Investigation of Parasitic Nematodes Detected in the Feces of Wild Carnivores in the Eastern Qinghai-Tibet Plateau, China

Wildlife shares grazing areas with herders in the eastern Qinghai-Tibet Plateau, and humans can be infected by zoonotic nematodes through direct contact with animals or contaminated water. In this study, fecal samples (n = 296) from wild carnivores were collected to explore the infection rate and molecular genetic characteristics of nematodes by stratified random sampling in the survey areas. Host species and the nematodes they carried were then identified using 16S rRNA and 18S rRNA gene sequencing, respectively. Statistical analysis, neutrality tests, genetic diversity analysis and Bayesian inferred trees were performed to complete the study. In total, 10 species of nematodes were detected in 240 feces from six species of carnivores identified (including dominant Vulpes ferrilata and Vulpes vulpes), namely Uncinaria stenocephala, Toxascaris sp., Crenosoma vulpis, Parapharyngodon bainae, Oesophagostomum muntiacum, Aspiculuris tetraptera, Mastophorus muris, Nematodirus spathiger, Muellerius capillaris, and Molineus patens. Among these nematodes, U. stenocephala (35.83%, 86/240) and Toxascaris sp. (14.58%, 35/240) were detected at higher rates than the other nematodes (χ2 = 516.909, p < 0.05). Of 17 and 18 haplotypes were found based on the ITS1 gene for U. stenocephala and nad1 gene for Toxascaris sp., respectively. For the first time, using molecular methods, we report the infection of V. ferrilata by U. stenocephala, a potential zoonotic parasite, and suggest Toxascaris sp. may be a newly discovered nematode that lives within the fox intestine.


Introduction
Nematodes are unsegmented helminths widely distributed in freshwater, brackish water, soil, plants, and animals [1]. According to the reports, over 25,000 species of nematodes have been described so far, 12,000 of which are vertebrate parasitic nematodes [2]. For humans, some of the most prevalent but neglected tropical diseases are caused by nematode infections (e.g., ascariasis, hookworm disease, whipworm disease, lymphatic filariasis, and onchocerciasis), which lead to a decline in health and productivity, adversely affecting socioeconomic development [3]. The statistics based on the global burden of diseases caused by soil-transmitted nematodes showed that ascariasis, trichuriasis, and hookworm disease, were estimated to result in 4.5 and 39 million disability-adjusted life-years (DALYs) [4]. Among them, certain nematodes are among the most common infectious pathogens in developing countries with poor sanitation, potentially affecting the psychological and physical development of children by causing anemia, malnutrition, stunting, and cognitive impairment [5,6]. According to the third national survey of important zoonotic diseases in China in 2015, Sichuan Province is a highly endemic area for soil-derived nematodes with a 23.55% infection rate, and herders are a key population experiencing soil-derived nematode infections [7].

Study Area
Shiqu County is located northwest of Ganzi Tibetan Autonomous Prefecture, Sichuan Province, the eastern part of the Qinghai-Tibet Plateau, at the junction of Sichuan Province, Qinghai Province, and the Tibet Autonomous Region, with an average altitude of above 4200 m, where animal husbandry is the main vocation in the alpine meadows covering the vast majority of territory [30,31]. It belongs to an area of subtropical climate, but the climate in the territory is influenced by the complex topography of the Qinghai-Tibet Plateau, showing the characteristics of a mountainous climate and continental climate of the Qinghai-Tibet Plateau [32].

Sampling
In October 2021, five townships, namely Derongma, Niga, Sexu, Luoxu, and Zhengke, in Shiqu County, were selected for investigation based on previous survey results and information on the local landscape distribution. Sampling points were selected via stratified random sampling based on the proportion of land-use area within the study area extracted from the 2020 China Land Use Remote Sensing Monitoring Data released by the Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences. Sixty-seven 15 m × 200 m plots were sampled. Each sampling point was spaced 5 km apart. All fecal samples of field carnivores were thoroughly searched at the selected sites, with the fresher samples collected and stored in 50 mL centrifuge tubes and numbered using new disposable tools to avoid cross-contamination.

