P. Roychoudhury

Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolate Possessing the Shiga Toxin Gene (stx1) Belonging to the O64 Serogroup Associated with Human Disease in India

Shiga toxin-producing Escherichia coli (STEC) causes a spectrum of human sufferings, like bloody diarrhea and even life-threatening conditions, such as hemolytic uremic syndrome (HUS) ([1][1]). In addition to the serotype O157:H7, several serogroups of STEC have been isolated from severe outbreaks

Multidrug-resistant Staphylococcus pettenkoferi isolated from cat in India

Background and Aim: Coagulase-negative staphylococci (CoNS) are considered to be one of the emerging pathogens in human and animals in recent times. Staphylococcus pettenkoferi, a novel pathogen under CoNS, is discovered in 2002 in humans with multiple clinical manifestations in various patients. To date, the pathogens have not yet been reported from any animals. The present study reported the first ever isolation, identification, and characterization of multidrug-resistant S. pettenkoferi from a cat with peritonitis in India. Materials and Methods: Peritoneal fluid was collected aseptically from 3 years old cat processed for bacteriological culture by standard techniques. Isolates were confirmed by BD Phoenix™ automated bacterial identification system and were subjected to plate and tube coagulase tests. All the isolates were tested for antimicrobial sensitivity profile by disc diffusion assay, extended-spectrum β-lactamase production by double disc diffusion assay, in vitro biofilm production ability by microtiter plate assay, and detection of virulence genes and mecA gene by polymerase chain reaction assay. Results: A total of five clonally expanded isolates of S. pettenkoferi were isolated from peritoneal fluid of the affected cat. All the isolates were resistant against 36 antimicrobial agents and were also methicillin-resistant staphylococci. Phenotypically, all the isolates were negative for biofilm production but were carrying multiple biofilm-producing genes (icaA, IS257, nuc, and mecA). Conclusion: Although S. pettenkoferi was previously reported once from animal (cat) environment, this is probably the first ever report of isolation of the organism directly from any animals. This is also probably the first report from any species in India.

Extended-spectrum β-lactamases producing multidrug resistance Escherichia coli, Salmonella and Klebsiella pneumoniae in pig population of Assam and Meghalaya, India

Aim: The present study was conducted to record the prevalence of extended spectrum β-lactamases (ESBLs) producing Escherichia coli, Salmonella spp., and Klebsiella pneumoniae from pig population of Assam and Meghalaya and to record the ability of the resistant bacteria to transfer the resistance genes horizontally. Materials and Methods: Fecal samples (n=228), collected from pigs of Assam (n=99) and Meghalaya (n=129), were processed for isolation and identification of E. coli and Salmonella spp. All the isolates were tested for ESBLs production by double disc synergy test (DDST) followed by screening for ESBLs producing genes (blaTEM, blaSHV, blaCTX-M, and blaCMY) by polymerase chain reaction (PCR). Possible transfer of resistance encoding genes between enteric bacterial species was carried out by in vitro and in vivo horizontal gene transfer (HGT) method. Results: A total of 897 enteric bacteria (867 E. coli and 30 Salmonella) were isolated and identified. Altogether 25.41% isolates were confirmed as ESBL producers by DDST method. Majority of the isolates were E. coli followed by Salmonella. By PCR, 9.03% isolates were found positive for at least one of the target resistance genes. blaSHV was absent in all the isolates. blaCMY was the most prevalent gene. All the E. coli isolates from Assam were negative for blaTEM. A total of 2.76% isolates were positive for blaTEM + blaCMY. On the other hand, 0.67% isolates were positive for blaCTX-M + blaCMY genes. Only 0.33% isolates carried all the three genes. Altogether, 4.68% bacteria carried the resistance encoding genes in their plasmids. blaTEM gene could be successfully transferred from Salmonella (donor) to E. coli (recipient) by in vitro (5.5-5.7×10−5) and in vivo (6.5×10−5 to 8.8×10−4) methods. In vivo method was more effective than in vitro in the transfer of resistance genes. Conclusion: The pig population of Assam and Meghalaya are carrying multidrug resistance and ESBLs producing E. coli and Salmonella. The isolates are also capable to transfer their resistance trait to other bacterial species by HGT. The present finding could be considered as a serious public health concern as similar trait can also be transmitted to the human commensal bacteria as well as pathogens.

