Trichinella spiralis Infecting Wild Boars in Southern Chile: Evidence of an Underrated Risk

Abstract

Trichinellosis in Chilean wild animals has scarcely been documented. The introduction of wild boars into the wild environment represents a viable new host with a potential risk of infection for human health. The aim of this study was to determine the presence and prevalence of Trichinella in wild boars. Two hundred seventy eight wild boars from of the Southern Chile were examined by compression and artificial digestion techniques. The larvae in the positive samples were collected for taxonomic analysis through polymerase chain reaction–inter-simple sequence repeats and to calculate the parasitic burden. A prevalence of 1.8% (5/278) of infected animals and an average parasitic burden of 6.8 ± 2.1 larvae per gram were estimated. The only species identified by molecular techniques was Trichinella spiralis. Prevalence of T. spiralis in wild boars was similar to those described around the world. T. spiralis infection rate and parasite burden detected in Chilean wild boars represent a certain food-borne risk for human population.

Introduction

The taxonomy of the genus Trichinella currently considers nine species and three genotypes distributed in two clades: nonencapsulated with three species evolutionarily older and infecting reptiles (Trichinella papuae and Trichinella zimbabwensis) and birds and mammals (Trichinella pseudospiralis); and six capsulated species (Trichinella britovi, Trichinella murrelli, Trichinella nativa, Trichinella nelsoni, Trichinella patagoniensis and Trichinella spiralis) and three genotypes (Trichinella T6, Trichinella T8 and Trichinella T9) infecting only mammals (Murrell et al. 2000, La Rosa et al. 2003, Zarlenga et al. 2006, 2009, Pozio et al. 2009, Krivokapich et al. 2012).

In Chile, trichinellosis is considered an endemic zoonosis, and only T. spiralis has been identified in humans and animals (Fonseca-Salamanca et al. 2006).

Although pig raised and slaughtered domestically are the main source of infection in Chilean population (Schenone et al., 2002), the introduction of wild boars constitute a new way of infection for humans due to the lack of sanitary control (Meng et al. 2009).

The wild boar is an agriotype of the domestic pig, native of the palearctic region and introduced to South America at the beginning of the 20th century in Argentina. In Chile, it was introduced around 1938 on a hunting reserve by the Allipén River in La Araucanía where some animals escaped from. Later, the remaining animals were released in Puesco (Andean area) during 1948 (Skewes et al. 2007).

Subsequently in 1956, several natural invasions were registered from Argentina and in the Chilean localities of Panguipulli and Palena (Jaksic et al. 2002).

The wild boar is considered a harmful species, therefore, hunting is allowed throughout the year without restriction on the animals per excursion (Agricultural and Livestock Service of Chile—SAG 2018).

The analysis of gastric contents of hunted specimens in these environments has shown the prevalence of native plants such as the nalca (Gunnera tinctoria) and several vertebrate and invertebrate organisms, including arthropods, amphibians, birds, and rodents, which allow them to compensate for the shortage of protein in their diet (Skewes et al. 2007).

The inclusion of rodents in the wild boars diet suggests one of the most likely sources of Trichinella transmission. In southern regions of Chile and Argentina, a phenomenon known locally as ratada (rat invasion) takes place occasionally, caused by the flowering of South American bamboo known as quila and colihue (Chusquea spp.), promoting the uncontrolled reproduction of wild rodent (Jaksic and Lima 2003).

The presence of Trichinella spp. in wild boars has been described in various European, Asian, and North American countries (Boadella et al. 2012, Szell et al. 2012, Bilska-Zajac et al. 2013, Vu Thi et al. 2014).

In South America, it has been identified in wild boars from Argentina (Cohen et al. 2010), while in Chile, the only report is linked to a case of human infection by ingestion of wild boar meat (Garcia et al., 2005); however, there is no published information on the prevalence and diversity of the Trichinella spp. in this mammal.

Morphological identification of Trichinella spp. does not allow to discriminate among them due to the high similarity of the anatomical structures (Dunn 1978). Currently, the identification of Trichinella spp. is achieved by applying molecular techniques such as sequencing (Mitreva and Jasmer 2008) or protocols based on polymerase chain reaction (PCR) such as random amplified polymorphic DNA, single-strand conformation polymorphism, multiplex PCR, simple sequence repeat (SSR), and inter-simple sequence repeats (ISSR) among others (Gasser et al. 1998, Zarlenga et al. 1999, Fonseca-Salamanca et al. 2006, Perteguer et al. 2009).

