Prevalence and molecular identification of gastrointestinal nematodes in Qinghai‐Tibetan Plateau of China

Abstract Background Gastrointestinal nematodes (GINs) have seriously affected the production and earnings of animal husbandry in various countries, while some species of GINs infect humans. At present, little is known about the species and prevalence of GINs in Qinghai‐Tibetan Plateau (QTP). Methods In this study, 528 fresh faecal samples were collected from typical areas in different altitudes with seven species of livestock in Qinghai, Tibet, Gansu and Yunnan Provinces. ITS‐2 rRNA gene of nematodes was employed to detect by PCR and sequencing analysis. Phylogenetic analysis of related sequences was performed using MEGA 6.0 software. Results The overall prevalence of GINs was 80.3% with 20 species of GINs detected, while Teladorsagia circumcincta was the dominant one, and four of which were zoonotic species such as Trichostrongylus colubriformis, Trichostrongylus axei, Trichostrongylus vitrinus and Oesophagostomum stephanostomum. Conclusion The study provided panoptic information on the prevalence and species diversity of GINs in QTP area, which is useful and valuable for reference of measure formulation in livestock husbandry and public health concerns. The parasites of T. circumcincta, Cylicocyclus nassatus, Strongylus edentatus, Cylicostephanus longibursatus, Telephlebia brevicauda, Cyathostomum catinatum, Mecistocirrus digitatus, Cooperia punctata, Cylicodontophorus bicoronatus, Nematodirus oiratianus and Oesophagostomum asperum were firstly reported the presence in QTP area. The study also showed that horse could be infected by T. circumcincta, goat could be infected by C. nassatus, cattle could be infected by S. edentatus and C. bicoronatus,and O. stephanostomum could infect yak, cattle and Mongolian sheep in worldwide. Nevertheless, more investigations are needed, such as microscopic examination, to accurately determine the species in the region.


INTRODUCTION
Gastrointestinal nematodes (GINs), as a common parasite are widely distributed in the worldwide (Saidi et al., 2020), giving rise to reduced production and economic losses in husbandry (Barghandan et al., 2020), are a major constraint to the survival and productivity of animals (Yuan et al., 2019).There have been many reports on the host of domestic or wild animals infected with GINs, such as sheep (Ploeger & Everts, 2018), European bison (Demiaszkiewicz et al., 2020) and chimpanzees (McLennan et al., 2017).Some species of GINs can also infect humans through ingested infective-stage larvae (Gholami et al., 2015) and cause zoonotic infections due to the wide range of human activities, which pose a challenge to public health concerns.
The Qinghai-Tibetan Plateau (QTP), with its average elevation over 4000 m, low annual temperature and oxygen content, and changeable climate, is the highest and largest plateau in China (Tang et al., 2018;Zhang et al., 2019).As the source of three rivers (the Yangtze River, Yellow River and Mekong River) (Jian et al., 2018), there is a large proportion of livestock husbandry in economy, with yak and Tibetan sheep as the primary livestock breeds.Due to the primitive breeding manners in the area, the livestock management is mainly family-running with extensive husbandry in small group rearing and lower productivity (J.-Z.Liu et al., 2016).When grazing in steppe of the Plateau, the animals may be infected with water and grass contaminated with eggs of nematodes.Once infected, these animals continuously excrete the faeces containing the eggs due to the failure of timely intervention, resulting in mutual infection within the same herd, which further aggravates the pollution of grassland and water, and poses a threat to the health of residents and livestock in downstream.
Although there are a variety of livestock and wild animals living in QTP area, few investigations have been carried out on the species and prevalence of GINs in the region.In this study, we conducted an epidemiological investigation of GINs in seven species of asymptomatic domestic animals from the QTP in order to provide references for animal husbandry and public health.

Sample collection
A total of 528 fresh faecal samples were collected from seven species of (Figure 1).The fresh faeces samples were collected quickly as soon as large animal excreted and placed into sterile tubes immediately.For small ruminants, the samples were directly acquired from the rectum of sheep or goat by hand covered with a sterile glove then placed into sterile tubes.Each of the tube was given a unique number, properly frozen with ice packs and immediately sent to the laboratory for further analysis.

