Endoparasites Infecting Domestic Animals and Spectacled Bears (Tremarctos ornatus) in the Rural High Mountains of Colombia

Simple Summary The spectacled bear (Tremarctos ornatus) is a threatened species, a member of the Ursidae family that lives in the Andes rural high mountain territories of Colombia, Venezuela, Ecuador, Peru, and Bolivia near livestock areas. Parasites in the spectacled bear are a relevant area of interest to preserve this species and understand its habitats and interactions with farm animals. The present work aimed to evaluate the presence of endoparasites in both T. ornatus and domestic animals in these areas, by copro- parasitological examination. The results indicate that some parasites have zoonotic potential in wild endangered species and domestic animals in Colombian regions. More sensitive molecular techniques are needed for further identification of the parasite species. Abstract This research described the co-infection prevalence of endoparasites in Tremartus ornatus and domestic animals in the rural high mountains of Colombia by copro-parasitological examination. Some parasites have a zoonotic potential in wild endangered species and domestic animals in Colombian regions. T. ornatus had a notable infection with Eimeria spp., Ascaris spp., Ancylostoma spp., and Baylisascaris spp. Cryptosporidium spp., Balantidium coli, Anoplocephala spp., and Acanthamoeba spp. In B. taurus, Eimeria spp. is coinfecting with Cryptosporidium spp. (6.6%) and represents 18% of the total parasitism. In E. caballus and B. taurus. Eimeria spp. coinfecting (34.7%), with the Strongylus spp. (21.9–25%). In T. ornatus, Eimeria spp. is coinfecting with Ancylostoma spp. (36.2%), Cryptosporidium spp., Ascaris spp., Baylisascaris spp., and B. coli.

T. ornatus is a threatened and endangered species according to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and the International Union for Conservation of Nature [4]. This species has been systematically studied in its taxonomy, genetics, reproduction, distribution, habitats, diets, behavior, status, and conservation [5] as well as livestock-based conflicts in Colombia, Ecuador, and Bolivia [5,6].
In Venezuela, Colombia, Ecuador, Peru, and Bolivia, Andean bears occupy more than 260,000 km 2 of forested habitat [7]. These specimens are believed to number over 20,000 adults in these countries [7,8]. Unfortunately, T. ornatus' population has been reduced by 30% to 42% in South America in the last years [9]. These areas are insufficient to guarantee T. ornatus' preservation [7].
Tenerife is located 1750 to 2750 m.a.s.l, with temperatures of 2 • C-14 • C. The climate is dry, with a relatively well-defined dry season from January to June and a rainy season from July to December. On the other hand, Combia is located in the rural area of Cerrito, where their inhabitants have reported bear attacks. With fewer than ten animals per owner, Combia's residents have a small production system that guarantees food security through the production of poultry, eggs, milk, and meat [14].

The Village of Gabriel Lopez, Municipalities of Totoró (Cauca)
This is located in the Valle de Malvazá, 20 km east of the capital of the department of Cauca, at 3000 m.a.s.l. Its temperatures range between 9 • C and 19 • C. The economic activity of its inhabitants revolves around agricultural products such as potatoes, fique, coffee, and aromatics [17]. It borders the Paramo de las Delicias (central mountain range) and the upper basin of the Cauca River, where several water sources of importance are born, such as the Palace River [18]. There are numerous reports of bears attacking and eating cattle and horses in these areas ( Figure 1). This is located in the Central Mountain system, within the Parque Nacional Natural los Nevados, on the way to the Nevado del Tolima, at 2600 m.a.s.l. Its waters are essential to sustain the production of Colombian coffee, rice, sorghum, cotton and corn. The wild animals that require preservation in this area are Tapirus pinchaque (mountain tapir), T. ornatus (spectacled bear), Pudu mephistophiles (northern Pudu), Odocoeilus virginianus (white-tailed deer), Silvilagus andinus (Andean tapeti), Leopardus tigrinus (oncilla) and Puma concolor (puma) [15,16].
2.1.3. The Village of Gabriel Lopez, Municipalities of Totoró (Cauca) This is located in the Valle de Malvazá, 20 km east of the capital of the department of Cauca, at 3000 m.a.s.l. Its temperatures range between 9 °C and 19 °C. The economic activity of its inhabitants revolves around agricultural products such as potatoes, fique, coffee, and aromatics [17]. It borders the Paramo de las Delicias (central mountain range) and the upper basin of the Cauca River, where several water sources of importance are born, such as the Palace River [18]. There are numerous reports of bears attacking and eating cattle and horses in these areas ( Figure 1). The geographic location. High altitude of the central Andean mountains. The farms are located at the border of T. ornatus territory, 2600 to 4100 m.a.s.l. Valle del Cauca, Tolima, and Cauca (Colombia). Generated with ArcGIS, 10.8.1 version of SIG laboratory, Universidad Nacional-Palmira.