The aim of this study was to report on the first molecular identification of T. spiralis, prevalence, and parasitic burdens in wild boars from Chile through ISSR-PCR.

Materials and Methods

Samples collection and Trichinella spp. detection

Diaphragm samples from 278 wild boars hunted in numerous rural areas from La Araucanía (n = 226) and Los Ríos (n = 52) were sent for inspection to a private veterinary clinic center during 2009–2014.

The information obtained from the samples was registered in a record book considering essential data such as reception date, geographic localization, and results of the analysis.

Samples were analyzed in the clinic center by compression technique, accepted by the Chilean standards for meat inspection. After reporting the cases, the infected animals were seized and incinerated by the sanitary authority.

Individualized samples from the infected and noninfected animals were donated and analyzed by artificial digestion at the laboratory.

The meat samples were isolated from fat tissue, weighed and grinded individually in a meat grinder, and added solution for enzymatic digestion, composed of 15 mL of hydrochloric acid (37%), 15 g of pepsin (diastatic activity 1:10,000), and 1500 mL of distilled water (38°C), considering a ratio of 10 mL per gram of meat.

Each mixture was stirred in a magnetic shaker during 1 h at 38–40°C and transferred to a funnel to decant for 15 min. Later, 40 mL was collected in conic tubes of 50 mL to decant for 15 min.

Supernatant was discarded and the sediment washed in phosphate-buffered saline buffer and transferred to plates. The larvae were collected individually using micropipette and stereoscopic magnifying glass (40 × magnification) and stored in tubes with 70% ethanol at −20°C.

DNA was extracted from the individual larvae using commercial kit E.Z.N.A^® (Omega Bio-Tek) according to the manufacturer's instructions and stored indefinitely at −20°C. To quantify the DNA concentration, an analysis was made by spectrophotometry at 230/260 nm and electrophoresis in 1% agarose gel to evaluate integrity.

ISSR-PCR assay

A total volume of 20 μL of reaction mixture was prepared using 10 μL of Taq PCR Master Mix^® (Qiagen), 2 μL of primer (10 pmol/μm), 6 μL of nuclease-free distilled water, and 2 μL of DNA. The primer sequence used was 5′CAC ACA CAC ACA CAC AT3′, and the amplification by ISSR-PCR was applied following the protocol proposed by Fonseca-Salamanca et al. (2006). Using a MultiGene^® OptiMax (Labnet International, Inc.) thermocycler, the tubes with the reaction mixture were incubated in an initial step for 5 min at 94°C, followed by 40 cycles of 30 s at 94°C, 45 min at 52°C, 2 min at 72°C, and a final step for 6 min at 72°C to extent the amplified products.

The amplified fragments were run by electrophoresis for 1 h at 100 V in 2% agarose gels (UltraPure, Invitrogen^®) stained with GelRed^® to visualize the bands. A 100 pb molecular weight marker (BioTools), a positive control of T. spiralis, and a negative control with the reaction mixture were used.

Data analysis

The study was descriptive and the findings were located on a map by georeferencing. The prevalence of infection was expressed in percentage considering the number of infected samples divided into the total samples. A comparison of prevalence in groups of both regions was made through the chi-square test (p < 0.05).

The parasitic burden was expressed as larvae per gram (lpg) dividing the number of collected larvae by the sample weight (in grams).

A comparison of the parasitic burdens was made using the Shapiro–Wilk normality test and analysis of variance, considering a value of p < 0.05.

Results

Five infected animals were diagnosed by both techniques, representing a prevalence of infection of 1.8% (5/278). The distribution of the findings was individualized by georeferencing and located on a map (Fig. 1).

FIG. 1.

Map of the Andean area of La Araucanía and Los Ríos in southern Chile showing the location (numbers 1–5) of wild boars infected by Trichinella spiralis (Source: Map data 2018 Google Earth).

From the 226 samples from La Araucanía, 3 were infected representing 1.3% of this group. While in the group from Los Ríos, 2 samples were infected out of 52 representing 3.8%. Despite this calculation, no significant differences were detected (p > 0.05).

Comparing the parasitic burdens (lpg), it was similar among infected samples and no significant differences were recorded (p > 0.05). The highest was 9.3 and the lowest 3.5, and the estimated average burden for the animals was 6.8 ± 2.1 lpg (Table 1).

Table 1.

