Molecular identification of Toxoplasma gondii in roadkill wild animals in Yucatan, Mexico

Toxoplasma gondii is an obligate intracellular protozoan parasite, recognized as the etiologic agent of toxoplasmosis, a zoonotic endemic disease in several countries, including Mexico. In the Yucatan State of Mexico, Toxoplasma infection has a high impact in both human and domestic animal health. Wild animals can also host zoonotic pathogens such as Toxoplasma gondii . The presence of Toxoplasma gondii DNA in roadkill wild animals in Yucatan was detected using a nested Polymerase Chain Reaction. Toxoplasma gondii DNA was identified in several organs retrieved from a Yucatan squirrel ( Sciurus yucatanensis ), a coatimundi ( Nasua narica ), and a greater grison ( Galictis vittata ). The amplified fragments of Toxoplasma gondii DNA were purified, sequenced, and certified by BLAST analysis. Our results confirm that Toxo-plasma gondii can infect wild mammals from Yucatan, which could act as intermediate hosts and contribute to the transmission of the disease to humans and domestic animals, as well as other wild animal species. We present the first molecular evidence of Toxoplasma gondii in a squirrel and a coati-mundi from Yucatan, and quite possibly in a greater grison at a global level.


Introduction
Toxoplasma gondii is an obligate intracellular protozoan parasite that infects a large variety of warm-blooded animal species, including humans.One-third of the human population has likely been exposed to this parasite.Moreover, Toxoplasma infection has been recognized in some countries as an important concern for animal and public health. 1Infection occurs mainly by accidental ingestion of oocysts present in the environment, the consumption of bradyzoites found in contaminated tissues or organs of intermediate hosts, or by transplacental transmission of tachyzoites (vertical infection). 2 The T. gondii infection (clinically named toxoplasmosis) affects several organs, especially the lungs, the central nervous system (CNS) and the eyes. 3The prevalence rate varies between countries (10%-80%) due to diverse climate conditions that impact oocyte viability.Human factors such as personal hygiene, sanitary conditions, feeding habits, drinking water quality and management practices in livestock production systems can also affect infection rates. 4any wild animal species are susceptible to be infected by T. gondii.In fact, several studies have identified high infection rates in zoo animals and wild birds. 5oreover, T. gondii has a high level of genetic variation in these animals 6 .Recognizing the distribution of T. gondii in wild animals is essential to understand the transmission cycle of this parasite. 5Circulation of T. gondii in wild ecosystems is a result of environmental contamination with oocysts disseminated by wild or domestic felids (final hosts). 6n Mexico, human toxoplasmosis has been detected in different regions of the country, particularly in tropical areas where the parasite remains infectious for long periods of time, due to the prevailing environmental conditions (i.e.optimal temperature and humidity).Several studies have also been performed in diverse mexican animal populations (wild and domestic), which have reported different infection rates. 7he aim of this study was to identify T. gondii DNA in tissue samples from roadkill wild animals in Yucatan, Mexico, to contribute with the epidemiologic understanding of toxoplasmosis in the region.We present the first molecular evidence of Toxoplasma gondii in a squirrel and a coatimundi from Yucatan, and quite possibly in a greater grison at a global level.

Animal sampling
Four roadkill wild animals were studied: a coatimundi, a greater grison, a Yucatan squirrel, and a tayra (Eira barbara).This last species is also known in the region as "cabeza de viejo".Species identification was performed by veterinarians, according to information found in the book "A Field Guide to the Mammals of Central America and Southeast Mexico". 8ll animals were collected from October to December 2016, at different points of the "Merida-Cenotillo" highway.Carcasses were inspected before retrieval to ascertain post-mortem changes and were later transported to the laboratory inside a plastic cooler with ice.Upon arrival, specimens were submitted to a second inspection, and a necropsy was performed.All animals seemed to have had a good body condition before death.Nematodes inside cardiac cavities and trachea were found only in the coatimundi carcass (data not shown).
Different tissues were collected during the necropsy (care was taken to only sample tissues that were inside body cavities and that did not contact the floor at any time, to avoid possible environmental cross-contamination).Samples were kept frozen at -70 °C in 1.5 mL centrifuge tubes containing 96% ethanol, until processed for total DNA extraction.

Total DNA extraction and Toxoplasma gondii molecular identification
All biological samples were washed with bidistilled water for five minutes to eliminate alcohol excess, before the total DNA extraction process.
For total DNA extraction a QIAamp DNA Mini Kit® was used (QIAGEN; Hilden, Germany).DNA purification from tissues followed manufacturer specifications.The DNA extraction process was performed inside a laminar flow hood (LABCONCO®; Kansas City, United States) to prevent contamination.Extracted DNA was quantified by spectrophotometry (NanoDrop 2000 TM , Thermo Sci-entific®, Wilmington, United States) and preserved at -70°C until used for PCR.
A fragment of T. gondii B1 gene was identified by nested polymerase chain reaction (nested PCR).
All reactions included positive (total DNA from a brain of a [BALB/c] mouse infected with T. gondii) and negative controls (sterile water).Electrophoresis was performed in 8% agarose gels stained with ethidium bromide.To visualize bands, a Bio-Rad® photodocumentation system was used (Bio-Rad®, California, United States).

