Evaluation of an indigenous ELISA for diagnosis of Johne’s disease and its comparison with commercial kits

Abstract

Country lacks sensitive and indigenous diagnostic kits for the screening of goats and sheep against Johne's disease. Therefore an indigenous ELISA kit was developed using protoplasmic antigen from native Mycobacterium avium subspecies paratuberculosis 'Bison Type' strain of goat origin (Kit 1). In the present study, kit 1 and two commercial kits (kit 2 and 3) were evaluated with respect to 'Gold Standard' fecal culture in 71 animals (55 goats and 16 sheep). Kit 1 using indigenous antigen (protoplasmic antigen) was sensitive at very low concentration (0.1 μgm / well) as compared to purifi ed commercial protoplasmic antigen (4 μgm / well) used in kit 2, in the Type 1 reactors (strong positive as positive). Screening of 71 animals by fecal culture detected 38.0% animals (goats-40.0%, sheep-31.2%) as positive (clinical shedders of bacilli) from these farm animals. Of the farm animals located at Central Institute for Research on Goats, herds of goat were endemic whereas, sheep fl ocks were comparatively resistant to Johne's disease. The 29.5 and 61.9, 15.4 and 57.7 and 4.2 and 14.0% animals (goats and sheep) were in the category of sero-reactors type 1 and 2 of the ELISA kits 1, 2 and 3, respectively. In the type 1 sero-reactors, sensitivity and specifi city of kit 1, 2 and 3 was 53.7 and 86.0, 17.8 and 86.0 and 3.5 and 94.7%, respectively. Indigenous ELISA test (kit 1) was signifi cantly superior for the screening of goatherds and sheep fl ocks against JD as compared to commercial ELISA kits (Kit 2 and 3).

Diagnostic Potential of Three Antigens from Geographically Different Regions of the World for the Diagnosis of Ovine Johne’s Disease in India

“…Using 3 antigens from geographically distinct regions of the world showed that it was prudent to use species specific antigen prepared from native strain (S 5) of MAP, the novel dominant bio-type ('Indian Bison Type') reported first time in the world from India (Sohal et al, 2009;Singh et al, 2013a). Similar findings have been reported in our earlier studies (Singh et al, 2007b). Present study also used semi-purified protoplasmic antigen from strain 'S 5' of MAP of goat origin in 'Indigenous g-ELISA' which detected highest percentage (90.0%) of sheep as positive and sheep was positive independently in the test.…”

“…Sheep detected positive in 'Indigenous g-ELISA' were also detected by other tests in 2 and 3 tests combinations. Previous studies by Singh et al (2007b) using Purified Protoplasmic Antigen (PPA) from Allied Monitor Inc., USA exhibited poor cross reactivity with sheep sera. However, in the present study purified PPA of bovine origin from Allied Monitor Inc., USA (supplied by ID Vet, France) exhibited better sensitivity and detected 77.5% sheep positive.…”

“…Like our study in sheep, JTC-ELISA developed by Shin et al (2008) was particularly sensitive at detecting low level fecal shedders of MAP (sub-clinical infection) and works effectively both on serum and milk samples for the detection of cattle infected with sub-clinical MAP infections, providing a cost-effective diagnostic tool to support JD control programs in cattle herds. Unlike Cho and Collins (2006) reporting that protein antigens of sero-diagnostic potential were more abundant in culture filtrates than cellular extracts from liquid culture of MAP, they used protein based species specific semi-purified whole cell PPA from cellular part of novel native MAP biotype (Indian Bison Type) cultured on solid HEY medium and was highly sensitive and specific (Singh et al, 2007b(Singh et al, , 2009a. Kumar et al (2006) reported that use of species specific antigens from 'Indian Bison Type' MAP showed better cross reactivity i.e., using PPA from sheep isolate in sheep rather than goat origin PPA in sheep.…”

“…Sensitivity and specificity of 'Indigenous ELISA kit' as per (Singh et al, 2007b) was 83.3 and 90.0%, respectively.…”

“…Protein estimation of the dilution of antigen used, showed that 100 μL of coating buffer contained 0.4 µg per well of the microtiter plate (b-ELISA) and other steps were like 'Indigenous ELISA kit' (Singh et al, 2007b).…”