Anti-Diarrhoeal Activity and Toxicity Trial of Methanolic Fruit-Pulp Extract of Aegle Marmelos (L.) Correa in Sprague-Dawle Rats

To determine the toxicity as well as anti-diarrhoeal effect of the unripe methanolic fruit-pulp extract of Aegle marmelos in SD rats. Qualitative Phytochemical analysis of methanolic un ripe fruit-pulp extract of Aegle marmelos was done as per standard method. Extracts of methanolic unripe fruit-pulp extract of Aegle marmelos were evaluated at doses of 15mg/kg, 30mg/kg, 120mg/kg and 1600mg/kg in SD rats for anti diarrhoeal effect in castor oil induced diarrhoea. For Toxicity effect, 5000 mg/kg of methanolic extract of Aegle marmelos was administered orally to three female rats at first with 0.02% tween 80 vehicles and observed for any abnormal condition up to 14days .Statistical analysis was conducted by ANOVA following post hoc test. Qualitative analyses of different phytochemicals of methanolic extract of Aegle marmelos showed that the presence of Tannin and Terpenoids were highly positive whereas Alkaloid was moderately positive and Flavonoid was slightly positive. The study revealed that the dose of 30mg/kg BW and 1600 mg/kg BW of methanolic extract of A. marmelos showed same treatment response with Loperamide against castor oil induced diarrhoea in rats. Safety study did not show any side effects of high dose of A. marmelos therapy during rat model study. It can be concluded that methanolic extract of Aegle marmelos may be effective in reducing diarrhoea in animals

Bacteriological Quality of Raw Pork Sold in Retailed Butcher Shops of Aizawl and Imphal

Food is the major need for the survivability of the all living organisms. Thus it acts as a major route of transmission for all types of contamination by chemical and microbiological contaminants. Bacterial contamination of food is the most common cause of food-borne illness resulting 70% of deaths associated with food-borne diseases (Adak et al., 2002; Lynch et al., 2006). Meat is considered as one of the most important food items for human consumption from the ancient time and a major proportion of the worldwide population chiefly relies on meat as a potent source of good quality protein (Bradeeba and Sivakumaar, 2013). All over the world, pork shares about 38% of meat production (Jeffries 2012). Pork, being the most important meat in North-Eastern Region of India, the pig population shares 38% of total pig population of India with contribution of 18.77% of India’s total pork production and in hike with every passing year. International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 05 (2018) Journal homepage: http://www.ijcmas.com

Evaluation of specific humoral immune response in pigs vaccinated with cell culture adapted classical swine fever vaccine.

Aim: To determine an efficient vaccination schedule on the basis of the humoral immune response of cell culture adapted live classical swine fever virus (CSFV) vaccinated pigs and maternally derived antibody (MDA) in piglets of vaccinated sows. Materials and Methods: A cell culture adapted live CSFV vaccine was subjected to different vaccination schedule in the present study. Serum samples were collected before vaccination (day 0) and 7, 14, 28, 42, 56, 180, 194, 208, 270, 284 and 298 days after vaccination and were analyzed by liquid phase blocking enzyme-linked immunosorbent assay. Moreover, MDA titre was detected in the serum of piglets at 21 and 42 days of age after farrowing of the vaccinated sows. Results: On 28 days after vaccination, serum samples of 83.33% vaccinated pigs showed the desirable level of antibody titer (log10 1.50 at 1:32 dilution), whereas 100% animals showed log10 1.50 at 1:32 dilution after 42 days of vaccination. Animals received a booster dose at 28 and 180 days post vaccination showed stable high-level antibody titre till the end of the study period. Further, piglets born from pigs vaccinated 1 month after conception showed the desirable level of MDA up to 42 days of age. Conclusion: CSF causes major losses in pig industry. Lapinised vaccines against CSFV are used routinely in endemic countries. In the present study, a cell culture adapted live attenuated vaccine has been evaluated. Based on the level of humoral immune response of vaccinated pigs and MDA titer in piglets born from immunized sows, it may be concluded that the more effective vaccination schedule for prevention of CSF is primary vaccination at 2 months of age followed by booster vaccination at 28 and 180 days post primary vaccination and at 1 month of gestation.

