Toxoplasma gondii (Nicolle & Manceaux, 1908)

Fancourt, Bronwyn A., Nicol, Stewart C., Hawkins, Clare E., Jones, Menna E. & Johnson, Chris N., 2014, Beyond the disease: Is Toxoplasma gondii infection causing population declines in the eastern quoll (Dasyurus viverrinus)?, International Journal for Parasitology: Parasites and Wildlife 3 (2), pp. 102-112 : 105

2.4. Testing for T. gondii IgG antibodies

Serum samples were defrosted and tested for the presence of T. gondii -specific IgG antibodies using a commercial modified agglutination test (MAT) (Toxo-Screen DA, bioMérieux, Marcy-l’Etoile, France). IgG antibodies are usually detectable within 2 weeks of initial infection and remain detectable for the life of the host ( Remington et al., 2004; Dubey, 2010). Accordingly, MAT-derived titres are not indicative of recency of infection or clinical status ( Dubey, 2010) but rather an exposure to the parasite at some time at least 2 weeks before sampling. Of the agglutination tests that do not require species-specific reagents, MAT is considered to be the most sensitive for detecting T. gondii specific-IgG antibodies in marsupials ( Munday, 1972; Dubey, 2010). Haemolysis does not interfere with the test, so it can be used with serum, blood plasma or even whole blood ( Dubey, 2010).

Samples were treated with 2-mercaptoethanol to denature any IgM antibodies and suppress any non-specific agglutination ( Desmonts and Remington, 1980; Dubey and Desmonts, 1987). Each sample was tested at serial fourfold dilutions of 1/16, 1/64 and 1/256 together with positive and negative controls supplied in the MAT kit. A positive reaction was observed when agglutination of toxoplasma formed a mat covering about half of the well base. Titres were expressed as the inverse of the highest dilution at which a positive reaction was observed. A titre of À 64 was used for determining a sample as positive for T. gondii infection ( Dubey and Desmonts, 1987).

To validate the results obtained using these protocols, a subsample of sera underwent retesting by the Tasmanian government Animal Health Laboratories. Where longitudinal samples were collected from individual quolls over multiple sampling periods, further validation was obtained by checking that seroconversion occurred only once in each quoll’s life, and that seroconversion occurred only in one direction (from seronegative to seropositive).

To validate the reliability of results using blood from frozen cats, 20 samples were collected from cats at the time of death in 2012, and matched to samples from the same cats after the body had been frozen for around 12 months.