Genotyping of Toxoplasma gondii and Sarcocystis spp . in road-killed wild mammals from the Central Western Region of the State of São Paulo , Brazil

Introduction: Road-killed wild animals host zoonotic pathogens such as Toxoplasma gondii, offering a new opportunity for the epidemiological study of these infectious organisms. Methods: This investigation aimed to determine the presence of T. gondii and other apicomplexan parasites in tissue samples of 64 road-killed wild animals, using polymerase chain reaction (PCR). Positive samples were then typed by PCR-restriction fragment length polymorphism (RFLP) using 7 markers: SAG1, 5′-3′SAG2, SAG3, BTUB, c29-6, PK1, and Apico. PCR-RFLP targeting 18S ribosomal RNA (rRNA) genes was also performed on all samples to detect other apicomplexan parasites. Results: T. gondii DNA was detected in 16 tissue samples from 8 individual animals, as follows: 1 Cerdocyon thous (crab-eating fox), 1 Didelphis albiventris (white-eared opossum), 1 Lutreolina crassicaudata (lutrine opossum), 2 Myrmecophaga tridactyla (giant anteater), 1 Procyon cancrivorus (crab-eating raccoon), and 2 Sphiggurus spinosus (Paraguay hairy dwarf porcupine). Seven different T. gondii genotypes were identified, 6 of which were novel. Typing by 18S rRNA verified these 16 T. gondii-infected samples, and identified 1 Sarcocystis spp.-infected animal [Dasypus novemcinctus (nine-banded armadillo)]. The amplified T. gondii (GenBank accession No. L37415.1) and Sarcocystis spp. 18S rRNA products were confirmed by sequencing. Conclusions: Our results indicate that T. gondii is commonly present in wild mammals, which act as sources of infection for humans and animals, including other wild species. The approach employed herein proved useful for detecting T. gondii and Sarcocystis spp. in the environment and identifying their natural reservoirs, contributing to our understanding of host-parasite interactions.


INTRODUCTION
Several pathogens derive from wild animals, the study of which is becoming increasingly restrictive, especially when euthanasia is required.Thus, road-killed wild mammals offer an alternative source of such animals for research involving molecular detection of parasites.Although microbiological culture and histopathological analysis using tissue samples from road-killed wild animals are challenging, the identification and typing of pathogens can be achieved through molecular methods (1) .
Apicomplexan parasites, principally Toxoplasma gondii, are very common among domestic and wild animals.T. gondii is an obligate intracellular protozoan parasite, prevalent in animals worldwide, and commonly infecting humans.Infection can occur by transplacental transmission, oral ingestion of contaminated soil, raw vegetables, fruits, or water containing sporulated oocysts shed by definitive hosts in their feces, or by ingestion of tissue cysts in raw or undercooked meat or viscera of intermediate hosts (2) (3) .This parasite exhibits a highly complex clonal genetic population structure that has been extensively studied in recent years (4) .Several regions of the T. gondii genome have been used for the identification of this organism.The 529-base pair (bp) repetitive sequence, which repeats 200-300 times per genome, provides high sensitivity and specificity, representing an important target for identification (5) (6) .Toxoplasma gondii comprises several clonal lineages whose pathogeneses in humans and animals may differ in progression and severity (7) .
Thus, research concerning the identification of T. gondii by molecular techniques in novel hosts is crucial to clarify its interactions with hosts and molecular epidemiology, and may also provide a good indicator of environmental contamination.Wild animals act as reservoirs of T. gondii infection affecting humans and food animals, necessitating the adoption of epidemiological and sanitary control measures.With this in mind, the present study aimed to identify new hosts of T. gondii and other apicomplexan parasites in tissue samples of roadkilled wild mammals using molecular techniques.In addition, parasite genotypes in circulation were determined.

Sampling
All animals were transported at 4°C to the FMVZ.Lung, spleen, liver, kidney, heart, and mesenteric lymph node samples were collected from each animal, finely chopped, and stored at −80°C in 1.5-mL centrifuge tubes containing sterilized ultrapure water (Life Technologies, Carlsbad, CA, USA), until needed for Deoxyribonucleic acid (DNA) extraction.
Toxoplasma gondii typing was performed using seven genetic markers (SAG1, 5′-3′SAG2, SAG3, BTUB, c29-6, PK1, and Apico), as previously described (8) (9) (10) (11) .Reference strains (GT1, PTG, CTG, TgCgCa1, MAS, and TgCatBr5) were used as reaction controls.The multiplex PCR products were used in nested PCRs specific to each marker, and restriction fragment length polymorphism (RFLP) was then applied to obtain a profile for each sample.All products were visualized by electrophoresis on a 2.5 or 3% agarose gel (depending on the marker under examination) stained with SYBR Safe DNA gel stain, and imaged using a digital gel documentation system as above.
The identification of apicomplexan parasites other than T. gondii in the studied samples was carried out by molecular methods developed to target the T. gondii 18S ribosomal ribonucleic acid (rRNA) gene, as described by Da Silva et al. (12) .A nested PCR was performed using 25µM external primers Tg18s48F (5′-CCATGCATGTCTAAGTATAAGC-3′) and Tg18s359R (5′-GTTACCCGTCACTGCCAC-3′), and 50µM internal primers Tg18s58F (5′-CTAAGTATAAGCTTTTATACGGC-3′) and Tg18s348R (5′-TGCCACGGTAGTCCAATAC-3′) (Integrated DNA Technologies), expected to amplify products of 290bp for Neospora caninum, Hammondia hammondi, and T. gondii, and ~310bp for Sarcocystis spp.(except Sarcocystis neurona).All nested PCR products were confirmed by RFLP (12) and sequencing.Reactions were carried out on a MasterCycler ep Gradient instrument, and 1.5% agarose gel electrophoresis was used to gauge the quantity and quality of the resulting products.Sequencing was carried out at the facilities of the UNESP Biosciences Institute.

