Introduction
The tapeworm Echinococcus granulosus, responsible for cystic echinococcosis, is highly endemic in the Magallanes region in southern Chile (Alvarez Rojas et al., Reference Alvarez Rojas, Fredes, Torres, Acosta-Jamett, Alvarez, Pavletic, Paredes and Cortes2016; Deplazes et al., Reference Deplazes, Rinaldi, Alvarez Rojas, Torgerson, Harandi, Romig, Antolova, Schurer, Lahmar, Cringoli, Magambo, Thompson, Jenkins, Thompson, Deplazes and Lymbery2017). This area, characterized by its archipelago extending to the west and south, along with its mountainous terrain and elements of the Patagonian Ice Sheet, hosts the largest sheep population at national level, of approximately 2 million. Such a demographic provides ideal conditions for the transmission of E. granulosus, with canids serving as the definitive hosts. Recent data indicate a prevalence of 2.9% in sheep (Alvarez Rojas et al., Reference Alvarez Rojas, Fredes, Torres, Acosta-Jamett, Alvarez, Pavletic, Paredes and Cortes2016) and between 1.8 and 18% in dogs (Alvarez et al., Reference Alvarez, Ruiz, Ríos and Alvarez Rojas2021; Eisenman et al., Reference Eisenman, Uhart, Kusch, Vila, Vanstreels, Mazet and Briceno2023) across various areas of Magallanes. In this landscape, guanacos (Lama guanicoe), co-inhabiting with sheep on expansive private grasslands known as ‘Estancias’, are also susceptible to E. granulosus infection. Reports from the 1980s and 1990s showed prevalence rates between 3.1 and 12% in guanacos (Valdebenito Díaz, Reference Valdebenito Díaz2008; Carmanchahi and Lichtenstein, Reference Carmanchahi and Lichtenstein2022).
Following a decline in the 1970s and 1980s, the guanaco population has rebounded to over 150 000 animals in Magallanes (WCS, 2022). The legalization of seasonal guanaco hunting since 2003 has promoted sustainable resource utilization, contributing to a growing trade in guanaco meat, leather and fibre. During meat inspection of hunted guanaco, the parasitic lesions in liver and lungs have been attributed to E. granulosus at meat inspection (SAG, Reference SAG2022). However, some metacestodes, initially presumed to be E. granulosus, were morphologically identified as cysticercus of Taenia spp. This study aims to clarify the identity of these metacestodes, identify the Taenia species infecting guanacos and estimate the prevalence of both Taenia spp. and E. granulosus in guanacos from Chilean Patagonia.
Materials and methods
Sample collection
During the year 2022, a total of 2704 animals were legally hunted in the Magallanes region: 36.8% (996 animals) from San Gregorio in the continental part of the region and 63.2% (1708 animals) in Tierra del Fuego Island (Fig. 1). Sex was recorded and the age of guanacos was estimated following dental examination (Kaufmann et al., Reference Kaufmann, Álvarez and Pérez2017). Out of 2704 inspected carcasses, 73 animals (2.7%) showed parasitic lesions in liver and lungs initially attributed to E. granulosus. Out of 73 guanacos, 37 animals (50.6%) were hunted in San Gregorio and 36 animals (49.3%) in Tierra del Fuego. For molecular analysis we collected metacestodes from 56 guanacos: 27 from San Gregorio and 29 from Tierra del Fuego. Larval stages of cestodes were externally measured and the presence or absence of protoscolices of E. granulosus or a single scolex in the case of Taenia spp. was verified through microscopic examination of the metacestode content.
Map of the Magallanes region showing the locations where guanacos were captured, 1: San Gregorio in the continental part of the region and 2 in Tierra del Fuego Island divided in the north of the island Primavera and in the south of the island Timaukel.
DNA extraction and cox1 PCR
DNA was extracted from each metacestode with the EZNA® Tissue DNA Kit (Omega Bio-Tek) following manufacturer instructions. An initial PCR was performed to differentiate between Taenia spp. (12S rDNA, 267 bp) and E. granulosus (12S rDNA, 117 bp) using the primers Cest3, Cest4 and Cest5 designed by Trachsel et al. (Reference Trachsel, Deplazes and Mathis2007). DNA tested positive for Taenia spp. underwent a second PCR for the amplification and sequencing of a section of the cox1 gene (366 bp) (Bowles et al., Reference Bowles, Blair and Mcmanus1992) to identify the species through comparison with GenBank entries. Both PCR were performed using Green GoTaq Master mix (Promega). The thermocycler conditions were 95°C for 2 min, and 35 cycles at 95°C for 30 s, 55°C for the initial PCR (Trachsel et al., Reference Trachsel, Deplazes and Mathis2007) and 50°C for cox1 (Bowles et al., Reference Bowles, Blair and Mcmanus1992) for 30 s and 72°C for 30 s. The PCR products were visualized through electrophoresis in 1.5% agarose and sequenced bidirectionally using the same primers (Macrogen, Chile).