Description Area
Most farms in the rural high mountains are centered on the production of beef and dairy cattle. It is a traditional system without technical support in which calves are allowed to graze freely or are stocked and brought in for lactation twice a day. Diarrhea in calves was reported in some farms. Most animals drink water from rivers or small ponds without water treatment (non-potable).
Some areas have small farms with pigs, cattle, horses, sheep, and pets such as dogs and cats. T. ornatus transits through livestock lands to find food, for instance, "piñuelas" Puya furfuracea (Willd.). The Valle del Cauca is rich in "frailejones" Ruilopezia cardonae (Cuatrec.), Speletia steyermarkii Cuatrec and Hesperomeles goudotiana "mortiño colorado" [19]. (Figure 2). Some cattle owners move animals to high altitudes for them to graze in T. ornatus' land, invading and affecting this bear's territory, while also contaminating rivers and water sources (  The geographic location. High altitude of the central Andean mountains. The farms are located at the border of T. ornatus territory, 2600 to 4100 m.a.s.l. Valle del Cauca, Tolima, and Cauca (Colombia). Generated with ArcGIS, 10.8.1 version of SIG laboratory, Universidad Nacional-Palmira.

Description Area
Most farms in the rural high mountains are centered on the production of beef and dairy cattle. It is a traditional system without technical support in which calves are allowed to graze freely or are stocked and brought in for lactation twice a day. Diarrhea in calves was reported in some farms. Most animals drink water from rivers or small ponds without water treatment (non-potable).
Some areas have small farms with pigs, cattle, horses, sheep, and pets such as dogs and cats. T. ornatus transits through livestock lands to find food, for instance, "piñuelas" Puya furfuracea (Willd.). The Valle del Cauca is rich in "frailejones" Ruilopezia cardonae (Cuatrec.), Speletia steyermarkii Cuatrec and Hesperomeles goudotiana "mortiño colorado" [19]. (Figure 2). Some cattle owners move animals to high altitudes for them to graze in T. ornatus' land, invading and affecting this bear's territory, while also contaminating rivers and water sources (

Type of Study
This cross-sectional study sought to assess the associations between the disease or health-related traits and other variables of interest in a specific population and time. The presence or absence of the disease and its variables were examined in a sample and without considering the temporal sequence of cause and effect [14]. The prevalence of gastrointestinal parasites was estimated using prevalence (p) = the number of total cases divided by the sum of the population at the moment (×100). The data were expressed in percentages (%). We used at least three stool samples to accurately diagnose parasitic intestinal infections (IPI) with a 95% confidence interval (CI). The value for significance of the association and allowable error was 0.05 [20]. We collected 58 stool samples from T. ornatus, but we estimated the populations of bears to range from 40 to 60 specimens.

Type of Study
This cross-sectional study sought to assess the associations between the disease or health-related traits and other variables of interest in a specific population and time. The presence or absence of the disease and its variables were examined in a sample and without considering the temporal sequence of cause and effect [14]. The prevalence of gastrointestinal parasites was estimated using prevalence (p) = the number of total cases divided by the sum of the population at the moment (×100). The data were expressed in percentages (%). We used at least three stool samples to accurately diagnose parasitic intestinal infections (IPI) with a 95% confidence interval (CI). The value for significance of the association and allowable error was 0.05 [20]. We collected 58 stool samples from T. ornatus, but we estimated the populations of bears to range from 40 to 60 specimens.

Samples
Stool samples (10 g) were obtained from domestic animals, horses, and cattle on the border of the reserve forest, directly from the rectum. Between 13 July and 6 December 2021, we collected fresh feces in the morning (6-12 h old), which were identified with the aid of an experienced park ranger. Fresh samples were recognized by their brown or green color. Saline wet mounts were made by mixing approximately 2 mg of stool with a drop of physiological saline on a microscope glass slide and placing a coverslip over the stool suspension. Samples were also analyzed using iodine wet mounts and microscopically examined with the afore mentioned method. The wet mounts were studied microscopically with a low power objective (10×) followed by switching to a high-powered one (40×). Each stool sample was screened by an experienced microscopist before reporting negative results. Additionally, the Zieh Nielsen technique was employed using 10 g of fuchsine diluted in 100 mL of ethanol and a 5% of phenol solution (5 mL of phenol and 95 mL of water). Then, 10 mL of basic fuchsine was filtered, and 100 mL of phenol solution was added in order to form the mother solution. Excess alcohol was removed with tap water and discolored with 7% H 2 SO 4 until the plate was pale pink, forming a sulphuric acid solution (7% H 2 SO 4 , 7 mL of sulphuric acid mixed with 93 mL of Ethanol). Excess colorant was also removed with tap water, and then we added methylene blue or malachite green, spreading it for 3 min. 10 g of methylene blue was diluted in 95% ethanol, and then 30 mL was filtered from the 100 mL of the mother solution; afterwards, 70 mL of water was added. The malachite green solution was conformed of 5 g malachite green diluted in 10% ethanol, 100 mL). The excess colorant was eliminated with tap water and left to dry in order to visualize the plate with immersion oil, using the 100× objective. The parasite analysis was performed with direct microscopic examination using a ZEISS AxioCam ICc 1 microscope, with flotation using the Sheather technique and sedimentation methods, as well as fixation and coloring techniques of Zieh Nielsen [10,14]. Samples were stored at −20 • C for future molecular studies.