DNA extraction and PCR amplification
Two hundred milligrams of faecal samples were taken out from the sterile tube to extract the genomic DNA by using TIANamp Stool DNA Kit (TIANGEN Biotech, Beijing, China) following the manufacturer's instruction.One of the steps was modified with lysis temperature increased from 70 to 95°C to obtain high quality and yield of DNA.
The extracted DNA was divided into three tubes, one of which was used for PCR amplification, and the other two tubes were stored at −80°C for further validation.A pair of primers from checked literature and with validation in pre-experiment (forward primer: TAGCTTCAGC-GATGGATCGGT; reverse primer: CTTTTCCTCCGCTAAATGATATGC) which could amplify the products of approximate 500 base pairs were used for targeting the ITS-2 rRNA gene to detect GINs as described previously (Bisset et al., 2014).The PCR amplifications was performed in a 25 µl reaction system with 12.

Sequencing and phylogenetic analysis
The obtained positive PCR products were sent to BGI (Beijing) for sequencing.All received sequences were searched by BLAST first, and the nematodes species corresponding to the matched homologous sequence (E-value = 0, identity > 96%) was obtained and identified as the species infected by the sample of the collected animal.
The representative nucleotide sequences acquired in the study have been submitted to the GenBank database under the accession numbers MT193646 and MT193665.Phylogenetic tree of related nematodes was constructed using the neighbour-joining algorithm and Kimura 2parameter model in MEGA 6.0 software, and the robustness of the tree topology was assessed using 1000 bootstrap replicates (Zhang et al., 2019).

Data analysis
The data were analyzed by the chi-squared test using SPSS 17.0 software to calculate the infection rates of GINs in different animals, The location of the sampling site selected for this study.♦ Sampling site elevations and regions.p-Values < 0.05 were considered a significant difference criterion.

Prevalence and distribution
The total infection rate of GINs against seven species of livestock was 80.3% (424/528) (Table 1), which ranged from 75.0% to 89.9% for different regions and altitudes (

Molecular identification
Twenty nematodes species were detected and identified by sequence analysis.Among them, the parasites Teladorsagia circumcincta was the dominant one with a total infection rate of 22.9% (121/528), which

Phylogenetic analysis
The phylogenetic relationship of the GINs species was established, and their reference strains were acquired form Nucleotide Database in NCBI.All of the species of GINs identified and the original reference strains were clustered into a same clade, and the clustering results of the study were consistent with the literature (Sun et al., 2020) The two strains between the evolutionary relationships was also close (Fig- ure 2), indicating that the results of our identification of GINs were credible.

