For samples collected from a variety of naturally

For samples collected from a variety of naturally infected animals, test results for serum and/or whole blood samples obtained from two domestic (cat and sheep) and 20 wildlife species and tested by in-house MAT (1:50 dilution) and commercial MAT kit (1:40 dilution) showed comparable results (Table 1). Samples obtained from domestic cats showed a relatively high rate of positive results by both in-house MAT and commercial MAT (38.05% and 36.06%, respectively) with excellent agreement between the two tests (k=0.841). All samples from sheep tested negative by both tests, indicating no exposure to T. gondii. A total of 668 serum and/or whole blood samples collected from 20 diverse species of wildlife animals in the northern and arctic regions of Canada were tested. Results of all wildlife species, except wolf, indicated substantial to excellent agreement between the two test methods.
Discussion In the present study, an in-house MAT using tachyzoites antigen of T. gondii cultivated in tissue culture instead of the peritoneal cavity of mice was developed and its performance compared with that of a commercial MAT kit (Toxo-Screen DA, Biomerieux, France) as the gold standard. The commercial MAT test kit is widely accepted as a reliable test for screening animals for Toxoplasma infection regardless of host species. Commercial MAT kits for T. gondii are only intermittently available and there is difficulty in obtaining the antigen to perform the test which is usually prepared in the peritoneal cavity of mice. Whole tachyzoites grown in cell culture are available commercially (Kerafast-USA), but there are no published data on the validity of these cultured derived tachyzoites. Currently, we are aware of only a single commercial supplier of MAT kit (Toxoplasma Microwell Kit-New Life Diagnostics-USA). The in-house MAT using cultured tachyzoites was shown to be effective and specific in detecting anti-Toxoplasma IgG order Tacrolimus in serum, blood, and meat juice samples from experimentally and naturally infected animals. Also, culture conditions were improved using cell culture multi-flasks instead of conventional cell culture flasks to scale-up tachyzoites productivity and yield. Under these culture conditions, the yield of harvested tachyzoites increased approximately 10 fold from 1.6×109/ml (using conventional cell culture flasks) to 1.2×1010/ml when multi-flasks were used. Therefore, a significant increase in tachyzoite production was achieved with improvement in cell culture conditions for large batch reagents. The modified agglutination test (MAT) developed by Fulton and Turk (1959) and improved by others (Desmonts and Remington, 1980; Dubey and Desmonts, 1987; and Dubey, 2010) was designed for screening serum and blood samples from multiple host species for T. gondii infection. The present in-house MAT was adapted from the direct agglutination test described by Dubey (2010) with modifications involving changes in the preparation of antigen and incubation period of test plate. The T. gondii antigen used in the in-house MAT was prepared from tachyzoites harvested from MDBK cells grown in tissue culture. To preserve the parasite integrity and reduce cell culture debris, the tachyzoites were drawn through a sephadex column instead of using needles and millipore filters to break up and separate intact cells. Dubey (2010) suggested using 2×104/μl as the final concentration of tachyzoites for the MAT, whereas in the in-house MAT, the antigen concentration was optimized to 3×104/μl to detect positive or negative reactions. The negative results (blue button) were not clearly detected if the antigen concentration was too low, and appear to be inconclusive rather than true negative when the antigen concentration was too high. The incubation temperature affects the pattern of agglutination and sensitivity of the plate. Results from the in-house MAT could be read after incubating the test plate at room temperature (23±5°C) overnight, which facilitated the performance of the test without the requirement of incubation equipment. However, previous studies recommended overnight incubation at 32°C or 37°C, respectively Desmonts and Remington (1980) and Dubey (2010). Adjustment of test conditions was required to optimize test performance when using in vitro cultured antigen. Similar observations were reported when using Neospora caninum tachyzoites harvested from Vero cells grown in tissue culture to develop a MAT for detecting infection in various animal species (Packham et al., 1998).