Application of a competitive ELISA for the detection of bluetongue virus antibodies in llamas and wild ruminants.

A competitive enzyme-linked immunosorbent assay (C-ELISA), using a group-specific monoclonal antibody against bluetongue virus (BTV), was applied to detect anti-BTV antibodies in serum samples from two llamas (Llama glama) experimentally infected with BTV serotype 10. Antibodies were detected in both llamas by 1 wk or 2 wk post-infection. Antibodies to BTV increased exponentially during the first 4 wk in both llamas and stabilized at an elevated level during the remaining 5-wk-period of the experiment. We evaluated the C-ELISA for 1,442 field sera from bluetongue-free areas, collected from 398 llamas in New Zealand as well as 451 elk (Cervus elaphus canadensis), 323 bison (Bison bison) and 270 reindeer (Rangifer tarandus tarandus) in Canada. Based on the frequency distribution of the C-ELISA values, we propose that the current negative cut-off value of 50% inhibition established for bovine field sera also can be applied to the sera from these wild ruminants. The C-ELISA values for other wild ruminant field sera collected in bluetongue-free areas of Canada from 98 native caribou (Rangifer tarandus caribou), 32 white-tailed deer (Odocoileus virginianus), 14 moose (Alces alces), and nine musk-oxen (Ovibos maschatus) and 15 yak (Bos grunniens) also were less than 50%, with the exception of three caribou samples. Based on our results, we propose that the C-ELISA be used as a rapid and specific test for serodiagnosis of BTV infection in llamas and possibly other wild ruminants.

Suitability of autoclaved tap water for preparation of ELISA reagents and washing buffer.

The suitability of autoclaved tap water for the preparation of ELISA reagents and washing buffer was compared with that of ultrapure water, in a standard indirect ELISA for the detection of antibodies to pseudorabies virus (PRV). The performance of the assay, using autoclaved tap water (AT-ELISA) compared favourably to that of the standard assay, using ultrapure water (UP-ELISA) in detecting anti-PRV antibodies in sequential serum samples from a pig experimentally infected with PRV. While both the UP-ELISA and AT-ELISA proved reliable in detecting anti-PRV antibodies in a coded proficiency serum panel (n = 60), the AT-ELISA detected fewer positive sera than the UP-ELISA in evaluating a limited number (n = 80) of field samples. The results suggest that autoclaved tap water may be substituted for ultrapure water for the preparation of ELISA reagents when or where ultrapure water may not be available.

Evaluation of a commercial competitive ELISA test kit for the detection of group-specific antibodies to bluetongue virus.

The performance of a competitive (c) enzyme-linked immunosorbent assay (ELISA) test kit, Blueplate Special (BPS), commercially produced by DiagXotics (Wilton, CT) for detection of group-specific antibodies to bluetongue virus (BTV) was compared with that of an internationally endorsed cELISA-I. A total of 1,026 serum samples were tested in this study: 133 samples from 23 calves and 3 sheep experimentally infected with South African isolates of 19 BTV serotypes and US isolates of 5 BTV serotypes and 7 calves infected with US isolates of 2 epizootic haemorrhagic disease of deer virus (EHDV); 102 paired sera from cattle, sheep, and goats experimentally infected with the Australian isolates of BTV, EHDV, and Palyam virus; 229 bovine and ovine samples of Canadian origin (BTV free); and 562 bovine and ovine field samples from the USA and Barbados (BTV endemic). Seroconversion was demonstrable by the BPS cELISA 10 days postinfection in all experimental animals inoculated with BTV, with the exception of 4 calves in which there was a delay of 10-20 days. Similar to the cELISA-I, none of the sera from calves inoculated with US and Australian isolates of EHDV and Palyam viruses cross-reacted with the BTV antigen in the BPS cELISA. The total agreement between the two assays for all the total bovine and ovine field sera was 98.1%. The overall results substantiate the usefulness of the BPS cELISA test kit for monitoring animal sera for group-specific antibodies to BTV.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive ELISA for serodiagnosis of bluetongue: evaluation of group-specific monoclonal antibodies and expressed VP7 antigen.

The performance of 2 competitive enzyme-linked immunosorbent assays (C-ELISA) was compared with the reference C-ELISA I for the detection of antibodies to bluetongue virus (BTV). One of the assays (C-ELISA II) used a group-specific monoclonal antibody (MAb) to BTV, obtained from the American Type Culture Collection (8A3B-6) and tissue culture (TC)-derived BTV antigen (Ag), and the other assay (C-ELISA III) used BTV core protein VP7 (expressed in yeast) and the reference MAb (Pirbright Laboratory, 3-17-A3). Test sera were obtained by sequential blood samples from 22 calves, each inoculated with a different serotype (T) of BTV (South African [SA] T-1-T-16 and T-18-T-20 and USA T-11, T-13, and T-17). Sera were also obtained from 4 calves and 4 sheep inoculated with USA BTV T-10 and from several groups of calves exposed to single or multiple doses of epizootic hemorrhagic disease virus (EHDV) T-1-T-4 grown in TC (BHK-21) or suckling mouse brain (SMB). A total of 618 bovine and ovine field sera collected from BT-free and BT-endemic areas were also tested. The C-ELISA III was more sensitive than the C-ELISA II in the detection of anti-BTV antibody in sera from cattle and sheep early after infection with BTV. Seroconversion was demonstrated by the 3 C-ELISAs in all animals inoculated with BTV by 20 days postinfection (DPI), except in calves that received SA T-3 or USA T-13, which became positive at 40 DPI.(ABSTRACT TRUNCATED AT 250 WORDS)