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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.1 Infection 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.3 The 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.4
Many 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.5 Moreover, T. gondii has a high level of genetic variation in these animals6. Recognizing the distribution of T. gondii in wild animals is essential to understand the transmission cycle of this parasite.5 Circulation of T. gondii in wild ecosystems is a result of environmental contamination with oocysts disseminated by wild or domestic felids (final hosts).6
In 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.7
The 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.
Material and methods
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”.8
All 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 2000TM, Thermo Scientific®, 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).
For the first reaction, the primers described by Sroka et al (2009) were used: Pml/S1 (5’-TGTTCTGTCCTATCGCAACG-3 ‘) and Pml/AS1 (5’-ACGGATGCAGTTCCTTTCTG-3’). For the second reaction, the following primers were used: Pml/S2 (5’-TCTTCCCAGACGTGGATTTC-3’) and Pml/AS2 (5’-CTCGACAATACGCTGCTTGA-3’). Both reactions amplified a 560 base pair (bp) segment. Reagents used in both reactions had the following concentrations in a final volume of 25µL: 5X PCR Buffer, 25mM MgCl2, 1mM dNTP´s, 10µM of each primer, 5U Taq polymerase (Thermo Scientific® Inc.; Waltham, Massachusetts, United States), 3ml template DNA, and distilled water. The thermocycler conditions were: initial denaturation at 95 °C for three minutes, followed by 35 cycles at 95 °C for 30 seconds, 64.2 °C for 30 seconds, and 45 °C for 45 seconds; the final extension was 72° C for five minutes.
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 ZymocleanTM Gel DNA Recovery kit (Zymo Research, The Epigenetics CompanyTM, 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 National Institute of Health (NIH, United States; http://blast.ncbi.nlm.nih.gov/Blast.cgi), to determine identity similarity and coverage percentages.9-12
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.
Figure 1 Agarose gel presenting PCR amplicons (560 bp) positive to Toxoplasma gondii. 1) C+: positive control; A: Yucatan squirrel liver sample; B: great grison femoral muscle sample; C: coatimundi kidney sample; C-: negative control. 2) A: Yucatan squirrel brain sample; B: great grison lung sample; C-: negative control.
Table 1 Species identification and food habits, sampled tissues, PCR results and recovery site coordinates of roadkill wild animals from Yucatan, Mexico
| Species common name | Scientific name | Feeding habits | Sampled tissue | T.gondii PCR results | Recover y site coordinates |
| Yucatan squirrel | Sciurus yucatanensis | Frugivore, insectivorous |
Brain Lung Liver Kidney Spleen Femoral muscle Heart Masseter muscle |
+ + + + + - - - |
21°05’24.5”N, 88°30’56.0”W |
| Cabeza de viejo | Eira barbara | Omnivorous |
Brain Tongue Liver Kidney Spleen Femoral muscle Heart Masseter muscle |
- - - - - - - - |
21°11’04.5”N, 88°33’07.1”W |
| Coatimundi | Nasua narica | Omnivorous |
Brain Tongue Liver Kidney Spleen Femoral muscle Heart Masseter muscle |
- - - + - - - - |
21°11’33.1”N, 88°47’17.9”W |
| Greater grison | Galictis vittata | Omnivorous |
Brain Lung Liver Kidney Spleen Femoral muscle Heart Masseter muscle |
- + - - - + - - |
21°10’44.0”N, 88°29’33.4”W |
+: Positive
-: Negative
N: North
W: West
Identity and coverage percentages obtained in the BLAST analysis are shown in Table 2. All sequences were homologous to a previously sequenced T. gondii.
| Purified product name | Identity (%) | Coverage (%) | GenBank acc ession of the homologous sequence |
| T. gondii coatimundi (kidney) | 99 | 98 | AF179871.1 |
| T. gondii great grison (femoral muscle) | 100 | 99 | AF179871.1 |
| T. gondii great grison (lung) | 100 | 98 | AF179871.1 |
| T. gondii Yucatecan squirrel (liver) | 99 | 99 | AF179871.1 |
| T. gondii Yucatecan squirrel (brain) | 100 | 100 | AF179871.1 |
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.13
In Yucatan, Mexico, T. gondii is widespread. There are frequent reports of infection in domestic animals such as cats14 and pigs,15,16 as well as in wild animals such as opossums17 and synanthropic rodents,18 and also in humans.19-22 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.7
Intermediate 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.24
There 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 Ceballos28 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).29 Oocysts shed in cat faeces may contaminate nuts, fungi, plant shoots, or berries, all of which are part of the diet of squirrels.26 Omnivorous squirrels could also get infected through ingestion of contaminated animal tissues.27
The 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 al13 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.13
Rendón-Franco et al have described a T. gondii infection in a coatimundi from Mexico though a serological study.32 The 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.33 This 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.13 Further 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.13 Finally, 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.