Sequencing and alignment analyses
PCR positive amplicons were purified with the Zymoclean TM Gel DNA Recovery kit (Zymo Research, The Epigenetics Company TM , California, United States) and sent to a private laboratory for sequencing (DIMYGEN http:// http://www.dimygen.com/).
Obtained sequences were contrasted with data stored in the GenBank®, using the Basic Local Alignment Search Tool (BLAST), implemented by the

Results and discussion
Toxoplasma gondii DNA was detected in several tissues from a Yucatan squirrel, a coatimundi, and a great grison (Fig. 1).Table 1 summarizes wild animals studied, tissues sampled, and nested PCR results.
Identity and coverage percentages obtained in the BLAST analysis are shown in Table 2.All sequences were homologous to a previously sequenced T. gondii.
The use of wild animals for the molecular detection of T. gondii and other zoonotic pathogens in roadkill samples is an efficient alternative to live wild animal research.Moreover, molecular tools allow T. gondii identification with high sensitivity and specificity, without the need of arduous microbiological cultures and histopathological examinations. 13n Yucatan, Mexico, T. gondii is widespread.0][21][22] Additionally, T. gondii oocysts have been identified in drinking water sources, 23 which states the relevance of the toxoplasmosis in the region.However, T. gondii identification in other wildlife populations are very scarce. 7ntermediate hosts, such as domestic livestock (pigs, chickens, goats, and sheep) and wild animals (rodents, wild boars, foxes, and wild birds) get infected by ingesting sporulated T. gondii oocysts found in food or water, or by consuming infected tissue cysts. 24There are several reports of T. gondii infection in species of squirrels all around the world, with the infection attaining different organs and tissues.Indeed, Fayyad et al., 25 reported a fatal systemic T. gondii infection in a red squirrel (Sciurus vulgaris) and in a Swinhoe's striped squirrel (Tamiops swinhoei) from Germany.Also, Kik et al., 26 described that 20 of 37 red squirrels died of a disseminated T. gondii infection in the Netherlands, and Jokelainen and Nylund, 27 mentioned three cases of fatal toxoplasmosis in red squirrels from Finland.This stresses the importance of this species in the life cycle of toxoplasmosis.Conversely, Suzán and Ceballos 28 did not find serological evidence of T. gondii in two rock squirrels (Spermophilus variegatus) oror a gray squirrel (Sciurus aureogaster) in Mexico.However, this could be due to the small sample size (n = 3).
Mode of transmission of T. gondii in squirrels remains unclear, but it is known that the parasite's life cycle depends on infecting felids (domestic or wild). 29ocysts shed in cat faeces may contaminate nuts, fungi, plant shoots, or berries, all of which are part of the diet of squirrels. 26Omnivorous squirrels could also get infected through ingestion of contaminated animal tissues. 27he presence of T. gondii in Yucatan squirrels may indicate an important issue for human health nationwide since squirrel meat is consumed in various Mexican states; 30 especially in communities where subsistence hunting is common practice.However, further studies are needed to confirm this hypothesis.
Information about T. gondii in greater grison is very scarce worldwide.Khan et al., 31 described the isolation of a T. gondii strain from one individual in the French Guiana; and Richini-Pereira et al. 13 reported not to have found the infection in two animals from Brazil.It is thus possible that the results shown here are the first molecular evidence of T. gondii in this species.Evidence of T. gondii infection in carnivores other than the greater grison is extensive, so the mode of transmission for this species could be similar, i.e. by eating infected animals such as rodents and birds. 13endón-Franco et al. have described a T. gondii infection in a coatimundi from Mexico though a serological study. 32The identification of T. gondii infection in a coatimundi could have important zoonotic consequences, since this species is also hunted and consumed by several inhabitants of Mexico. 33This is the first molecular evidence of the presence of T. gondii in a coatimundi from Yucatan.
Our results contribute to ascertain that T. gondii has a worldwide distribution, and also underline that a broad diversity of intermediate hosts may take part in the epidemiological chain of toxoplasmosis. 13Further epidemiological studies are necessary to identify the T. gondii genotype(s) present in wild animals from Yucatan, Mexico. 7

Conclusions
Results of this work confirm that T. gondii is present in wild mammals in Yucatan.Also, the studied species could act as intermediate hosts, helping to spread the infection to humans and other animals. 13Finally, our study presents the first molecular evidence of Toxoplasma gondii in a squirrel and a coatimundi from Yucatan, and quite possibly in a greater grison at a global level.

Table 1 Table2Table 1 .
Species identification and food habits, sampled tissues, PCR results and recovery site coordinates of roadkill wild animals from Yucatan, Mexico

Table 2 .
Identity and coverage percentages obtained in BLAST analyses