Molecular Identification
Each fresh fecal sample collected was mixed well, and 2 g of each sample was used for DNA extraction. Deionized water (50 mL) was added to each fecal sample, followed by incubation in a water bath at 80 • C for 10 min. Thereafter, the sample was filtered through two layers of gauze, and the filtrate was added into another 50 mL centrifuge tube, then centrifuged at 3000× g for 30 min. The supernatant was discarded, and the precipitate was retained. DNA was extracted using the QIAamp Fast DNA Stool Mini Kit as per the manufacturer's instructions (QIAGEN, Hilden, Germany). Parallel polymerase chain reaction (PCR) tests were performed for each sample using six pairs of primer pairs. Primer synthesis was conducted by Sangong Biotech Co. Ltd. (Shanghai, China). Host universal primers (16S) were used to identify host species based on the 16S rRNA gene, and a primary screening was performed to detect nematodes using three pairs of universal parasitic primers (Nems A/B/C) targeting the 18S rRNA gene. Considering the lack of reference sequences for the amplified target fragment, the specific primers ITS1 and nad1 were used to enrich sequence information for Unicinaria sp. and Toxascaris sp., respectively (see Table 1 for details). All PCRs were carried out in a final reaction volume of 50 µL, consisting of 25 µL of Ex TaqTM PCR premix (RR902A; Takara, Dalian, China), 17 µL of RNase-free water (9012, Takara, Dalian, China), 1 µL of bovine serum albumin (2320, Takara, Dalian, China), 1 µL of each primer (10 pmol/µL) (Sangon Biotech Co. Ltd., Shanghai, China), and 2 µL template DNA. PCR reactions were performed on a Bio-Rad DNA Engine Dyad PCR instrument (Bio-Rad) with the following amplification reaction parameters: 94 • C for 5 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing for 30 s (annealing temperature selected according to primers), extension at 72 • C for 30 s and a final extension step at 72 • C for 10 min.
All PCR products were sequenced, and abnormal sequencing results with multiple peaks were cloned at Sangong Biotech (Shanghai) Co., Ltd. The final sequences obtained were compared with those in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov, accessed on 15 September 2022) to identify host and parasite species.

Statistics
SPSS (version 25.0; IBM Corp., Armonk, NY, USA) was used for the statistical analysis of data, with indicators such as frequency and rate used to describe host species identification and parasite detection. Comparisons between rates or composition ratios were performed using the chi-squared test at a significance level of p = 0.05. Comparisons between groups were performed using the Z test and Bonferroni correction. Fisher's exact test was used to compare data that did not meet the requirements for chi-squared tests.

Phylogenetic Analyses
Reference sequences for the nad1 gene of Uncinaria stenocephala, ITS1 gene of Toxascaris sp., and 18S rRNA gene of Crenosoma vulpis were searched in NCBI (see Additional file: Table S1 for details). All the sequences were first imported into MEGA 7 for alignment and trimming [37]. Thereafter, the ordered sequences were input into DNASP 6 to obtain haplotypes and perform neutrality tests, including haplotype diversity, nucleotide diversity, Tajima's D, and Fu's Fs [38]. The haplotype network graphs were mapped using Network 10.2.0.0 (http://www.fluxus-engineering.com) (accessed on 27 September 2022). Bayesian phylogenetic trees for U. stenocephala, Toxascaris sp., and Crenosoma vulpis were constructed to analyze the relationship between the nematodes obtained in this survey and other regions. The best nucleotide substitution models were screened using jModeltest 2.1.10 [39]. Phylogenetic trees were constructed using MrBayes 3.2.4 [40]. Markov chain Monte Carlo (MCMC) data simulation was used to estimate the posterior probabilities with 10,000 generations of operations, the sampling frequency was set to 10, and the first 25% of aging samples were discarded. Finally, Bayesian trees were compiled and processed using

Host Species Composition
Of the 296 fecal samples initially recognized by the investigators as wild canids, 240 were successfully identified after DNA extraction (81%). Six carnivorous species belonging to the families of Canidae, Mustelidae, and Felidae were identified, namely Vulpes ferrilata

Nucleotide Polymorphisms
Neutral tests showed significant negative results (Tajima's D = −2.20369, p < 0.05; Fu Fs = −12.411, p < 0.05) for U. stenocephala, suggesting that the population had a lar number of low-frequency mutant genes in its gene pool and underwent an expansion aft

Nucleotide Polymorphisms
Neutral tests showed significant negative results (Tajima's D = −2.20369, p < 0.05; Fu's Fs = −12.411, p < 0.05) for U. stenocephala, suggesting that the population had a large number of low-frequency mutant genes in its gene pool and underwent an expansion after a recent bottleneck effect under significant selection pressure. Moreover, U. stenocephala had lower genetic diversity (Hd = 0.470 and Pi = 0.00249) than Toxascaris sp. (Hd = 0.836 and Pi = 0.01836). In contrast, although the neutrality tests showed negative values, there was no significant difference for Toxascaris sp. (Tajima's D = −4.9277, p > 0.10; Fu's Fs = −2.610, p > 0.10), indicating that Toxascaris sp. population dynamics were closer to neutrality with random evolution (see Table 3 for details).