Results
From 264 fecal samples collected from domestic animals and T. ornatus, we identified that 98/264 specimens were positive to at least one parasite, with a total prevalence of 60.93%. 35 Most samples from domestic animals were collected from B. taurus, Equidae Equus caballus, Equus asinus and their crossing. Samples from calf and young bears were more soluble than adults' feces. None of them had blood, mucus, or clinical parasitic symptoms.
Most of the bear samples were soft with green and brown color due to the nature of the vegetable tissue in the animals' feeding area ( Figure 2E,F). Some samples had a red fruit smell.
We studied the prevalence of parasites associated with more than one species considering the total number of samples (264). We identified that the most frequent association in B. taurus was Eimeria spp. with Cryptosporidium spp. (4/60, 6.6%). Eimeria spp. represents 18% of the total parasite associations, followed by Cryptosporidium spp. In horses and cattle, Eimeria had a strong association with other parasites (34.7%), but most co-infections were associated with the Strongyle family (21.9-25%). In bears, there was a robust parasite co-infection with Eimeria spp., Ancylostoma spp., Cryptosporidium spp., Ascaris spp., Baylisascaris spp., and B. coli (36.2%) (Figure 3). Vet. Sci. 2022, 9,   Using microscopy techniques, we observed different parasites with morphological characteristics and compatible measures with Buxtonella sulcata, Eimeria bovis, Eimeria zuernii, Strongylus vulgaris, Ascaris lumbricoides, Baylisascaris venezuelensis, Ancylostoma ailuropodae and Eimeria pellita; however, further studies using molecular techniques are required to confirm these classifications (Figures 4 and 5).

