Nidhi Rawat

Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens

Aim: Present investigation was conducted to isolate and characterize bacteriophages with lytic activity against common bacterial pathogens. Materials and Methods: A total of 60 samples of animal waste disposal from cattle (42) and buffalo (18) farms were collected from three different strata, i.e., top, mid, and bottom of collection tank. Samples were primarily subjected to rapid detection methods, and then isolation of phage was done by double agar layer method using Bacillus subtilis (BsH) and Escherichia coli (EH) as host system. Phages were characterized on the basis of plaque morphology, temperature, pH susceptibility, and host range. Results: Recovery of phages was higher from dairy cattle farm waste (78.57%) as compared to buffalo farm waste (72.22%) and bottom layer of tank showed maximum recovery. Bacillus subtilis (91%) supported the growth of more phages as compared to E. coli (9%). Three different phage morphotypes were observed each against Bacillus subtilis (BsHR1, BsHR2, and BsHR3) and E. coli (EHR1, EHR2, and EHR3). Mean phage titer of above six phage isolates ranged between 3×1010 and 5×1012 plaque forming units/ml. Viability of phages was by, and large unaffected at 70°C within 2-3 min, and phage isolates were completely inactivated below pH 3 and above 11. Coliphage EHR1 had widest host range followed by BsHR1 and BsHR2 while EHR2, EHR3, and BsHR3 had low lytic activity. Conclusion: It could be concluded from the present study that the Bacillus and Coli phage has wide host range and thus exhibits the potential to be used as drug substitute tool against common bacterial pathogens.

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.