Phylogenetic Tree Results
The Bayesian tree showed that U. stenocephala isolates from the United States and the haplotypes in this survey were located on the same branch, with 99% support. However, some haplotypes were clustered into new sub-branches, such as Hap-Us-1, Hap-Us-6, Hap-Us-8, Hap-Us-12, Hap-Us-13, Hap-Us-15, and Hap-Us-16, which may have been formed by potential geographical divergence ( Figure 3c). As shown in Figure 3a, some parasitic gene sequences obtained from this study were clustered on a sister branch of Toxascaris leonina and were, therefore, considered a new local species of the Toxascaris genus. Figure 3b shows that the genes of Crenosoma vulpis from this study and from a study in Italy clustered together.

Primers Variability Comparison
Of the 10 nematode species detected, only one (U. stenocephala) was detected using all three primers, and Nem A detected the largest number of nematode species (Figure 4). Statistically significant differences were observed in the detection rates of parasites using different primers. The detection rate of Toxascaris sp. was higher for Nem A than for Nem B and C (χ 2 = 73.577, p < 0.05). The detection rates of U. stenocephala were higher for Nem B and Nem C than for Nem A (χ 2 = 16.620, p < 0.05), and the detection rate of Mastophorus muris was higher for Nem A than for Nem C (χ 2 = 9.520, p < 0.05, Fisher's exact test).

Primers Variability Comparison
Of the 10 nematode species detected, only one (U. stenocephala) was detected using all three primers, and Nem A detected the largest number of nematode species (Figure 4). Statistically significant differences were observed in the detection rates of parasites using different primers. The detection rate of Toxascaris sp. was higher for Nem A than for Nem B and C (χ 2 = 73.577, p < 0.05). The detection rates of U. stenocephala were higher for Nem B and Nem C than for Nem A (χ 2 = 16.620, p < 0.05), and the detection rate of Mastophorus muris was higher for Nem A than for Nem C (χ 2 = 9.520, p < 0.05, Fisher's exact test).

Discussion
This study provides molecular evidence for the presence of local nematode-infected carnivores in the Qinghai-Tibetan Plateau. In this study, six species of carnivores were identified through the analysis of fecal DNA collected throughout Shiqu County, Ganzi Tibetan Autonomous Prefecture, the eastern Qinghai-Tibetan Plateau. Foxes, especially V. ferrilata, were the dominant species within the region based on fecal analysis. In the present work, the zoonotic parasite U. stenocephala was reported to infect V. ferrilata for the first time through molecular detection. The findings suggest that Toxascaris sp. may be a new parasitic species affecting foxes, distinct from T. leonina and other Toxascaris sp. reported worldwide.
Uncinaria stenocephala belongs to the family Ancylostomatidae [41], which is widely parasitic in dogs, wolves, foxes, and badgers, as well as other canids, and is distributed in regions with colder climates throughout Asia, Europe, North America, South America, Australia, and New Zealand [42]. It is among the most common hookworms infecting dogs [43]. Cats can also be infected by this parasite, but their resistance is higher than that of canines [44,45]. U. stenocephala eggs are transmitted via the feces of definitive canine hosts, Larvae hatch in the superficial soil layer within one day and then develop into thirdstage larvae after approximately one week under ideal conditions [46,47]. Humans are infected through skin contact with infective third-stage larvae or through the consumption of raw food containing infectious third-stage larvae [48]. However, due to the lack of relevant investigations in China, only very limited data can be referred to. The earliest records were reported in Sichuan Province: U. stenocephala was observed in the intestines of V. ferrilata and Arctonyx collaris by morphological methods in Luhuo County and Hongyuan County in 1963 and 1981, respectively [49]. Since then, there were no reports in the literature until 2019, when U. stenocephala infection was determined morphologically in five of 10 dogs examined in Nangqian County, Qinghai Province [50]. In the present work, the overall detection rate of U. stenocephala in foxes was 36.9% (86/233), lower than rates reported from Denmark (60-86%) [51], Spain (58.2%) [52], Switzerland (64-78%) [53,54], and Italy (39-75%) [55], but comparable to that in Ireland (38%) [56]. Those data suggest that foxes are an important definitive host for U. stenocephala and play a key role in the process of transmission that cannot be ignored. This should be considered a warning because this parasite can cause disease in humans.

