N S Sharma

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis

Aim: The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. Materials and Methods: A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. Results: Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. Conclusion: It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Isolation, Identification and Molecular Detection of Brucella spp., in Cattle and Buffaloes

Transmission of the organism occurs mainly through contact with placenta, fetus, fetal fluids and vaginal discharges from an infected animal. Clinically, the disease is characterized by abortion in the third trimester of pregnancy. Infections may also cause stillborn or weak calves, retained placentas, reduced milk yield and orchitis and epididymitis in males. Confirmation in clinically affected animals is done by isolation and identification of the organism from aborted foetus, foetal membranes and vaginal mucus.

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.