Sanjay Shakya

Occurrence and characteristics of extended-spectrum β-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India

Aim: To assess the prevalence of antimicrobial resistance producing extended-spectrum β-lactamases (ESBL) (blaTEM, blaSHV, and blaCTX-M) genes in Escherichia coli isolated from chicken meat, chevon meat, raw milk, and human urine and stool samples collected from tribal districts of Chhattisgarh, viz., Jagdalpur, Dantewada, Kondagaon, and Kanker. Materials and Methods: A total of 330 samples, comprising 98 chicken meat, 82 chevon meat, 90 raw milk, and 60 human urine and stool samples, were processed for isolation of E. coli. Isolates were confirmed biochemically and further tested against commonly used antibiotics to know their resistant pattern. The resistant isolates were tested for ESBL production by phenotypic method followed by characterization with molecular method using multiplex-polymerase chain reaction technique. Results: Overall 57.87% (191/330) samples were found positive for E. coli, which include 66.32% (65/98) chicken meat, 46.34% (38/82) chevon meat, 81.11% (73/90) raw milk, and 25% (15/60) human urine and stool samples. Isolates showed the highest resistance against cefotaxime (41.36%) followed by oxytetracycline (34.03%), ampicillin (29.31%), cephalexin (24.60%), cefixime (16.75%), and ceftazidime (13.08%). Phenotypic method detected 10.99% (21/191) isolates as presumptive ESBL producers, however, molecular method detected 3.66% (7/191), 2.09% (4/191), and 0.00% (0/191) prevalence of blaTEM, blaCTX-M, and blaSHV, respectively. Conclusion: The present study indicates a high prevalence of E. coli in raw chicken meat, chevon meat, and milk due to poor hygienic practices. The antibiotic susceptibility test detected the presence of the resistance pattern against ESBL in E. coli isolated from raw chicken meat, chevon meat, milk, and also in human clinical samples is of great concern. The appearance of E. coli in the human food chain is alarming and requires adaptation of hygienic practices and stipulate use of antibiotics.

Rapid diagnosis of fowl pox with coagglutination assay

SummaryThe coagglutination test was standardised for detection of fowl pox antigen in infected scabs and chorioallontoic membrane of chicken embryos. TheStaphylococcus aureus Cowan I strain, containing large amounts of Protein A in their cell wall, coated with fowl pox antibodies was found specific and sensitive for detection of fowl pox antigen. The test is easy to perform and rapid as the positive results can be read within 15 seconds.RésuméLe test de co-agglutination fut standardisé pour la détection de l’antigène de la peste aviaire sur des jaunes d’oeufs infectés et des membranes du chorion chez des embryons de poulet. La souche Cowan I duStaphylococcus aureus, contenant de large quantité de Proteine A dans leur paroi cellulaire, fixée par des anticorps de la peste aviaire se révéla spécifique et sensible pour la detection de l’antigène de cette maladie. Le test est facile d’utilisation et rapide puisque les résultats peuvent ête obtenus en 15 secondes.ResumenSe estandarizó el test de coaglutinación para detectar antígenos de la viruela aviar en la membrana corioalantoidea de embriones de pollo y en muestras de piel de animales infectados. La cepa Cowan I deStaphylococcus aureus—que contiene grandes cantidades de Proteina A en su pared celular-, unida a anticuerpos contra el virus de la viruela aviar, resultó ser sensible y específica para detectar antígenos del virus de la viruela aviar. El test es rápido y fácil de realizar, y los resultados positivos pueden leerse en 15 segundos.

Isolation and molecular characterization of Salmonella spp. from chevon and chicken meat collected from different districts of Chhattisgarh, India