Sequence alignments, phylogenetic and haplotype analyses
Sequences of the cox1 gene region were trimmed (320 bp) and aligned with those of representative T. omissa along with T. hydatigena as the outgroup using Muscle (Edgar, Reference Edgar2004). Phylogenetic analysis of the alignment was conducted using the Maximum Likelihood method in the program MEGA11 (Tamura et al., Reference Tamura, Stecher and Kumar2021) with initial model selection performed in IQ-TREE (Kalyaanamoorthy et al., Reference Kalyaanamoorthy, Minh, Wong, Von Haeseler and Jermiin2017). Evolutionary distances were computed using the Hasegawa–Kishino–Yano (HKY) method with invariant sites. Each site, including gaps, were considered with a total of 2000 bootstrap replicates. The identification of haplotypes and the network analyses were computed with PopArt (Leigh and Bryant, Reference Leigh and Bryant2015).
Statistical analysis
Statistical analyses were performed to assess differences between parasite species and infection rates. An independent sample t-test was used to compare the sizes of E. granulosus and T. omissa metacestodes. Fisher's exact test was applied to evaluate the distribution of parasites across different geographic regions. Additionally, χ 2 tests were conducted to examine the relationship between sex and infection rates.
Results
Genetic characterization of metacestodes
The first PCR results, performed on DNA isolated from 27 metacestodes collected in San Gregorio, identified Taenia spp. (267 bp) in 22 samples whereas 4 were identified as E. granulosus (117 bp); no amplification was achieved in 1 sample. Of the 22 isolates identified as Taenia spp., PCR amplification of the cox1 gene was successful in all samples. However, only 20 samples produced a strong band enabling high-quality sequencing. Among these, 19 were identified as T. omissa and T. pisiformis (Table 1). Unique individual sequences of the cox1 gene from T. omissa acquired in this study were deposited in GenBank under accession numbers PP326076–PP326081. For Tierra del Fuego, PCR analysis, carried out on 29 metacestodes showed a band of 117 bp corresponding to E. granulosus in all samples. Sequencing of these PCR products showed 100% homology with E. granulosus sensu stricto (Table 1). No amplification was observed with primers targeting Taenia spp. DNA in samples from these areas.
Distribution of T. omissa and E. granulosus in animals from San Gregorio in the continent and Tierra del Fuego Island from the Magallanes region in Chile
Numbers show the animals infected. Statistical analysis via Fisher's exact test confirmed significant differences in the distribution of both parasites between the 2 provinces.
Prevalence of T. omissa and E. granulosus
We identified notable differences in the prevalence of T. omissa and E. granulosus infections between San Gregorio and Tierra del Fuego (Table 1). For San Gregorio, T. omissa was found in 73.1% of animals with metacestode lesions for which molecular identification was possible (19/26animals), whereas E. granulosus was identified in 15.4% (4/26) and T. pisiformis in 3.8% (1/26). Conversely, Tierra del Fuego showed a remarkable pattern, where all examined animals (25/25) were infected with E. granulosus.
Biological features of metacestodes from guanaco
The external aspect of a T. omissa cysticercus in liver tissue of a guanaco and a hydatid cyst of E. granulosus in the lung are shown in Fig. 2A and D respectively. Figure 2B and C shows the internal aspect of T. omissa cysticercus and a scolex of T. omissa from the same cyst, respectively. Figure 2E and F shows the internal aspect of a hydatid cyst and protoscolices from the same cyst, respectively. The average size of metacestodes was 8.72 mm (s.d. = 2.80 mm, range = 4.0–13.0 mm) for T. omissa and 21.88 mm (s.d. = 13.72 mm, range = 5.0–54.0 mm) for E. granulosus. Echinococcus granulosus parasites are significantly larger than those of T. omissa (t-statistic of −3.02 and a P value of 0.0049). While there is no significant difference in the age of guanacos infected with these 2 parasites (P value of 0.1305). Among the 29 cysts identified as caused by E. granulosus (4 from San Gregorio and 25 from Tierra del Fuego) protoscolices were observed in 10 samples, all of which were located in the lungs. None of the 12 cysts of E. granulosus found in the liver of a guanaco were fertile. Eight of the fertile cysts were found in male and only 2 in female guanacos.
(A) Taenia omissa sample collected from liver tissue measuring 6 mm. (B) Taenia omissa cysticercus open showing internal structure. (C) Microscopic observation (40×) of the scolex of a cysticercus of T. omissa collected in this study. (D) Echinococcus granulosus metacestode collected from the lung of a guanaco measuring 38 mm. (E) Echinococcus granulosus cyst open showing internal structure and ‘hydatid sand’ on the Petri dish. (F) Protoescolices of E. granulosus observed microscopically (40×) in fertile cysts collected in this study.