Geographic Characterization, Weight of the Samples, and Parasitic Burden of Five Wild Boars (Sus scrofa scrofa) from Chile Infected with Trichinella spiralis

Sample	Geographical origin	Georeference	Meat weight (g)	Larvae (n)	Larvae per gram
1	Reyehueico-Panguipulli	−39.690941/−71.898639	15	85	5.7
2	Hualapulli-Villarrica	−39.409402/−72.284827	22	157	7.2
3	Catripulli-Curarrehue	−39.361130/−71.696094	10	93	9.3
4	Llancahue-Panguipulli	−39.582775/−71.923575	8	68	8.5
5	Los Laureles-Cunco	−38.999513/−72.216276	12	42	3.5

The patterns obtained by ISSR-PCR in 100% (n = 445) coincide with the positive control for T. spiralis, which is a pattern consisting of three fragments of 600, 700, and 900 bp in each sample.

Discussion

Although the prevalence varies according to methodologies and geographic location, it represents a benchmark to assess the occurrence of trichinellosis in this study. Considering these conditions, this study shows values concordant within the prevalence ranges determined by several authors.

Bilska-Zajac et al. (2013) estimated an approximate prevalence of 0.8% in wild boars in Poland. In Spain, Pérez-Martín et al. (2000) identified 0.3% and 0.2% (Boadella et al. 2012).

Higher prevalence was detected by Moskwa et al. (2015) in Poland with 2.0%, warning that there is also an increase in the wild boars population.

Kirjušina et al. (2015) in a study in Latvia gathered information for 38 years, determining a prevalence of 2.5%, with an increase in the incidence through the last decade.

In Vietnam, Kang et al. (2013), through an enzyme-linked immunosorbent assay (ELISA) study, reported a prevalence of 1.7% as well as Vu Thi et al. (2013) estimated 3.2% by artificial digestion and ELISA.

Geographically the area where the infected animals were found corresponds to a Andean zone that presents continuity through biogeographic corridors formed by an interconnected network of fluvial valleys with humid temperate forests with areas fragmented by exotic plantations and fields used for farming activities. This environment permits the displacement and invasion by this species from and toward the eastern slope of Los Andes, which could be related to the presence of infected wild boars from Lacar in the Argentinean province of Neuquén (Tesón et al. 1997).

The diagnosis by compression is a technique still used in Chile for meat inspection (Ministry of Health of Chile 2009), despite the greater sensitivity of artificial digestion (Webster et al. 2006), this has not been massively incorporated into the diagnosis, and its application is still limited mainly to laboratories research. In this study, the results for both techniques were coincident.

The species can be identified from the larvae by different PCR-based molecular techniques, including PCR, which has been adapted to discriminate species of Trichinella, and its replication in the laboratory is fast and simple (Fonseca-Salamanca et al. 2006).

The electrophoretic profiles obtained from this technique are not limited and can provide information about new gene polymorphisms through the detection of allelic variations, compared to fixed detection techniques such as multiplex PCR-based in regions of the mitochondrial and nuclear DNA, which limit detection to only the known species and genotypes (Blaga et al. 2009).

This is particularly valuable when studies of this parasite are conducted in the wild, where the possibility of finding new species or genotypes cannot be ruled out.

The presence of T. spiralis in the wild boars studied could be related to the acquisition of the parasite from the domestic cycle (Pozio et al. 1996). In Argentina, T. spiralis has been identified in wild boars; one of these findings was made in Junín, which is very near geographically to the findings of the present study, considering the mobility of this mammal through the Andean corridors (Ribicich et al. 2010).

Despite there being records of the parasite in synanthropic hosts such as rodents of the genus Rattus (Schenone et al. 1967, Rojas et al. 1971) and two reports in Puma concolor (Hidalgo et al. 2013, Landaeta-Aqueveque et al. 2015), the knowledge of this parasitic disease in this country's wildlife is still scarce.

Conclusions

In Chile, T. spiralis infection rate and parasite burden detected in wild boars represent a certain food-borne risk for human population.

Footnotes

Acknowledgments

We thank Mr. Marco Lillo of the Veterinary Clinic Center “Veterinaria Araucanía” (Villarrica, Chile) for providing samples and records and Mr. Sebastian Campos Leyton for the translation of this article. This study was supported by International Cooperation Direction and Vice-Rectorate of Research and Graduate, Universidad de La Frontera, Temuco, Chile (DIUFRO PROJECT DI17–0081).

Author Disclosure Statement

No competing financial interests exist.

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