Detection of Paramphistomum cevi Antibodies in Rabbits using ELISA and DID

Abstract

Paramphistomum is a member of the family paramhistomatidae which constitutes one of the most common and abundant groups of digenetic trematodes of domesticated livestock, especially in tropical and subtropical regions of the world. The disease paramhistomiasis caused massive infection of the small intestines with immature paramphistomes, characterized by sporadic epizootic outbreaks of acute parasitic gastroenteritis with high morbidity and mortality ABSTRACT Sheep (Ovis aries) is infected with a variety of gastrointestinal helminths , of which notably Fasciola hepatica and Paramphistomum cervi are prominent and pathogen causing a lot of morbidity and mortality (Mukherjee and Chauhan, 1965). Immature stages of P. cervi are highly pathogenic (Horak 1971) but the routine parasitological diagnosis is difficult, under the circumstances immunological test applied to the present study, is aimed at investigating the relative immunodiagnostic reliability and sensitivity of the Ouchterlony gel diffusion test and ELISA in paramphistomiasis.Sheep blood was collected at the local abattoir naturally infected with Paramphistomum cervi. Using somatic antigen of whole worms derived from P.cervi was prepared by homogenisation, sonication and centrifugation at 10,000 r pm for 20 minutes at 4 o C. Rabbits were immunized with the antigen mixed with Freunds complete adjuvant (1:1 ratio) for raising hyperimmune sera. Blood was collected at regular intervals by puncturing ear vein of the rabbit. The naturally infected sheep sera were also collected from the slaughter houses. The Ouchterlony test and ELISA were found to be positive as early as 2-4 th weeks post-infection. By gel diffusion test, two precipitation bands were observed, and at 6 th -8 th weeks of post infection by ELISA. An indirect ELISA standardized for detection of anti-Paramphistomum antibodies using antigen concentration of 2µg / ml was used on coating buffer. Indirect ELISA revealed antibody titre as high as 1: 12,800 in rabbit sera where as in sheep sera 1: 6400.