Foxes (Vulpes vulpes) as sentinels for parasitic zoonoses, Toxoplasma gondii and Trichinella nativa, in the northeastern Canadian Arctic

Abstract

Outbreaks of Toxoplasma gondii and Trichinella spp. have been recurring for decades among Inuit of Nunavik, northeastern Canada. Contact with wildlife has been identified as a risk factor for Inuit exposure to T. gondii, but reservoirs have yet to be confirmed based on direct detection of DNA or organism. Similarly, little is known about the occurrence of Trichinella spp. in wildlife species of Nunavik other than walrus (Odobenus rosmarus) and bears (Ursus americanus, Ursus maritimus). Foxes (Vulpes vulpes) were targeted as possible sentinels for T. gondii and Trichinella spp. because of their high trophic position within the Arctic food chain as carnivorous scavengers. A total of 39 red foxes were sampled from four communities in southern and western Nunavik between November 2015 and September 2016. For the first time in wildlife, a novel magnetic capture DNA extraction and real-time PCR technique was used to isolate and detect T. gondii DNA from the heart and brain of foxes. A double separatory funnel digestion method followed by multiplex PCR was used to recover and genotype larvae of Trichinella spp. from tongues of foxes. Seroprevalence based on detection of antibodies to T. gondii was 41% (95% CI: 27–57%) using a commercially available modified agglutination test (MAT). Detection of DNA of T. gondii and larvae of Trichinella nativa (T2) occurred in 44% (95% CI: 28–60%) and 36% (95% CI: 21–51%) of foxes, respectively. Coinfection with both T. nativa and T. gondii occurred among 23% (95%CI: 13–38%) of foxes which can be attributed to co-transmission from prey and scavenged species in their diet. There was only moderate agreement between T. gondii serology and direct detection of T. gondii DNA using the MC-PCR technique (Kappa test statistic: 0.321), suggesting that using both methods in tandem can increase the sensitivity of detection for this parasite. These findings show that foxes are good sentinels for circulation of parasitic zoonoses in terrestrial northern ecosystems since they are highly exposed, show measurable indicators of infection and do not serve as exposure sources for humans.

Serological and molecular detection of Toxoplasma gondii in terrestrial and marine wildlife harvested for food in Nunavik, Canada

“…Since blood from marine mammals contains lipids that may interfere with the performance of agglutination assays [36], we removed lipid from seal samples using a chloroform method, re-tested using MAT, and compared serological test results with the ID Screen® Toxoplasmosis Indirect Multiple-Species ELISA kit used as per manufacturer instructions (IDVet Innovative Diagnostics, Montpellier). To ensure that lipid removal did not interfere with subsequent analyses using MAT, heart fluid from naturally-infected foxes and sera from experimentally-infected reindeer were included as positive controls, and these remained positive following lipid removal [32, 37]. ELISA results were measured as optical density percentages (OD %) as per manufacture instructions, where an OD% greater than 50% is positive, between 40–50% is ambiguous, and less than 40% is negative.…”

“…Real-time PCR amplification was done in a Bio-Rad CFX 96 DNA thermal cycler (Biorad, Hercules, California, USA) based on a published protocol for the detection of the 188 bp Toxoplasma sequence within the 529 repeat-element with the forward primer TOX 9 (5′-agg aga gat atc agg act gta g-3′) and backward primer TOX 11 (5′-gcg tcg tct cgt cta gat cg-3′) as per [28] and [38]. The final PCR assay reaction included 6.5 µl (0.5 M) of Itaq Supermix, 0.25 µl (20 µM) of TP1 probe, 1.25 µl (10 µM) of Tox 9F, 1.25 µl (10 µM) of Tox 11R, 0.5 µl (2 femtograms) of a competitive internal amplification control (CIAC), 1 µl (5 µM) of CIAC probe, 6.75 µl of PCR-grade water and 8 µl of template DNA [32]. A positive PCR reaction was defined as any reaction with a Ct-value smaller or equal to 35, a control negative PCR with a Ct-value of zero, a negative extraction control with a Ct value of zero and a control positive extraction control with a Ct-value smaller or equal to 40 [31].…”

“…Determination of the minimum number of DNA copies and tachyzoites detected by the MC-PCR technique has been described elsewhere [32]. Ct-values resulting from the amplified DNA recovered from the spiked beef samples for determining the lowest detection limit were then used to estimate the equation that predicts the log 10 (concentration) by fitting a generalized linear model in R statistical software version 3.4.4.…”

“…In Europe, the MC-PCR technique has also been used as a screening tool in food production animals for research purposes [31]. Recently, it has been used successfully in naturally-infected foxes of Nunavik [32]. There is clearly a need, and now a good method, to determine whether T. gondii DNA is present in tissues of wildlife commonly consumed by Inuit of Nunavik, and to compare these results with serological findings based on a commonly used agglutination assay.…”

Veterinary informatics: forging the future between veterinary medicine, human medicine, and One Health initiatives—a joint paper by the Association for Veterinary Informatics (AVI) and the CTSA One Health Alliance (COHA)

“…Specifically, researchers evaluated pets as possible reservoirs of MRSA within the patient population. 164–166 Other diseases have also been examined (eg, Toxoplasmosis, Rocky Mountain Spotted Fever, and Hantavirus), 141 , 167 , 168 but many require additional research efforts to allow for extended detection and treatment in animals and humans. Recently, there has been an increased focus on carbapenem resistance and its transmission both between humans and humans and animals.…”