Therapeutic evaluation of homoeopathic drug Crotalus horridus 200C against Ehrlichiosis-infected dogs in Mizoram

Objective: To study, the effect of a homoeopathic medicine Crotalus horridus 200C on ehrlichiosis in dogs in an endemic area of Aizawl district of Mizoram state of India. Materials and Methods: To evaluate the efficacy of Crotalus horridus 200C against ehrlichiosis dogs. 12 positive cases confirmed by polymerase chain reaction (PCR) were divided into two groups comprising six dogs in each group. One group was treated with standard therapy (doxycycline) and other group was treated with Crotalus horridus 200C at 4 pills orally for 20 days. Clinical improvement of affected dogs was recorded after therapy. Important haemato-biochemical parameters before and after therapy such as haemoglobin (Hb), packed cell volume (PCV), total erythrocyte count (TEC), total leukocyte count (TLC), differential leukocyte count (DLC), platelet count, total protein, albumin, globulin, A:G ratio, total bilirubin, serum creatinine, blood urea nitrogen (BUN), and liver-specific enzymes namely alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were assessed following standard protocol. All the parameters were compared with a control healthy group (T3). All experiment dogs were from different age with different breeds and bloods were collected at forenoon only. Results: PCR test yielded 13 dogs positive out of 67 suspected samples screened (19.40%) with an amplification of 387 bp fragment from 16S rRNA gene of E. Canis. Off total positive, only 8 (61.53%) could be detected in peripheral blood smear. Crotalus horridus-treated group of dogs showed clinical recovery from fever and temperature to normalcy by the 14th day posttreatment. Haemato-biochemical profiles of affected dogs such as Hb, PCV, TEC, TLC, DLC, platelet count, total protein, albumin, globulin, A:G ratio, total bilirubin, serum creatinine, BUN, and liver-specific enzymes namely ALT and ALP were turned to normalcy within 21 days of post-treatment. Conclusion: Nested PCR assay had been shown to be sensitive and specific for detection of Ehrlichia canis. Crotalus horridus 200C may be an effective and choice of drug for control of canine ehrlichiosis.

Dirofilaria repens in dogs from Assam, India

Abstract Objective To access the prevalence of Dirofilaria repens (D. repens) in dogs from Assam, India. Methods A total of 223 blood samples from local dogs were examined with conventional (wet film and Knotts concentration technique), serological (ELISA test using Snap4Dx kits) and molecular techniques (targeting internal transcribed spacer-2 region using panfilarial primers) in Guwahati, Assam, India. Results The study revealed 4 (1.79%) cases of asymptomatic canine dirofilariasis caused by D. repens. The blood samples were positive for D. repens with microfilaremia on wet blood film, at Giemsa stained smear and under Knotts concentration technique, but were negative at Snap®4Dx test (IDEXX Laboratory, Westbrook, USA) which is specific for Dirofilaria immitis. D. repens could be detected by molecular test. Further confirmation was obtained on the basis of DNA sequencing and homology searching by basic local alignment search tool. Sequence analysis revealed that the species prevalent in Guwahati was genetically distinct from the other D. repens reported from elsewhere. Conclusions Occurrence of D. repens in dogs from this part of India was recorded for the first time, confirming the presence of a autochthonous canine reservoir for the zoonotic filarial nematode in Assam, India, where three cases of human subcutaneous and ocular infection with D. repens (dirofilariasis) have been reported.

Characterization of Salmonella Enteritidis from two separate outbreaks of acute enteritis in Japanese quail (Coturnix coturnix japonica) and turkey (Melleagris gallopavo)

Two independent outbreaks of acute gastroenteritis in Japanese quails and turkeys were investigated and 44 Salmonella Enteritidis isolates were recovered from clinical samples. The isolates were subjected to pheno- typic and genotypic characterization. All of them produced amplicons of ~1103 bp, ~700bp and ~617 bp specific for sefC, pefA and stn genes, respectively, by a multiplex PCR. Molecular typing by ERIC-PCR, RAPD-PCR and REP-PCR techniques revealed genotypic simi- larity among all the 44 isolates. The investigation indicated the possible involvement of contaminated feed for both the outbreaks. The results also indicated that molecular techniques are rapid in comparison to conventional methods for epidemiological investigation of salmonellosis. Presence of highly virulent S. Enteritidis in food animals is a great public health concern, because poultry birds are one of the commonest sources of meat and meat products for human consumption.