antioxidant, in retarding lipid and protein oxidation during storage. Results The TPC in AFEs using different solvents ranged from 11.04 to 16.53 mg gallic acid equivalents/g and extracts exhibited appreciable scavenging activity ranging from 50.02% to 58.62%. Arjuna extracts significantly (p<0.05) improved the colour score of meat samples by reducing the formation of metmyoglobin during storage. Both the AFEs (WE and EH-WE) significantly (p<0.05) lowered the thiobarbituric acid reactive substances value, peroxide formation and formation of protein carbonyls in raw pork than control sample during storage. Upon sensory evaluation of all samples, it was found that AFE treatment could prolong the storage period of meat samples, without influencing the colour and odour score, up to 6 days. Conclusion AFEs used at 1% improved the oxidative stability, colour and odour score and prolonged the refrigerated shelf life of ground pork up 6 days. Therefore, AFE could be explored as an alternative natural antioxidant in retarding lipid and protein oxidation in meat products.