Multi-scale occupancy approach to estimate Toxoplasma gondii prevalence and detection probability in tissues: an application and guide for field sampling
Graphical abstract
Introduction
Knowing what tissue is best for testing can make the difference between clinical detection and non-detection of a pathogen or disease process (McClintock et al., 2010). In most settings, where space, time and financial resources are limited, sampling and testing for disease or pathogen presence from all possible tissues is not feasible. Further, tissue selection during post-mortem analysis is not always straightforward because reports regarding parasite tissue predilection sites can be conflicting and detection in a specific tissue type might be sensitive to laboratory methodology, the species involved, or the stage of infection. Tissue coccidian parasites such as Toxoplasma gondii can be difficult to detect because their distribution might not be diffuse throughout an organ or muscle (Berenreiterová et al., 2011). More information on tissue predilection sites and detection probability of coccidian tissue stages is needed to improve diagnostics, especially in wildlife and food animal species.
Toxoplasma gondii is ubiquitous among vertebrates including humans (Dubey, 2009). Hosts can become infected by three different parasite life stages: oocysts, tissue cysts (containing bradyzoites) or tachyzoites, and transmission occurs through both direct and indirect routes. Felids are the only known definitive hosts of T. gondii; sexual reproduction of the parasite occurs in the intestinal epithelium and oocysts are then passed in faeces. A wide range of vertebrates can serve as intermediate hosts, developing tissue cysts in their organs and musculature (Dubey, 2009).
Previous studies in mammals provide comparisons of detection frequency in tissues, suggesting that T. gondii tissue cysts are consistently found in the brain and heart of infected animals (Juránková et al., 2014), although these sites are often the only ones tested (Feitosa et al., 2014, Krijger et al., 2014). Despite this assertion, other reports provide evidence of tissue tropism (the tissues of a host that support the growth and propagation of a pathogen), and findings also differ by host species, parasite genotype or inoculation route (Bangoura et al., 2013, Juránková et al., 2013, Juránková et al., 2014, Zöller et al., 2013). Toxoxplasma gondii can also be detected in less traditionally tested soft tissue such as tongue and skeletal muscle (Burrells et al., 2013, VanWormer et al., 2014). Occurrence of T. gondii in wild or domestic birds is not reported as often as in mammals, but numerous avian species worldwide are recognised as intermediate hosts and little is known about tissue tropism in these animals (Dubey, 2002).
Another difficulty surrounding tissue diagnostics for cyst-forming coccidian parasites (e.g., T. gondii) is the size of the organ in relation to the amount of tissue screened in the laboratory analysis. Because it is not usually feasible to test an entire organ, uncertainty exists when an infectious organism (e.g., T. gondii cyst) is not detected. While some DNA extraction methods effectively extract DNA from up to 1 g of tissue, most commercial kits allow testing of much less (e.g., 25 mg for the Qiagen (Canada) Blood and Tissue Kit, following kit instructions), possibly decreasing the chances that the tissue sample will contain the parasite; T. gondii might be unevenly distributed in an organ and processing less tissue might reduce the probability of detection (Berenreiterová et al., 2011).
Occupancy modelling approaches are traditionally used to estimate the probability of occurrence of a wildlife species within a habitat, and are especially useful for rare or cryptic species (MacKenzie et al., 2006). Occupancy approaches also allow for the estimation of the detection probability (denoted p), or, the probability that a species of interest will be detected, given that an area is occupied by that species. These methods are increasingly used in wildlife disease ecology because pathogens can be difficult to detect or are unevenly distributed among different organs within a host and throughout the tissue of infected organs. If so, imperfect detection can add to uncertainty in prevalence estimates (e.g., McClintock et al., 2010, Lachish et al., 2012, Eads et al., 2014, Elmore et al., 2014). To address this concern of imperfect detection, we used a multi-scale occupancy approach (Nichols et al., 2008) to determine whether goose organs vary in their likelihood of containing T. gondii, and to determine which infected organ(s) have the highest detection probability of T. gondii using molecular methods. To accomplish this, we used an experimental goose model from which detection probability results could be applied to a wildlife field question – which tissues should we be collecting and testing?
Our first objective was to determine the probability of detecting T. gondii DNA in infected tissues from experimentally inoculated domestic geese. We hypothesised that T. gondii would be most likely to occur in the brain of experimentally inoculated birds. We also expected that each organ would have a different probability of T. gondii occupancy and detection because previous research has demonstrated variable tissue predilection and detection of T. gondii among tissues of both mammals and birds (Bangoura et al., 2013, Juránková et al., 2013, Juránková et al., 2014, Juránková et al., 2015). We also predicted that the sex of the geese would not influence occupancy probability in the primary site (a goose), given the absence of this effect in a previous serological study (Elmore et al., 2014).
The results from the first objective helped to guide our second objective, which was to collect and test tissue samples from wild geese for T. gondii. We hypothesised that free-ranging geese from Karrak Lake, Nunavut, Canada would be infected with T. gondii, based on previous serological data that showed that Ross’s Geese (Chen rossii) and Lesser Snow Geese (Chen caerulescens) were exposed to the parasite at some point in their lives (Elmore et al., 2014). We also examined whether the primary site (goose) occupancy probability varied by species, due to weak evidence for a species effect in a previous study (Elmore et al., 2014). Identifying T. gondii in wild geese would support the idea that migratory birds transport the parasite from temperate regions to Arctic ecosystems and would suggest that wild geese are a potential source of T. gondii for wildlife predators such as Arctic foxes. The demonstration of parasite DNA in the soft tissues of a migratory prey species would support the hypothesis that ingestion of T. gondii by predators could perpetuate the parasite’s asexual life cycle in the Arctic region.
