Trichinella in Wildlife in Chile With the First Record in Leopardus Guigna and Galictis Cuja

Background: Trichinellosis is a worldwide distributed disease caused by Trichinella species and also considered neglected and emerging. Trichinella spp. are transmitted by predation or carrion consumption and present a domestic and a sylvatic cycle. Human trichinellosis occurs due to the consumption of raw or poorly cooked infected meat, mainly associated with the household slaughter of pigs without veterinary inspection, a cultural practice that is dicult to resolve. Therefore, the knowledge of the reservoir of this parasite is relevant. Methods: The aim of this study was to assess the presence of Trichinella sp. in several carnivore and omnivore vertebrates in Central-Southern Chile. A total of 61 animals, either found run over or that died in rescue/rehabilitation centers, encompassing 15 species, were examined by articial digestion for detection of Trichinella larvae, and larvae were molecularly analyzed to identify the species. Results: Trichinella larvae were found in a Leopardus guigna and a Galictis cuja. Only those of L. guigna could be identied as Trichinella spiralis. Conclusions: This is the rst record of Trichinella in a native mustelid of South America and the rst record of T. spiralis in L. guigna. These results increase the number of hosts, enhancing the need to identify the role of these animals in the reservoir for humans, and highlighting the priority that the study of the rural-sylvatic interphase represents.


Background
Trichinellosis is a disease with worldwide distribution caused by Trichinella species [1], and also considered neglected and emerging [2,3,4,5]. Trichinella is transmitted by predation and carrion consumption and circulates among carnivore and omnivore mammals; thus, human trichinellosis is mainly associated to cultural factors, especially the household slaughter of pigs without veterinary inspection and the consumption of raw or poorly cooked meat [6], conditions that are di cult to improve. Another source of infection is the consumption of game animals [7,8,9,10]. Thus, the knowledge of the cycle is relevant for proposing control measures. Trichinella circulates among carnivore and omnivore vertebrates and in both the domestic (encompassing mainly pigs, rats, dogs and cats) and the sylvatic environments (encompassing free-range vertebrates); which can be connected by invasive rats and other synanthropic animals [11]. At present, there are 10 recognized species of Trichinella around the world, as well as three genotypes that have not yet been demonstrated to be distinct species [1,12]. Since most Trichinella species infect only mammals, the sylvatic cycle encompasses mainly mammal hosts [13,14], including marine mammals [9,15]. However, T. pseudospiralis also infects birds, and T. zimbabwensis and T. papuae infect reptile hosts [1]. Thus, the ecological and epidemiological knowledge of the cycle are relevant for reducing the incidence. Hence, the study of the hosts participating in the reservoir of Trichinella is valuable.
In South America, Trichinella is present in Bolivia, Argentina, Brazil, Ecuador and Chile, and most studies have focused on the domestic cycle [16]. Four Trichinella species have been reported in this continent, mainly in Argentina: T. spiralis, T. patagoniensis, T. britovi and T. pseudospiralis [17,18,19,20], as well as in cougars, wild boars, foxes, opossums, sea lions, armadillos and a cricetid rodent, Graomys centralis [16,21]. These are the described wild host of Trichinella in the sylvatic environment. In Chile, the domestic cycle is fairly well-studied [22,23], but the sylvatic cycle is mostly unknown. Trichinella spiralis is the sole species that has been reported in Chile with few larvae identi ed to the species level [23,24,25]. Cougars and wild boars are the sole reported wild/feral host species reported [24,25], and, in addition to those reports, other studies did not nd infected animals [26,27,28]. Thus, the aim of this study was to assess the presence of Trichinella sp. in several carnivore and omnivore vertebrates in central-southern Chile.
This study considered animals that were found dead, mainly run over by a vehicle, or that died in wild animal rescue/rehabilitation centers (Fauna Rehabilitation Center of the Universidad de Concepción; Wild Fauna Rehabilitation Center of the Universidad San Sebastián) from 2013 to 2020. At least 1 g of muscle (10 g when possible) of these animals were examined for Trichinella larvae. Muscles selected for parasitological examination were diaphragm, masseter, tongue, quadriceps (in mammals), pectoral (in birds) and intercostal.
Arti cial digestion of the muscles was performed in the Laboratory of Parasitology Dr. Luis Rubilar of the Facultad de Ciencias Veterinarias of the Universidad de Concepción following Gajadhar et al. [29] and larvae were conserved in 96% ethanol. For the molecular identi cation, the DNA was extracted from a pool of 10 Trichinella larvae with DNeasy Blood & Tissue Kit (Qiagen®) and 10 ng of DNA was used for identi cation at the species level by nested PCR following Pozio and Zarlenga [30]. Reactions were performed in 25 µl nal volume. Primers Ne forward (5'-TCTTGGTGGTAGTAGC-3') and reverse (5'-GCGATTGAGTTGAACGC-3') were

Results
Trichinella larvae was isolated only from one L. guigna (güiña. 52 larvae per gram) and one G. cuja (lesser grison. 0.3 larvae per gram), both from the Ñuble Region (Fig. 1). The DNA of the grison was degraded and did not output a PCR product. The DNA of the guiña output a PCR product of 173 bp, corresponding to T. spiralis.