Aim: The aim was to assess the prevalence of Salmonella in raw chevon and chicken meat sold in the retail meat shops situated in and around Durg, Rajnandgaon, Dhamtari, Raipur, and Bilaspur districts of Chhattisgarh. Studies were also conducted to find out the antibiotic resistance in Salmonella isolates. Materials and Methods: A total of 400 samples comprising of 200 chevon meat and 200 chicken meat samples were processed for isolation of Salmonella and all isolates were further confirmed on the basis of cultural and biochemical characters and by targeting invA gene of Salmonella. All Salmonella isolates were also examined for their antimicrobial drug susceptibility/resistance pattern against commonly used antibiotics. Results: Out of 400 samples, the prevalence of Salmonella in chevon and chicken meat was found 9% and 7% respectively, with an overall prevalence of 8%. Polymerase chain reaction targeting invA gene of Salmonella showed positive result with 31 isolates. All 32 Salmonella isolates were found to be highly sensitive to ciprofloxacin while 96.87%, 96.87% and 93.75% were sensitive to gentamicin, imipenem, and ceftazidime, respectively. 93.75% and 59.37% isolates were resistant to erythromycin and oxytetracycline, respectively. Out of 32, 14 isolates had multiple antibiotic resistance index equal to or more than 0.2. Conclusion: Salmonella in chevon and chicken meat samples is prevailing in the areas of sampling due to poor hygienic conditions and also demonstrated the varied spectrum of antimicrobial resistance, including several multiple drug resistance phenotypes. Therefore, the present study emphasizes the need for continued surveillance of zoonotic foodborne pathogens including antimicrobial-resistant variants throughout the food production chain.

Characterization of Salmonella Gallinarum from an outbreak in Raigarh, Chhattisgarh

Aim: The present investigation was conducted to isolate and characterize Salmonella Gallinarum from an outbreak of fowl typhoid in layer birds. Materials and Methods: Clinically ill and dead layer birds from an outbreak were investigated. History, clinical signs, and postmortem lesions were suggestive of fowl typhoid. Postmortem samples including heart blood, intestinal contents, pieces of ovary, and liver were collected and processed immediately for bacterial culture, serotyping and antibiotic sensitivity tests. Isolates were further screened for the presence of extended spectrum beta lactamase (ESBL) (blaTEM) gene by polymerase chain reaction. Results: On the basis of cultural, staining and biochemical characteristics; three bacterial isolates were confirmed as S. Gallinarum. On serotyping, somatic antigen O: 9 and 12 with nonflagellated antigen were detected in all three isolates. Isolates were intermediate sensitive to amoxycillin, amoxyclav, gentamicin and ciprofloxacin and resistant to most of the antibiotics including chloramphenicol, ampicillin, ceftazidime, cefexime, cefepime, azithromycin, nalidixin, tetracycline, oxytetracycline, and streptomycin. Two isolates were found to harbor ESBL (blaTEM) gene. Conclusion: Beta lactamase producer S. Gallinarum was confirmed as cause of increased mortality in layer birds during present investigation. Existence of multi drug resistant Salmonella poses serious threat to poultry industry in Chhattisgarh.

Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin-Darby bovine kidney cell line.

Aim: Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test. Materials and Methods: Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test. Results: Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test. Conclusion: BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test.

Seroprevalence of bovine herpes virus type 1 in cattle and buffaloes from Chhattisgarh

Present study was carried out to know the seroprevalence of BHV-1 in the population of cattle and buffaloes from Chhattisgarh, India. A total of 464 serum samples were collected from cattle and buffaloes of different districts in Chhattisgarh. The collected serum samples were screened by Avidin-Biotin Enzyme Linked Immunosorbent Assay kit that recorded an overall seroprevalence of 34.69%. Out of 422 cattle serum samples, 158 (37.44%) were found positive compared to 3 (7.14%) serum samples out of 42 from buffaloes. In different age groups, there was variability in prevalence of BHV-1. Animals above 9 years of age showed the highest seropositivity (45.9%) whereas young animals between 0 to 2 years of age showed the minimum seropositivity (6.89%). Crossbred cattle showed higher seropositivity (40.42%) followed by non-descript cattle whereas indigenous cattle showed the seropositivity of 39.77% and 22.03%, respectively. Murrah, Nagpuri and indigenous buffaloes showed seropositivity of 0%, 3.03% and 50%, respectively. In the present study, seropositivity of 36.53% and 37.56% was recorded in male and female cattle, respectively. Male and female buffalo showed 11.11% and 6.06% seropositivity, respectively. Seropositivity of 45.45% was recorded in animals without clinical signs whereas animals with history of different clinical conditions showed 24.46% seropositivity. Rhinotracheitis, pustularvulvovaginitis, mastitis and balanoposthitis were the main clinical fi ndings associated with the selected in research trial animals.