West Greenland harbour porpoises assayed for antibodies against Toxoplasma gondii: false positives with the direct agglutination method

Abstract

We assayed blood/tissue fluid samples from 20 harbour porpoises Phocoena phocoena from western Greenland coastal waters for antibodies against the protozoan parasite Toxoplasma gondii by the direct agglutination test (DAT). Nine individuals (45%) were interpreted to be seropositive at 1:40 dilution and 4 (20%) were seropositive up to 1:160. Samples from these individuals were assayed by an enzyme-linked immunosorbent assay (ELISA), and tissue samples of the DAT-positive animals were tested by a nested polymerase chain reaction (nPCR). Results from both methods were negative, suggesting the absence of infection in the tested animals. After chloroform clean-up, all were negative when re-assayed by DAT. We concluded that infection with T. gondii was absent in all 20 animals, despite the initially positive DAT results, and that the false positives resulted from non-specific adherence to tachyzoites in the DAT assay which could be removed by the chloroform clean-up method. Our results suggest that detecting antibodies against T. gondii using the DAT or the modified agglutination technique, particularly on samples from Arctic marine animals which often are rich in lipids, may lead to false positive results. For such samples, the use of ELISA or PCR on available tissue samples may be advocated as confirmatory tests in order to avoid false positives and overestimating seroprevalence.

Toxoplasma gondii in stranded marine mammals from the North Sea and Eastern Atlantic Ocean: Findings and diagnostic difficulties

“…In the current study, no chloroform clean-up was applied on the serum samples. In accordance to the findings of Blanchet et al (2014), the results obtained by IFA and ELISA indeed gave a lower number of positive animals compared to the MAT results. MAT might be useful as a first screening method, but the results must be interpreted with caution.…”

“…This could be due to difficulties in determining serological parameters such as sensitivity and specificity. Blanchet et al (2014) tested tissue fluid and blood (thawed and centrifuged) with MAT and ELISA. In the current study, no chloroform clean-up was applied on the serum samples.…”

“…This is particularly useful when testing wildlife, such as marine mammals (Cabezón et al, 2011;Santos et al, 2011;Jensen et al, 2012). Recently, however, Blanchet et al (2014) questioned the reliability of MAT in the serological diagnosis of T. gondii infection in harbour porpoises (Phocoena phocoena). This was based on a comparison of MAT on untreated serum samples with enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which suggested that MAT may give false positive results.…”

“…Adherence of components to tachyzoites other than specific antibodies, such as samples rich in particulate matter and lipids, may be responsible for these false positive reactions. A chloroform 'clean-up' of serum samples may prevent this problem (Blanchet et al, 2014). With the exception of harbour porpoises (Phocoena phocoena), however, this method has not been evaluated for other marine mammal species.…”

“…As the reliability of MAT in marine mammal samples has been questioned (Blanchet et al, 2014), it was decided to perform additional testing to evaluate and compare various serological detection methods.…”