No significant difference in T. omissa infection rates between females and males was found (χ 2 statistic = 0.0, P value = 1.0) while a significant difference was observed in E. granulosus infection rates between sexes (χ 2 statistic = 7.5625, P value = 0.006), suggesting that male guanacos are significantly more likely to be infected with E. granulosus than females (Table 2).
Number of guanacos divided by sex infected with T. omissa and E. granulosus in places where samples were collected
In assessing the distribution of E. granulosus and T. omissa across liver and lung organs (Table 3) a χ 2 test of independence indicated a statistically significant difference in the prevalence of these species between the 2 organs [χ 2 (1) = 11.96, P < 0.001]. This suggests that the distribution of E. granulosus and T. omissa is not random across the liver and lungs, highlighting potential biological or ecological preferences in their organ colonization with T. omissa found more commonly in liver and E. granulosus in lungs of infected guanacos.
Distribution of E. granulosus and T. omissa across metacestodes found in liver and lungs in guanacos
Cysts identified as Taenia spp and a single metacestode that could not be identified are also included.
Phylogenetic analysis of T. omissa
For comparative purposes, all cox1 sequences acquired in this study and similar T. omissa sequences in GenBank were trimmed to 320 bp. From the 19 sequences identified as T. omissa we found 6 different haplotypes. One of these haplotypes was previously described in Argentina (OQ921986) while the other 5 variants had not been identified before. Further analysis revealed 25 segregating sites and 9 parsimony-informative sites when these sequences were compared with 11 sequences from GenBank, representing the same gene section of T. omissa from Argentina, Canada, Colombia and Peru. The haplotype network (Fig. 3) has a disperse distribution with none of the variants being the centre indicating a considerable genetic diversity among the T. omissa haplotypes. The phylogenetic tree (Fig. 4) illustrates that the T. omissa sequences extracted from Chilean guanacos cluster together with sequences from other parts of South America. Notably, these sequences form a distinct group separate from the sequence derived from a Canadian puma (JX860631).
Haplotype network with the cox1 sequences from Taenia omissa in the present study and all similar sequences deposited in GenBank. The nucleotide diversity: π = 0.015875, Tajima's D statistic: D = −0.891768, P (D ≥ −0.891768) = 0.402249.
Relationship of the novel Taenia omissa taxa (in bold) from metacestode tissue found in guanacos with representative T. omissa sequences from GenBank, based on a phylogenetic analysis of sequence data from a portion of the mitochondrial cytochrome c oxidase 1 gene (cox1) employing the Maximum Likelihood method. Branch supports are represented by Maximum Likelihood bootstrap percentages. Taenia hydatigena was used as an outgroup.
Discussion
This study provides the first evidence of T. omissa metacestodes in guanacos from the Chilean Patagonia, establishing a new southern limit for the parasite's distribution. Taenia omissa is known circulating between pumas and wild deer in North America ranging from white-tailed deer in Texas (Stubblefield et al., Reference Stubblefield, Pence and Warren1987) and Florida (Forrester and Rausch, Reference Forrester and Rausch1990) to red brocket deer (Mazama americana) (Gomez-Puerta and Mayor, Reference Gomez-Puerta and Mayor2021; Arrabal et al., Reference Arrabal, Arce, Macchiaroli and Kamenetzky2023) and alpacas (Gomez-Puerta et al., Reference Gomez-Puerta, Yucra, Lopez-Urbina and Gonzalez2017) in South America. The present study shows a natural infection of guanacos as intermediate hosts, extending the known range of T. omissa over 4000 km south to Patagonia. This is a significant leap from its previous southern limit in Argentina's Iguazu National Park (25.7 SL) to Torres del Paine in Patagonia (50.9 SL) (Arrabal et al., Reference Arrabal, Arce, Macchiaroli and Kamenetzky2023). The distribution of puma ranges from northern British Columbia to southern Magellan strait in Patagonia. It is not present in Tierra del Fuego Island. Puma is the single known definitive host of T. omissa (Rausch et al., Reference Rausch, Maser and Hoberg1983; Forrester and Rausch, Reference Forrester and Rausch1990). Adult specimens of T. omissa have been reported from the intestine of pumas from Oregon (Rausch et al., Reference Rausch, Maser and Hoberg1983), southern Florida (Forrester et al., Reference Forrester, Conti and Belden1985), Texas (Waid and Pence, Reference Waid and Pence1988) and Washington (Rickard and Foreyt, Reference Rickard and Foreyt1992). In the southern hemisphere, adults of T. omissa have been found in pumas from Paraguay (Schmidt and Martin, Reference Schmidt and Martin1978), Brazil (Benatti et al., Reference Benatti, De Santi, Werther, Tebaldi and Hoppe2021), the Peruvian Highlands in the Cuzco region (Gomez-Puerta et al., Reference Gomez-Puerta, Alarcon, Pacheco, Franco, Lopez-Urbina and Gonzalez2016) and the Atlantic Forest in Argentina (Arrabal et al., Reference Arrabal, Arce, Macchiaroli and Kamenetzky2023).
