Abstract
Toxoplasmosis is an important parasitic zoonosis worldwide. Many human and animal surveys use serological assays based on Toxoplasma gondii antibody detection in serum, a matrix that is not routinely available from wildlife. Commonly used serological assays have rarely been validated for use with fluids other than serum, nor validated for their performance in wildlife species. New molecular assays, such as magnetic capture DNA extraction and real-time PCR (MC-qPCR), offer high sensitivity for detection of T. gondii DNA in tissues. The aims of this study were to (1) assess prevalence of T. gondii DNA based on MC-qPCR detection in brain and heart of naturally infected wolverines (Gulo gulo) from the Yukon, Canada (2) compare two matrices [heart fluid (collected from thawed heart) and filter eluate (eluted from blood soaked filter paper)] for antibody detection in the same species, and (3) evaluate the performance of three serological tests [modified agglutination test (MAT), enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT)] to detect naturally infected wolverines as determined by MC-qPCR. DNA of T. gondii was detected in heart and/or brain in 16 of 68 wolverines (24%, 95% CI: 15.0–34.8). Tissue prevalence and infection intensity was higher in heart [16 positives, mediantachyzoites equivalents per gram (TEG) =1221] compared to brain (10 positives, median TEG = 347). Heart fluid (HF) and filter eluates (FE) performed equally well in ELISA and IFAT in terms of relative sensitivity, but HF performed better with MAT. ELISA and IFAT had higher relative sensitivity (94%) and relative specificity (100%) compared to MAT (relative sensitivity 75% and relative specificity 92%). Overall, our findings indicate that the parasite burden in naturally infected wolverines was higher in heart compared to brain, heart fluid performed better than filter paper eluate for serological testing using MAT, and both IFAT and ELISA had higher relative sensitivity, relative specificity, and accuracy compared to MAT.
Highlights
Toxoplasma gondii infects almost all warm-blooded animals including humans, mammals and birds; one third of the global human population shows evidence of exposure to T. gondii
We observed no discordance in qualitative and quantitative results in group-3 (HP) using the modified agglutination test (MAT), but in group- 2 (LP) the MAT titer for Heart fluid (HF) was higher than for filter eluates (FE), and one magnetic capture- qPCR (MC-qPCR) positive wolverine (ID-33) showed a negative result with FE, indicating that HF may be a better specimen for the MAT and that a cut off value of 1:25, as recommended by the manufacturer, can be used for HF
HF and FE can be used at the recommended dilution of 1:2 for enzyme linked immunosorbent assay (ELISA), and HF is a better matrix than FE for detecting anti-T. gondii antibodies
Summary
Toxoplasma gondii infects almost all warm-blooded animals including humans, mammals and birds; one third of the global human population shows evidence of exposure to T. gondii Routes of transmission to people are (1) consumption of food or water contaminated with oocysts shed by felids, the only final hosts, (2) consuming raw/undercooked meat of infected animals containing bradyzoites in tissue cysts, and (3) via transmission of tachyzoites vertically from mother to fetus and (4) horizontally during blood transfusion and organ transplant (Dubey, 2010; Hide, 2016; Hill and Dubey, 2002; Robert-Gangneux and Darde, 2012; Ryning et al, 1979; Tenter et al, 2000). Wolverines (Gulo gulo), fur-bearing mesocarnivores, have the potential to act as a sentinel animal host species, and a previous study showed that 42% of the wolverines (n = 41) were seropositive to T. gondii in Nunavut, Canada based on the modified agglutination test (MAT) (Reichard et al, 2008)
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