RESULTS
Table 1 contains the taxonomy, sex, and geographic location of the road-killed wild animals for which positive PCR results for T. gondii and Sarcocystis spp.DNA were obtained, and Table 2 details the corresponding tissue samples and parasite identities.Genotyping results are presented in Table 3 (8) (10) (13) (14) (15) (16) (17) .

DISCUSSION
Utilizing road-killed wild animals for molecular detection of T. gondii represents a feasible and efficient alternative to the use of live animals in research, as indicated by animal research ethics committees.Notably, most studies having used road-killed wild animals have identified a large number of mammalian species.In addition, sensitive and specific molecular tools enable pathogen identification without the need for laborious microbiological cultures and histopathological examination.
In this paper, molecular detection of T. gondii in several wild species was attempted using PCR.A number of studies have reported the presence of this parasite in wild rodents and members of Carnivora, Didelphimorphia, and Xenarthra (18) (19) (20) .These findings confirm the worldwide distribution of T. gondii, and highlight the wide variety of intermediate hosts that form part of the epidemiological chain responsible for transmission of this infection and the associated disease.
Here, 22 specimens were from members of the order Carnivora, with C. thous predominating.These animals can be found in several environments, from Cerrado savanna to the Atlantic Forest (21) .Their abundance may be due to their generalist and, of preference, nocturnal feeding habits, moving through tracks at forest edges and surviving in degraded and anthropic areas (22) .They are frequently seen on roadsides searching for food, which may include other road-killed animals, meaning that, as a carnivorous species, C. thous has a high road-kill rate (23) .T. gondii DNA was detected in samples from 1 C. thous and 1 P. cancrivorus.In the literature, similar results have been obtained using molecular assays (20) (24) .
Toxoplasma gondii DNA was not detected in animals of the orders Artiodactyla (2 M. gouazoubira), Lagomorpha (3 L. europaeus), and Primates (1 C. apella and 1 C. penicillata), but these groups are nevertheless important in the epidemiology of this parasite, since several reports of T. gondii infection in cervids, lagomorphs, and primates have been published (25) .
Toxoplasma gondii DNA was detected in 2/13 (15.4%) specimens of the order Rodentia.The positive S. spinosus samples emphasize the importance of this species as a carrier of T. gondii and several other pathogens with zoonotic potential (26) .Although the number of infected animals of this order was small, further assessment of this group is needed, since Truppel et al. (27) and Yai et al. (28) successfully isolated this parasite from capybaras (H.hydrochaeris), detecting its presence by serology.
Members of Didelphidae, represented here by D. albiventris and L. crassicaudata (10 specimens), are generalists and inhabit areas close to human dwellings, including farms, backyards, and urban centers (29) .Due to the destruction of their habitat, L. crassicaudata seeks shelter and food in urban areas (28) .This group is considered a reservoir of several potentially zoonotic organisms (30) .In our study, T. gondii DNA was detected in 1 L. crassicaudata and 1 D. albiventris.
Of the 12 animals belonging to the superorder Xenarthra, 3 (25%; 95%CI: 9.1-53.8%)gave positive PCR results, 1 for Sarcocystis spp.This reinforces the importance of this taxon in the epidemiology of T. gondii infection.The fact that this parasite was not detected in E. sexcinctus may reflect the differences between this animal's feeding habits and habitat and those of the other species examined.E. sexcinctus feeds on carrion found on the ground, and constructs its burrows in drier environments and open fields (21) .
Toxoplasma gondii samples from 8 animals were genotyped, 7 of which yielded previously unreported marker combinations (Table 3), and 1 of which demonstrated a profile similar to that already reported in RFLP studies performed by Dardé (13) , Su et al. (8) , Sousa et al. (15) , Dubey et al. (10) (16) , and Velmurugan et al. (17) .Two of these genotypes, TgCatBr38 and TgCatBr44, were identified in cats from Araçatuba and Conchas, both in the State of São Paulo (14) .In contrast to Pena et al. (14) , in this study, typing data was obtained from only 7 of the markers tested.It is likely that the remainder were negative due to low parasite loads.Having complete typing data for all 11 markers would certainly provide a more accurate picture of the present study sample.However, the 7 unique results obtained emphasize the importance of wild animals and the utility of road-killed specimens to the study of pathogens causing infectious diseases.The distinctiveness of these genotypes demonstrates that T. gondii is constantly adapting to its environment, as observed by Su et al. (8) , Pena et al. (14) , and Da Silva et al. (4) , with mutations and adaptive changes in clonal populations.Most of the animals identified in this work become infected through different routes.Therefore, further study of these species may provide valuable epidemiological information, supplying answers to the many questions concerning the adaptation and transmission of T. gondii to new hosts, its resistance, and the development of future vaccines.
Thus, road-killed wild animals may serve as an important T. gondii reservoir, contributing to its transmission to domestic and wild animals, as well as humans.

TABLE 1
Taxonomy, sex, and geographic location of PCR-positive road-killed wild animals.

TABLE 3
Genotypic profiles of Toxoplasma gondii identified in tissue samples from road-killed animals.