Discussion
This study provides molecular evidence for the presence of local nematode-infected carnivores in the Qinghai-Tibetan Plateau. In this study, six species of carnivores were identified through the analysis of fecal DNA collected throughout Shiqu County, Ganzi Tibetan Autonomous Prefecture, the eastern Qinghai-Tibetan Plateau. Foxes, especially V. ferrilata, were the dominant species within the region based on fecal analysis. In the present work, the zoonotic parasite U. stenocephala was reported to infect V. ferrilata for the first time through molecular detection. The findings suggest that Toxascaris sp. may be a new parasitic species affecting foxes, distinct from T. leonina and other Toxascaris sp. reported worldwide.
Uncinaria stenocephala belongs to the family Ancylostomatidae [41], which is widely parasitic in dogs, wolves, foxes, and badgers, as well as other canids, and is distributed in regions with colder climates throughout Asia, Europe, North America, South America, Australia, and New Zealand [42]. It is among the most common hookworms infecting dogs [43]. Cats can also be infected by this parasite, but their resistance is higher than that of canines [44,45]. U. stenocephala eggs are transmitted via the feces of definitive canine hosts, Larvae hatch in the superficial soil layer within one day and then develop into third-stage larvae after approximately one week under ideal conditions [46,47]. Humans are infected through skin contact with infective third-stage larvae or through the consumption of raw food containing infectious third-stage larvae [48]. However, due to the lack of relevant investigations in China, only very limited data can be referred to. The earliest records were reported in Sichuan Province: U. stenocephala was observed in the intestines of V. ferrilata and Arctonyx collaris by morphological methods in Luhuo County and Hongyuan County in 1963 and 1981, respectively [49]. Since then, there were no reports in the literature until 2019, when U. stenocephala infection was determined morphologically in five of 10 dogs examined in Nangqian County, Qinghai Province [50]. In the present work, the overall detection rate of U. stenocephala in foxes was 36.9% (86/233), lower than rates reported from Denmark (60-86%) [51], Spain (58.2%) [52], Switzerland (64-78%) [53,54], and Italy (39-75%) [55], but comparable to that in Ireland (38%) [56]. Those data suggest that foxes are an important definitive host for U. stenocephala and play a key role in the process of transmission that cannot be ignored. This should be considered a warning because this parasite can cause disease in humans. Fourteen U. stenocephala isolates were detected from the feces of 128 patients in Iran [57]; further, 22% of prisons in Madrid, Spain, had reported U. stenocephala infections [58] and compared to a prevalence of 7.9% from 38 residents tested in a suburb of Buenos Aires, Argentina. While no U. stenocephala infection in humans has been reported in China thus far, given the high detection rate of foxes in the present investigation, it is rather likely for transmission to the local residents to have occurred. Furthermore, when patients have hookworm-related cutaneous larva migrans, the location of the larvae is usually more distant than the visible area of the lesion and cannot be diagnosed via biopsy or based only on clinical features and epidemiological history [59]. Thus, we recommend relevant diagnostic training for local physicians to raise vigilance for U. stenocephala infection.
It is worth mentioning that a species of Toxascaris sp., suspected to be a new species of the genus Toxascaris, was identified in this study, with a detection rate of 14.58%. The phylogenetic tree revealed that it was closely related to Toxascaris leonina, currently the only known species in this genus [60]. The eggs of T. leonina can adapt to cold climatic conditions, survive for 40 days at −15 • C, and continue to develop to the infectious stage when the temperature returns to 25 • C, after which it infects animals via the oral route [61]. In general, canines and cats are the definitive hosts of T. leonina; small rodents can be their intermediate hosts, while rabbits and chickens can act as reservoir hosts [62,63]. In particular, T. leonina migrates into the intestinal wall only during the larval stage and has a long incubation period (48-72 days). A young host can complete growth and development before its health is affected, and thus, T. leonina is considered to have low pathogenicity [64]. Current research suggests that T. leonina is a potentially zoonotic parasite that may be responsible for eosinophilia among people in the St. Lawrence Island region of Alaska [60,63]. Whether the Toxascaris sp. found in this study has the same pathogenic potential as T. leonina requires further study.