Section snippets
Experimental infection of domestic geese with T. gondii
We obtained domestic goose goslings (Anser anser domesticus) on the day of hatching from a local hatchery in Saskatoon, Saskatchewan, Canada. To avoid contamination with coccidian oocysts in the environment, goslings were kept from contact with the ground until they were released in a Biosecurity level 1 room in the Animal Care Unit at the University of Saskatchewan Western College of Veterinary Medicine, Saskatchewan, Canada. Goslings were offered water and chick starter feed (without
Experimental inoculation
Of the 25 experimentally inoculated domestic geese, 24 geese displayed positive serological results following inoculation with T. gondii. We included PCR-MCA results from all inoculated geese in the final data analysis including two geese that were seropositive but tissue-negative (i.e., all PCR-MCA tissue results were negative for T. gondii), and one goose that was both seronegative and negative in tissue PCR-MCA. The two negative control geese stayed both seronegative and tissue PCR-negative
Discussion
Our study suggests that domestic geese are a good experimental host for T. gondii. We observed very few detections of T. gondii in some organs (none from the spleen and few from the kidney), making it difficult to differentiate whether T. gondii occurs less often in those organs (lower value for θ), or whether it occurs but is very difficult to detect (as shown in Fig. 2). Regardless, our finding suggests that the parasite is most likely to occur and be detected in the brain or heart of
Acknowledgements
We thank the following: G. Samelius, R. Kerbes, J. Guy, N. Sanchez, W. Tan, K. Likos, B. Malloure and K. Anderson for help with sample collection in the field; B. Wagner, M. Pawlik, M. Bal and technicians in the Animal Care Unit at the University of Saskatchewan, Canada, for help with the experimental study; D. Kellett and Environment Canada for field logistics; D. Stern for hospitality in Cambridge Bay, Canada; L. Lalonde, B. Al-Adhami, J. Benjamin and the Centre for Food-Borne and Animal
References (50)
- et al.
A new multi-host species indirect ELISA using protein A/G conjugate for detection of anti-Toxoplasma gondii IgG antibodies with comparison to ELISA-IgG, agglutination assay, and Western blot
Vet. Parasitol.
(2014) - et al.
Experimental Toxoplasma gondii oocyst infections in turkeys (Meleagris gallopavo)
Vet. Parasitol.
(2013) - et al.
Toxoplasma gondii: inconsistent dissemination patterns following oral infections in mice
Exp. Parasitol.
(2007) - et al.
First identification of Sarcocystis tenella (Railliet, 1886) Moulé, 1886 (Protozoa: Apicomplexa) by PCR in naturally infected sheep from Brazil
Vet. Parasitol.
(2009) - et al.
Genotyping of Toxoplasma gondii isolates in meningoencephalitis affected striped dolphins (Stenella coeruleoalba) from Italy
Vet. Parasitol.
(2011) A review of toxoplasmosis in wild birds
Vet. Parasitol.
(2002)- et al.
Genetic characterization of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type
Int. J. Parasitol.
(2011) - et al.
High prevalence and genotypes of Toxoplasma gondii isolated from organic pigs in northern USA
Vet. Parasitol.
(2012) - et al.
Endoparasites in the feces of arctic foxes in a terrestrial ecosystem in Canada
Int. J. Parasitol. Parasites Wildl.
(2013) - et al.
Toxoplasma gondii in arctic-nesting geese: a multi-state occupancy framework and comparison of serological assays
Int. J. Parasitol. Parasites Wildl.
(2014)
Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis
Int. J. Parasitol.
Toxoplasma gondii and Neospora caninum in slaughtered pigs from Northeast Brazil
Vet. Parasitol.
Identification of a 200-to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii and its use for diagnostic and quantitative PCR
Int. J. Parasitol.
Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR
Vet. Parasitol.
Brain is the predilection site of Toxoplasma gondii in experimentally inoculated pigs as revealed by magnetic capture and real-time PCR
Food Microbiol.
Predilection sites for Toxoplasma gondii in sheep tissues revealed by magnetic capture and real-time PCR detection
Food Microbiol.
Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR
Int. J. Food Microbiol.
Serosurvey for Toxoplasma gondii in arctic foxes and possible sources of infection in the high Arctic of Svalbard
Vet. Parasitol.
Direct high-resolution genotyping of Toxoplasma gondii in arctic foxes (Vulpes lagopus) in the remote arctic Svalbard archipelago reveals widespread clonal Type II lineage
Vet. Parasitol.
Characterization of a repetitive DNA fragment in Hammondia hammondi and its utility for the specific differentiation of H. hammondi from Toxoplasma gondii by PCR
Mol. Cell. Probes
Impact of traditional practices on food safety: a case of acute toxoplasmosis related to the consumption of contaminated raw pork sausage in Italy
J. Food Prot.
Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife
Vet. Parasitol.
Isolation and genotyping of Toxoplasma gondii from domestic rabbits in China to reveal the prevalence of Type III strains
Vet. Parasitol.
Northern wetland ecosystems and their response to high densities of lesser snow geese and Ross’s geese in North America
Model-based inference in the life science: a primer on evidence
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