Discussion
Trichinella larvae in Chile is di cult to study in wild fauna because most carnivore vertebrates are protected by law [31], either due to conservationist concerns or because they support pest control. Only invasive animals can be hunted for assessing the Trichinella infection and the larvae isolation. Given that, few studies have assessed the presence of Trichinella infection in native carnivores in Chile: Alvarez et al. [26] included two güiñas and 24 lesser grisons, in addition to other mammals, and González-Acuña et al. [27] included two güiñas, with negative results in both cases. Other studies in Argentina included another wild cat (Leopardus geoffroyi) and the lesser grison with negative results [32,33]. Thus, this is the rst record of Trichinella larvae in a native mustelid in South America, and the rst record of T. spiralis in the güiña, with the güiña being the second South American felid host for this parasitic species. Previously, other mustelids have been reported to host Trichinella elsewhere, as Neovison vison (American mink) hosting T. spiralis, T. britovi and T. pseudospiralis in Poland [34], as well as Meles meles (European badger) hosting T. britovi in Romania [35]. Similarly, other felids have been reported harboring Trichinella in Chile, as Puma concolor harboring T. spiralis [25], and elsewhere, as Puma concolor harboring T. patagoniensis in Argentina [20], Puma concolor coryi harboring T. spiralis and T. pseudospiralis in USA [36], Puma concolor cougar harboring Trichinella nativa, Trichinella pseudospiralis, Trichinella murrelli and Trichinella T6 [37], Lynx rufus harboring T. britovi and Felis silvestris harboring T. britovi and T. spiralis [38].
The güiña is one of the smallest felids in the world, with a restricted distribution in Chile and Argentina between 33º and 48º of latitude S [39]. This felid consumes micromammals preys as primary items [40,41], in such a way that this prey, especially rodents, can be the source of infection. However, rodents have been recognized as hosts of T. spiralis mainly in the domestic environment in Chile [23,42]. This record is in accordance with the fact that guiñas have been frequently infected by micro-organisms spilled from free-range domestic animals [43,44]; and, although T. spiralis is not an important pathogen for the health of non-human animals, its presence in the güiña highlights the need of surveyance of pathogens in the rural-sylvatic interphase.
The lesser grison is a Neotropical mustelid that inhabit an area from southern Peru, Uruguay and Paraguay to southern Chile and Argentina, encompassing several environments [45], and is a generalist predator, including rodents as an important part of its diet [46,47]. Given that, this species most likely can be infected in the domestic environment; however, a further identi cation of the Trichinella species that the lesser grison harbors allows to better understand the source of infection, given that not all Trichinella species identi ed in South America have been reported in the domestic cycle. For instance, T. patagoniensis has been reported only in cougars [20,48].
To the best of our knowledge, there is no report of the guiña as prey of larger predators while B. magellanicus (Magellanic horned owl) is the sole predator reported for the lesser grison [45]. Regarding that, T. pseudospiralis, also zoonotic, is the only species of the genus reported to infect birds. This species has not been reported in Chile with the record of a single pig from Argentina being the sole report in South America [18]. Thus, it is most unlikely that this owl could be a way for Trichinella to be transmitted from the grison to another species. Hence, whether guiña and lesser grison participate in the reservoir or constitute dead-end hosts is unknown, and the most likely way for Trichinella larvae to be transmitted from these hosts seems to be their consumption by carrion-consuming mammal. Therefore, further studies are necessary to test this hypothesis. On the other hand, human trichinellosis after the direct consumption of a free-range mammal has been also reported worldwide [7,8]; however, neither guiñas nor grisons are typical prey for hunters to eat, nor is their hunting permitted by law in Chile [31].
It is noteworthy that the two host species herein reported correspond to the mammal species with the larger sample size, which suggests that larger samples in the other mammals could output new hosts for Trichinella. The contrasting lack of nding of Alvarez et al. [26] could be due to a real absence of larvae in their samples, as well as to the parasitological technique, trichinoscopy, which is of lower sensitivity [49].

Conclusions
This is the rst record of Trichinella larvae in a native mustelid in South America, and the rst record of T. spiralis in the güiña. Thus, this study raises the number of mammals infected with Trichinella larvae, enhancing the need of identify the role of these animals in the reservoir for humans, and underlying the priority that the study of the rural-sylvatic interphase represents.
Abbreviations DNA deoxyribonucleic acid PCR polymerase chain reaction Declarations Ethic approval: The Comité de Ética of the Universidad de Concepción approved the study (Certify 112017).
Competing interests: The authors declare that they have no competing interests.
Funding: This study was funded by the Fondo Nacional de Desarrollo Cientí co y Tecnológico, Chile ANID/FONDECYT grant no. 11170294.