Parasites in feces of the endemic rattlesnake, Crotalus triseriatus (Serpentes: Viperidae), from Mexican highlands

There are few studies about parasitic infections in Crotalus triseriatus (Wagler, 1830), an endemic rattlesnake from the highlands of central Mexico. This species occupies several habitats, from conserved forested regions to heavily impacted landscapes. To increase the parasitological knowledge of this reptile species without impacting populations, we obtained fecal samples of 16 rattlesnakes between 2012 and 2014 from Toluca Valley, Mexico. By using flotation technique, we found oocysts of Eimeria sp. and eggs of Capillariidae sp. The most frequent parasite was Eimeria sp. (62.5%). This study provides the first records of occurrence of parasites in reptilian feces from Mexico. Our results may be important for wildlife conservation purposes, for example, they could indicate of the population health of this species during processes of translocation.

Reptiles are hosts of a wide variety of protozoan and metazoan parasites (Wilson and Carpenter 1996). Most of these parasites have been recorded through necropsies of specimens, although the diagnosis could be achieved by revealing parasitic elements in feces (e.g., eggs, larvae, oocysts). Even though, the latter non-invasive method only allows detecting intestinal parasites, may be useful for conservation projects, which involve knowing the health status of a reptile population in wildlife (Jorge et al. 2013).
In Mexico, 864 reptile species have been recorded (i.e., 8.7% of the reptiles of the world), of which 57% are endemic (Flores-Villela and García-Vazquez 2014). However, the inventory of their parasites is far from complete, since only a few more than 200 Mexican reptile species have been recorded as hosts of helminths, acari and crustaceans (Paredes-León et al. 2008).
Mexico holds the highest species richness of rattlesnakes, with 42 species (Heimes 2016), but wild populations have declined due to direct hunting and habitat loss caused by the ex-pansion of croplands and urban areas. Particularly, the Mexican dusky rattlesnake, Crotalus triseriatus (Wagler, 1830), is restricted to the Trans-Mexican Volcanic Belt, where occupies forested regions heavily impacted (Bryson et al. 2014). This species is considered in the category of "least concern" in the IUCN and it has not received any level of protection by Mexican government due probably to a lack of knowledge in demographic and ecological aspects (Bryson et al. 2014, Sunny et al. 2015. Translocations have increased for wildlife conservation, including the herpetofauna. One concern associated with conservation translocations is that the released individuals may suffer from parasitic diseases resulting in establishment failure. However, infectious hazards may be mitigated when diseases and/or parasites of translocated species or species at the destination are known (Bobadilla Suarez et al. 2017). Unfortunately, for C. triseriatus little is known about its parasites (Paredes-León et al. 2008), even though this information is essential to know the population health of this species and potentially useful in future translocation processes of species from one population to another. Our objective was to investigate the occurrence of parasites in feces of C. triseriatus to increase parasitological knowledge without affecting wild populations.
This study was conducted in Toluca Valley located in the State of Mexico, a highly anthropized area and considered the fifth largest metropolis in Mexico, with 2.1 million inhabitants (SEDESOL, CONAPO, INEGI 2004). The region has a humid temperate climate, with an annual precipitation of 500-600 mm and a range of average annual temperature between 12 and 15 °C (García 2004, Gobierno del Estado de México 2012. The sampling sites were El Cerrillo located at 5.1 km north of metropolitan area of Toluca City (19°24'27"N; 99°41'40"W; 2615 masl) and the Sierra Morelos Park located on the edge of the metropolitan area of Toluca City (19°18'39"N; 99°41'33"W; 2630 masl).
To capture individuals of C. triseriatus and collect individual fecal samples, during 2012 to 2014 we made 24 monthly visits at each study site, where five observers simultaneously walked five linear transects of 800 m long and separated by 300 m in a schedule of 09:00 am-13:00 pm. We capture the rattlesnakes with herpetological hooks and once subjected, we recorded total length (snout-vent length) in centimeters and weight in grams only with descriptive purposes. After we obtained fecal samples of each specimen place them in Eppendorf tubes and we released the rattlesnakes at their capture sites.
