Deepti Narang

Isolation of a bacteriophage against Salmonella Dublin and determination of its physical resistance under varied in vitro conditions

°C and direct sunlight beyond 5 days was found to be deleterious for survival of the phage.

Multiplex real-time PCR for identification of canine parvovirus antigenic types.

Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types.

ISMap02 element targeted nested polymerase chain in the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples of cattle and buffaloes

Background and Aim: Johne’s disease is chronic granulomatous enteritis which affects ruminants. There are many diagnostic approaches for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) of which molecular detection methods using various elements are less time consuming and more accurate. The present study was conducted using ISMap02 element for nested polymerase chain reaction (nPCR) based detection of MAP in fecal samples. The aim was to test the sensitivity and specificity of the ISMap02 element and also to use this element for the detection of MAP in fecal samples of cattle and buffaloes. Materials and Methods: A total of 211 fecal samples of cattle and buffaloes from different herds around Ludhiana aged between 2 and 13 years were collected, and DNA extraction was done from these samples. The nPCR was carried out for the detection of MAP in fecal samples. Results: The ISMap02 element was specific for the detection of MAP only and showed a sensitivity of detection of 7.6 fg/µL of the standard genomic DNA. Among the 211 fecal samples of cattle and buffaloes tested for the ISMap02 element, 18 samples (8.5%) were positive for MAP. Conclusion: The ISMap02 element is specific and sensitive for the detection of MAP in various samples, and when used in nPCR format, it can increase the sensitivity of detection.

Faecal Culture and IS900 PCR Assay for the Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Faecal Samples

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes johne’s disease in animals and is implicated in Crohn’s disease in humans. Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from faeces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct faecal polymerase chain reaction (PCR) is becoming more widely used, demonstrating similar sensitivity and specificity to culture. In the present study, faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was done on 200 clinically suspected cases of Johne’s disease in dairy cattle. One isolates appeared only on the mycobactin J supplemented media at 8–16 weeks post-inoculation. A total of 7 faecal samples out of 200 samples were detected positive by IS900 PCR assay for Mycobacterium avium subsp. paratuberculosis (MAP) yielding an expected product of size 229 bp. The sensitivity of the IS900 PCR was assessed by making ten fold serial dilutions of the known concentration (5 ng/μl) of the standard genomic DNA of MAP. The detection limit of the IS900 PCR was upto5 pg/μl.

Molecular Detection of Mycobacterium bovis Targeting esxB (CFP-10) in Blood Samples and Lymph Node Aspirates by Conventional PCR and qRT-PCR TaqMan Assay

Bovine tuberculosis, a chronic infectious disease is caused by an intracellular acid-fast bacilli Mycobacterium bovis that affects broad range of mammalian hosts. CFP-10 is a 10 kDa secreted antigen coded by esxB gene located on RD1 region of genome and is responsible for virulence of Mycobacterium bovis. DNA extraction of blood (n=48) and lymph node aspirates (n=48) was done and extracted DNA was subjected to PCR by targeting esxB gene with band size of 302 bp. None of the blood sample and lymph node aspirates was positive for M. bovis with esxB gene by PCR. The sensitivity of esxB was 8 pg/μl by conventional PCR. Among 48 blood samples targeted for esxB (CFP-10) gene using In house developed primer probe mix, one sample (2.1%) whose CT was 34 was considered positive by real-time PCR. Out of 48 animals (lymph node aspirates), four samples (8.3%) whose CT was between 29-34 were considered positive by real-time PCR. Remaining samples whose CT values were equal to or greater than 35 were considered negative. The sensitivity of esxB was 800 fg/μl by real time PCR. This study indicates the diagnostic potential of esxB by using real time PCR TaqMan Assay.