EMPLOYMENT OF INDIRECT DOT-ELISA FOR THE DETECTION OF ANTI-PARAMPHISTOMUMEPICLITUM ANTIBODIES IN RUMINANTS

Abstract

Paramphistomosis is one of the major parasitic diseases causing heavy economic losses to livestock production. Diagnosis of disease in early stage is very important for minimizing the losses through effective treatment. The conventional methods of diagnosis such as detection of eggs in faeces by sedimentation and floatation techniques have limitations while modern serology based tests like enzyme linked immune-sorbent assay requires well equipped laboratory. The immature and adult Paramphistomumepiclitum were collected from rumen of slaughtered goat and buffaloes in slaughterhouses fromBareilly, Delhi, Dehradun and Ludhiana. Collected parasites were thoroughly washed in normal saline and processed separately for antigen preparation. Parasites were homogenized in 0.1M PBS pH=7.4, sonicated in Soniprep-150 for 8 min (4 cycles of 2 min each) and centrifuged at 15000 rpm for 15 min at 4C. The supernatant obtained was filtered through 0.22m Millex–GV filter (Millipore, France) and stored in small aliquots of 0.5 ml each at -20C to be used as somatic antigen for ELISA test. Sera were collected at monthly interval from rabbits immunized by somatic P. epiclitum antigen for determining the titre variation with time and for Dot-ELISAtests. Indirect Dot-ELISA was standardized using antigen concentration ranging from 5μg/μl – 10ng/μl and goat anti-rabbit HRPO conjugate dilutions. P. epiclitum antigen was used for coating nitro cellulose membrane (NCM) pad on combs for DotELISA and kept overnight at 4˚C. The combs were then incubated in 3% lactogen in PBS, pH=7.4, at 37˚C for 1 hr for blocking the non-specific antigen binding sites. Subsequently, the combs were incubated in rabbit anti-P. epiclitum sera in dilution range 1:50 – 1: 90000 at 37˚C for 1 hr followed by three washings in PBS of 2 min each. The combs were then incubated in goat anti-rabbit HRPO conjugate at 37˚C for 1 hr again followed by 4 washings in PBS of 2 min each. The combs were then incubated in substrate 3, 3'- Diamino-benzidine hydrochloride (5 mg/10 ml PBS + 10μl 0.06% H O ) for 2 2 5- 15 min. Development of dark brown spot indicated positive reaction after using various control. The optimum antigen concentration was found to be 100ng/μl and optimum conjugate dilution was found to be 1:500. Anti-sera collected from rabbits at the interval of 30 and 60 days showed a maximum titre of 1:40,000. The experimentally infected sheep sera were taken at weekly interval from four sheep, which were examined against adult somatic P. epiclitum antigen following concentrations 50-75 ng/µl and conjugate dilution 1:500 to 1:1000 and sera dilution at 1:600 and 1:800. Sheep sera showed reaction after 2 weeks post infection. A total of 200 sera samples were examined by indirect Dot-ELISA. Overall percent positive incidence rate was recorded to be 33 percent. The highest percent positivity (38.66%) was found in buffaloes followed by 31.7% in sheep, 26.66% in cattle and 25% in goats. In the present study, a rapid and simple test (Dot-ELISA) was developed for the diagnosis of paramphistomosis. The findings are helpful for detection of paramphistomum antibodies in naturally infected animals and can be used under field conditions.