Crenosoma vulpis, a parasite species with foxes as the common definitive host, also infects stray and domestic dogs as well as various other carnivores such as Canis lupus, Canis aureus, Nyctereutes procyonoides, Urocyon cinereoargenteus, Lutra lutra and Meles meles. Further, it is an important causative pathogen of chronic respiratory disease in canids [65]. Infected animals suffer from chronic respiratory diseases, exhibit poor growth, and may even die due to bronchopneumonia in severe cases [66]. Its larvae reside in gastropods, with third-stage larvae (L3) infecting the definitive host. Adult nematodes reside within the bronchi and bronchioles where they produce first-stage larvae (L1) that can be coughed up within three weeks of infection or passed from the feces of definitive hosts through swallowing [67]. The high infection rate of Crenosoma vulpis in V. vulpes has been reported in Europe and America, including 78.4% in Canada [68], 8.7-15.8% in Italy [69], and 24.6% in Hungary [70]. We found only one infection case in V. ferrilata in this study, which may be related to the fact that the samples collected are feces rather than lung tissues.
In addition to the above three parasites with foxes as definitive hosts, some parasites detected in this study may not be common intestinal species of foxes. Parapharyngodon bainae is an intestinal parasite affecting carnivorous reptiles [71]. Oesophagostomum muntiacum usually lives in the intestinal tract of muntjacs and can also be parasitic in primates, such as macaques [72,73]. Aspiculuris tetraptera, a common parasite in the digestive system of mice, is usually non-pathogenic or slightly pathogenic in mice with a normal immune system [74]. Mastophorus muris is a common parasitic nematode in the stomachs of rodents, and its intermediate hosts are beetles, locusts, earwigs, cockroaches and other insects [75]. Muellerius capillaris is a common pulmonary parasitic nematode in small ruminants, such as goats and sheep, causing respiratory distress and coughing in small ruminants, making their lungs more susceptible to bacterial infection [76,77]. Molineus patens is a common intestinal parasite of mustelids; it can impair the health of skunks, affecting their population size [78,79]. This observation may be related to the feeding habits of carnivores. Vulpes ferrilata identified in this survey mainly prey on pika and voles [80], its prey also includes Pantholops hodgsoni, Lepus oiostolus, Phrynocephalus spp., Marmota himalayana, Moschus spp., Pseudois nayaur, and young domestic animals [81,82]. While foxes might not be the most suitable host, the current study indicates the local presence of these parasitic nematodes.
All results of this study were obtained by molecular methods and were not supported by morphological analysis. Because, V. ferrilata is protected in China [83], only non-invasive methods, such as fecal sample testing, can be applied in survey studies. Further in-depth study of parasitology and host-parasite relationships should be carried out in the future. However, even with the use of molecular detection, it should be noted that the experimental results of three pairs of nematode primers showed differences in detection rates. Therefore, we suggest that parallel experiments using multiple primer pairs would be a prudent approach for future studies.
The livestock industry is the major source of income within the Ganzi Prefecture on the eastern Tibetan Plateau, and its development has led to considerable overlap between the living areas of local herders and wild habitats [84,85]. Shared grazing areas and water sources are key factors in facilitating the transmission of nematodes between wildlife and humans [86,87]. The current surveillance of foxes and humans on the Tibetan Plateau for zoonotic nematode infection focuses mainly on echinococcosis [88,89], with the transmission of some zoonotic nematodes, such as U. stenocephala, potentially being overlooked. Monitoring and investigation of human-animal nematodes in the eastern Tibetan Plateau need to be improved to determine the disease burden of zoonotic nematodes in the area. This would require the combined efforts of ecologists, wildlife specialists, veterinarians, human physicians, and relevant disease control personnel from a "One Health" perspective (encompassing humans, domestic animals, wildlife, and the changing ecosystems in which they live [90]) in order to better prevent the spread of zoonotic nematodes.

Conclusions
Foxes are dominant among wildlife populations on the eastern Qinghai-Tibet Plateau, especially Vulpes ferrilata, which exhibits a very high nematode infection rate. Uncinaria stenocephala was the most common zoonotic nematode, detected for the first time using molecular methods, in V. ferrilata at a rate of 35.83%. The Toxcascaris sp. found in this survey may represent a new species of the genus Toxascaris. Taken together, we suggest strengthening parasite monitoring among foxes on the eastern Tibetan Plateau in the future to prevent the local transmission of zoonotic nematodes.