Each fecal sample was conserved in 70% alcohol and examined in the laboratory using the flotation technique (Bowman et al. 2004). Approximately 0.5 g of each sample was macerated and mixed with 9.0 ml of 0.8% saline solution. Each homogenized sample was filtered and placed in a vial, which was centrifuged for three minutes at 1500 rpm. After centrifugation, the supernatant was discarded, and a saturated glucose solution was used to re-suspend the pellet. The suspension was centrifuged for five minutes at 1000 rpm and three subsamples of top part of the flotation were obtained and examined by light microscopy. Parasitic elements (i.e., eggs and oocysts) were counted in each subsample to obtain an average per sample and to determine the levels of infection according to Rodríguez-Vivas and Cob-Galera (2005). Eggs and oocysts were photographed and measured using a light microscope at 40 and 100X magnifications (Leica DFC 490) and identified in accordance with Mehlhorn et al. (1993) and Wolf et al. (2014).
Knowledge about the parasites of Mexican rattlesnakes is still far from complete. For example, in Crotalus spp. only 21 taxa of metazoan parasites (1 Acanthocephala, 3 Cestoda, 9 Nematoda, 3 Acari, and 5 Crustacea) have been recorded through necropsies of bred or wild specimens (Table 2). Of them, Ophiotaenia perspicua La Rue, 1911, Kalicephalus inermis coronellae (Ortlepp, 1923), Ixobioides sp., and Ophionyssus natricis (Gervais, 1844) were recorded in C. triseriatus. Thus, findings of Capillariidae sp. and Eimeria sp. represent new host records and this study is the first on the parasites of wild reptilians in Mexico using fecal samples.
The eggs of Capillariidae sp. found in C. triseriatus were not identified at specific level, since capillariid eggs are morphologically almost identical even among species of different genera (see Moravec 1994). Therefore, we cannot know if the nematode is a parasite or a pseudoparasite for Mexican dusky rattlesnakes, i.e., a parasite of the rodents on which hosts feed (personal observation of hair follicles in all fecal samples). In this context, Fuehrer (2014) recognized that endo-and ectoparasites of potential prey animals can be recorded as transiting parasites in the intestinal tract of reptiles, for example eggs of Capillaria hepaticum (Bancroft, 1893), a parasite species in mammals, have been documented in fecal samples from reptiles fed with infected rodents. On the other, Šlapeta et al. (2018) pointed out that the nematodes of Capillaria (Zeder, 1800) are the only known trichurids affecting reptiles, infecting primarily the intestine and secondarily other organs as the liver and gonads. It is known that their life cycle is direct, unfortunately the life cycles of snakes capillarids have not been investigated (Anderson 2000).
An examination of fecal samples of captive reptiles, including specimens of Crotalus Linnaeus, 1758, from Campania region in southern Italy revealed the most frequent parasites were species of groups of Oxyurida, Rhabdias Stiles & Hassall, 1905, Kalicephalus Molin, 1861, Capillaria and Eimeriidae Minchin, 1903(Rinaldi et al. 2012. Analysis of the fecal samples in the present work confirmed that capillariids and eimeriids are the most frequent parasites of the reptiles, but they did not show the presence of any of the helminths recorded of rattlesnakes in Mexico (Table 2).
The oocysts of Eimeria sp. were not identified at specific level, because we did not observe some taxonomic characteristics. But it is known that Eimeria Schneider, 1875 is the most numerous group of snake coccidian with more than 80 species, which differ in both the morphology of exogenous stages (oocysts) and in endogenous development (Modrý 2004). Most species of Eimeria inhabit the intestine of reptiles, which become infected after ingesting oocysts. Schizogony occurs in the epithelial lining of intestine, depending on parasite species. Oocysts are shed with the feces following gametogony. The pathologic effects of coccidian appear mild for most reptiles; however, more severe cases resulting in epithelial ulceration and fibrosis, and septicemia from the loss of epithelial integrity, may occur (Mitchell 2007). Jorge et al. (2013) found the detectability of nematodes was significantly lower in feces than in the intestine of reptiles, suggesting a large number of fecal samples and temporal replication to increase the likelihood of detection of intestinal parasites. Undoubtedly, parasitological studies based solely on feces most probably underestimate the true species richness present in a given host population and/or locality. However, such studies may be important for wildlife conservation purposes mainly when the translocations are employed, since the released individuals or individuals within the destination ecosystem may suffer from parasitic disease linked to the translocation process (Bobadilla Suarez et al. 2017). Specifically, it has been shown the vulnerability of populations of C. triseriatus in the Toluca Valley, due to the proximity to urban areas (Sunny et al. 2015), therefore, a complete parasitological record would aid wellbeing of snakes by receiving appropriate treatment plan and/or quarantine procedures. That allow to move the rattlesnakes outside the urban area, without the danger of infecting other populations with any diseased.  Ali et al. (1984), Klauber (1972)