The detection of T. omissa in Chilean Patagonia contrasted with its absence in Tierra del Fuego, where pumas are absent providing insight into the complex transmission dynamics of this cestode, offering an ecological perspective on the distribution of its definitive host in low-density or cryptic habitats. Currently, no pumas have been observed on Tierra del Fuego Island; only feral domestic cats have been reported (Schuttler et al., Reference Schuttler, Saavedra-Aracena and Jimenez2018) This aligns with the results of the present study, underscoring the role of T. omissa as a potential bioindicator for puma presence. This insight is particularly valuable in understanding puma ecology, its interactions with prey like the guanaco (Fernández and Baldi, Reference Fernández and Baldi2014) and informing conservation strategies in these regions.
The distinct organ tropism exhibited by T. omissa, favouring the liver, in contrast to E. granulosus, which predominantly infects the lungs, introduces an additional layer of intricacy to the differential diagnosis of cestode infections in guanacos. This preference for specific organs, coupled with differences in metacestode sizes between the 2 parasites, allows differential diagnosis during guanaco meat inspection. Such refined diagnostics are crucial for public health, ensuring the safety of meat products while advancing the comprehension on the role of guanacos in perpetuation of life cycle of these tapeworms. This knowledge not only could aid in the effective management of these infections but also contributes to broader epidemiological insights, facilitating improved disease control and prevention strategies in affected regions. It has been reported that the G7 genotype of E. granulosus appears to infect preferentially the liver (Sanchez et al., Reference Sanchez, Mayta, Jara, Verástegui, Gilman, Gómez-Puerta and Gavidia2022).
The identification of E. granulosus in guanacos from both continental Patagonia and Tierra del Fuego island underscores the widespread prevalence of this zoonotic parasite in the region. The presence of E. granulosus in guanacos not only raises concerns about the potential zoonotic risk to humans and other animals but also highlights the intricate ecological interactions that facilitate the parasite's transmission across different hosts and environments.
The detection of T. pisiformis in a guanaco marks a rare finding that expands our understanding of the host range and ecological breadth of this parasite. While T. pisiformis is commonly associated with canids as definitive hosts (and rarely with felids), and lagomorphs as intermediate hosts, its presence in guanacos could indicate a broader spectrum of intermediate host species than previously recognized. Furthermore, the infection of guanaco with T. pisiformis could reflect a high level of environmental contamination with dog feces (Arona and Schiavini, Reference Arona and Schiavini2023).
This study significantly adds further data to the understanding of T. omissa, emphasizing the importance of enhanced monitoring and precise identification of parasitic infections in wildlife, which are crucial for both veterinary public health and conservation biology. The findings highlight the potential for T. omissa to serve as an indicator of ecosystem health and predator–prey dynamics. Future research should aim to uncover the ecological factors influencing T. omissa's distribution, delve into its genetic variability and evaluate its effects on the health and sustainability of intermediate host populations and their habitats. Such investigations will not only broaden our knowledge of T. omissa's ecological niche but also inform conservation strategies and public health policies, ultimately contributing to the preservation of biodiversity and ecosystem resilience.
Conclusions
Our findings reveal that T. omissa involves guanacos as intermediate hosts in Patagonia, where they represent the primary prey for pumas. The role of pumas in the life cycle of T. omissa in Magallanes remains to be clarified. We also report T. pisiformis for the first time in guanacos. Additionally, the presence of E. granulosus in guanacos accentuates the need to address the public health concerns posed by feral domestic dogs in the Patagonian regions of Chile and Argentina.
Data availability statement
Unique individual sequences of the cox1 gene from T. omissa acquired in this study were deposited in GenBank under accession numbers PP326076–PP326081.
Acknowledgements
We acknowledge Fernanda Peña, Ignacio Rivas, Valentina Cabello and David Morales, students at the School of Veterinary Medicine, Pontificia Universidad Católica, for their work in designing the graphical abstract.
Author contributions
C. A. A. R. and J. F. A. conceived and designed the study; C. A. A. R. and J. I. performed the morphological and molecular analysis; A. V. K. performed the phylogenetic analysis; C. A. A. R., J. F. A. and C. B. wrote the article.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The authors declare there are no conflicts of interest.
Ethical standards
Not applicable.
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