Discussion
In this study, 264 faecal samples using coproparasitological examinations techniques help to identify gastrointestinal parasites in Tremartus ornatus and domestic animals in the rural high mountains of Colombia. This technique has a lower operational cost and moderate sensitivity and specificity. These techniques are biologically useful, but they need to be complemented with biomolecular technologies in future studies to better understand the biological relations between host and the biology of parasites due to the difficulties in obtaining samples from these animals and optimize the effort in undeveloped countries, where there is limited knowledge available and research investment [14,26,27].
In our study, we found that Eimeria spp. was also the most prevalent (33.08%) in horses. We also found cyst of Eimeria spp. (53.89%) in B. taurus, which had a similar prevalence to the reports of other studies in low and high altitudes (17.4-77.9%) [30,31]. Parasite species such as Eimeria spp. might be transmitted from cattle to bear and vice versa, and probably, as stated previously, the host specificity of this parasite might be caused by adaptive rather than cophylogenetic processes [32,33].
In horses, we found a 4.2% higher prevalence in this species than in other countries, where the value is 2.3% [37]. We also identified that, in horses, there is association with Microsporidium spp. (6.25%). This data is consistent with previous studies [37]. In our research, Eimeria spp. was found circulating in B. taurus, E. caballus, and T. ornatus. Cryptosporidium spp. is circulating in B. taurus, E. caballus, and T. ornatus. Microsporidium spp. is infecting B. taurus and E. caballus, and finally, Buxtonella spp. was identified in B. taurus, E. caballus, and T. ornatus ( Figure 6).
Giardia in horse was not detected in this study with coprological techniques, but G. duodenalis (17.4%) has been previously reported in Colombia's horses using PCR [38]. Giardia spp. in cattle and T. ornatus has been previously reported in domestic animals and wildlife, particularly G. duodenalis in livestock [39,40]. This parasite was reported in T. ornatus by Figueroa, in Peru [41]. G. duodenalis is a common anthropozoonotic parasite [42].
Although this is the first evidence of Giardia spp. in both species (B. taurus and T. ornatus), it is essential to know the level of parasites impacting their health. This information may have consequences for conservation, associated with nutritional stress, parasitism, and the human-cattle-T. ornatus conflict. As such, intervention may be needed to prevent further damage [43,44]. Genetic characterization of Giardia isolates from humans and T. ornatus and the water used in a closed environment will help to understand the transmission routes and the level of association of this parasites in farms where cattle-horses and bears share common spaces in Colombia at high altitude [45]. migrans in humans and animals requires further research.
Finally, further research is needed to better understand parasitic dynamics in different seasons and the parasites' effects on these populations in the high rural mountains of Colombia, where farms located at this altitude increase the likelihood that the mountain bear T. ornatus, under low food conditions, extreme climate events, and deforestation and fragmentation processes, is forced to increase its interaction with domestic animals, which will continue to drive human-bear conflicts [11,67]. The microsporidia are obligate intracellular parasites consisting of at least 200 genera and 1400 species, infecting a broad range of animals (vertebrates and invertebrates). They infect fish, insects, farm animals, humans, and companion pets, leading to zoonotic transmission and affecting immunocompetent and immunocompromised humans [46][47][48]. In giant panda (Ailuropoda melanoleuca), Enterocytozoon bieneusi has been identified through PCR techniques with a positive rate of 35.5% [49].
E. bieneusi is the most common human-infecting microsporidian species, which includes pathogens of diverse companion animals and livestock [50]. Fast evolutionary rates, host switching using distant related hosts and habitats, as well as habitats destruction, environmental stress, extensive animal farming, and human encroachment on wild ecosystems may drive these new host-parasite interactions [50].
Microsporidium spp. in T. ornatus was not reported in our study, but we encourage further research using more sensitive molecular techniques on biological evidence, considering that Microsporidios spp. showed a prevalence of 16.66% in a study developed in the Chingaza National Park [10].
Interestingly, we did not find previous reports of B. sulcata in horses. Probably the parasite was introduced to America by the Spanish conquistadors, who obtained their horses in northern Africa, where they had been in contact with camels infected with Infundibulorium cameli syn of B. sulcata. Future studies are required to test the association with horses [60].
Regarding nematodes identified in T. ornatus during this study, we found Ascaris spp.  [10]. In Ecuador, T. ornatus in captivity were found infected by Ancylostoma spp. and Ascaris spp. [29].
Baylisascaris spp. has been previously published as B. venezuelensis, since it has already been characterized in T. ornatus using molecular techniques and compared with Baylisascaris transfuga, which has shown a 52.9% prevalence in brown bears [12,62].
Baylisascaris spp. has a monoxenous life cycle [63] and high potential to cause visceral, ocular, and neural migratory larvae in a range of different hosts, such as mammals and birds; therefore, they represent a zoonotic risk [62]. It is critical to warn tourists to prevent a zoonotic outbreak, considering that B. procyonis, B. columnaris and B. transfuga are described as etiological agents of migratory larvae [12,61]. Regarding B. venezuelensis, its level of pathogenicity in bears it is unknown, even though B. schroederi in pandas is a significant cause of morbidity and mortality. Additional research on the potential risk of B. venezuelensis to spectacled bears is needed [64].
Regarding other nematodes, we report Ancylostoma spp. (15%) in T. ornatus. This species has also been found in Colombia with a prevalence of 5.55% in T. ornatus [10]. Uncinaria sp. has also been documented in the American black bear, Ursus americanus, brown bears and polar bears, Ursus maritimus [65].
The interaction or multiple associations between wild animals and domestic animals and humans are not completely understood [66], and the potential role of hosts for transmission of zoonotic diseases in rural high mountains is not completely explored, as well as other wild animals that can trigger different dynamics. Zoonotic parasites such as Uncinaria spp., Strongyloides spp., Baylisascaris spp. and Cryptosporidium spp. are present in T. ornatus and domestic animals. This environment can cause potential larval migrans, skin problems as well as enteric human, domestic and wild infections.
Previous reports in humans at high rural mountains by Peña-Quistial shows that Toxocara canis and Toxocara cati had a prevalence of 24% and 44% [14] indicating that these parasites might be circulating in domestic animals that are able to cause larva migrans [14,66]. In the case of Baylisascaris (Ascarididae family, Ascaridida order, phylum Nematoda), its potential role to infect other animals as well as the agent that can cause larva migrans in humans and animals requires further research.
Finally, further research is needed to better understand parasitic dynamics in different seasons and the parasites' effects on these populations in the high rural mountains of Colombia, where farms located at this altitude increase the likelihood that the mountain bear T. ornatus, under low food conditions, extreme climate events, and deforestation and fragmentation processes, is forced to increase its interaction with domestic animals, which will continue to drive human-bear conflicts [11,67].

Conclusions
Endoparasites such as Eimeria spp. in T. ornatus and Cryptosporidium spp. and Buxtonella sulcata are common parasites in T. ornatus, B. taurus and E. caballus that require further studies around the clinical effects in these populations.
We recommend developing seasonal parasites studies as well as research regarding the population dynamic of each parasite to know the levels of exposition throughout the year. Future studies are also needed to identify other parasites species association among T. ornatus, wild and domestic animals.