DISCUSSION
GINs are the most common and economically destructive parasite in ruminants (El-Alfy et al., 2019).Because of their diversity and different susceptibility to anthelmintic (T.Han, Wang, et al., 2017;Rashid et al., 2018), it is important to identify their species, hosts and infection sites to clarify which animals can be easily infected in specific areas.
As known, the eggs of different species of GINs are very similar in size and shape.In addition, the identification for third stage of infective larvae requires the assistance of experienced specialist, which makes The molecular methods have the characteristics of rapidity and accuracy, which have great advantage for specific identification for different species of nematodes (Callaghan & Beh, 1994;Roeber et al., 2011).
Former investigations on GINs in QTP were mostly on morphological ways (Cao et al., 2020;J.-Z. Liu et al., 2016), few with specific molecular identification.In this study, the ITS-2 sequence was used to identify the species of GINs, which has been proved suitable and effective (Bisset et al., 2014;Dallas et al., 2000;Santos et al., 2020;Tan et al., 2018).
Before the present study, there were a few reports on GINs infection among animals limited in one district or single province of the QTP area.For example, the infection of GINs on yaks, cattle, sheep, goats, horses, donkeys, Tibetan pigs and other domestic animals in Tibet district with 63.8% (Wu et al., 2017), which is lower than the infection rate of 80.3% detected in this study, and the infection in wild female Tibetan antelope in Qinghai Province with 93% (Cao et al., 2020), which is similar to 92.5% infection rate of camels measured in this study, and little related with GINs infection against all species of livestock in large areas of QTP region.Hence, we sampled whole typical area in different elevation distributed with domestic animals in the QTP and detected 20 species of GINs from the faeces of seven species of livestock with 80.3% infection rate.Further, we also found that the parasites of T. circumcincta were the dominant species (28.5%, 121/424) in this region, which is common in sheep and goats in other areas (Costa-Junior et al., 2021;Hrabok et al., 2006) and also detected in large animals such as cattle, yaks and horses in this study.Up to now, there was a report on T. circumcincta-infected cattle, which was a detection of antibodies against the parasite in cattle faeces and serum (no microscopic examination done) (Cooke et al., 2019).One document reported the isolation of the parasite from the small intestine of yaks (L.Han, Zhou, et al., 2017) but no report that the infection case in horses.Our study demonstrated that T. circumcincta could not only infect cattle and yaks but may infect horses, and then expended the host range of this parasite.
Outside of QTP, there were some reports on livestock infection with GINs in other areas of China (Bhuiyan et al., 2017;T. Han, Wang, et al., 2017;Lv et al., 2016;Shen et al., 2019;Yuan et al., 2019), such as the sheep in inner Mongolian with 79.2% infection rate (T.Han, Wang, et al., 2017), which is consistent with the 78.8% ( 126 From a previous study, it was widely believed that T. axei, T. colubriformis, T. vitrinus and O. stephanostomum could infect humans (Bundy et al., 1985;Cibot et al., 2015;Elseadawy et al., 2021;Ghadirian & Arfaa, 1975;Phosuk et al., 2013) as zoonotic parasites, and there were already documents showing the infection with Trichostrongylus spp. in Henan (0.13%), Jiangsu (0.07%) and Jilin (0.06%) in local residents (Guan et al., 2015;Lee et al., 2017;Wei-Qi et al., 2017).Except for horses, these four species of zoonotic nematodes were all detected in six species of livestock in the QTP.Although there have been many reports on O. stephanostomum infecting animals and human in other countries (Cibot et al., 2015;Gasser et al., 1999;Krief et al., 2008;Terio et al., 2018), there is no case about the parasite infecting human in China at present.The infection rate of T. axei and T. colubriformis on Tibetan sheep detected in this study was 5.8% and 0.8%, respectively.Considering the similar investigation in sheep conducted in Heilongjiang Province with the infection rate of 10.4% and 36.8%(C.R. Wang et al., 2006), which was much higher than our results, indicating that the parasites of T. axei and T. colubriformis might be more suitable to live in regions at lower elevations than at higher ones while the management level in the former would not be much lower than in the QTP area.Till now, there was only one case about the infection of O.
stephanostomum in China reported in Tibetan pigs (Li et al., 2017).This study identified that O. stephanostomum infected yaks, cattle and Mongolian sheep.Besides, the infection status on parasites of Trichostrongylus spp.evaluated in healthy yak in Gansu Province previously (Li et al., 2018) is the same as what we detected, suggesting that nomads in this region need to be vigilant for potential infection.
In addition, there were other four species of nematodes detected in the study, such as T. circumcincta, Marshallagia marshalli, Haemonchus contortus and Ostertagia ostertagi, considered as zoonotic potential as well.Looking through literatures, there have been two reports on human infection with these four parasites and the both occurred in Iran (Ashrafi et al., 2020;Ghadirian & Arfaa, 1973).Evidently, the scope and severity of zoonosis caused by these four parasites need to be further studied and determined.The nematodes of Marshallagia spp., Haemonchus spp.and Ostertagia spp.usually live in the abomasum and small intestine of ruminants such as camels, cattle and sheep (Besier et al., 2016;Lichtenfels et al., 1988).In this study, we also detected marshalli infection of yaks (Sun et al., 2018) and wild animals, such as M. marshalli infection of Tibetan antelope and Przewalski's Gazelle in the QTP region (Cao et al., 2020;Sun et al., 2018;Y. Wang et al., 2016).As for H. contortus and O. ostertagi, both parasites have been widely reported their infections in ruminant livestock such as goats, yaks and sheep (including Tibetan sheep) in the QTP (Khan et al., 2019;Z. Liu et al., 2018).Another nematode infected yaks and cattle with relatively large amounts (n = 60) was C. oncophora, which is frequently parasitic in the small intestine of ruminants (Sun et al., 2020) and has been identified in yaks before in the QTP region (L.Han, Zhou, et al., 2017).
Beyond the above-mentioned parasites, we have also found 11 species of nematodes in the QTP area but with a few infection numbers less than 10 individuals.Except for C. ovina detected in the goats, Tibetan sheep and yaks which had been reported several times in literatures infected yaks and Tibetan sheep in the QTP (Xu et al., 2016;L. Zhao et al., 2015).The rest of the 10 parasites, either some nematodes identified in the QTP for the first time, such as Cylicostephanus longibursatus (n = 5), Triodontophorus brevicauda (n = 2) and C. catinatum (n = 1) which are commonly found in equine animals (Gao et al., 2017;Tzelos et al., 2020) and detected in horse in this study, Mecistocirrus digitatus, C. punctata living in the abomasum and small intestine of ruminants (Paguem et al., 2020;von Son-de Fernex et al., 2014) and also detected in cattle in the study.Nematodirus oiratianus and Oesophagostomum asperum commonly had been found in ruminants in other countries (Santos et al., 2020;G.-H. Zhao et al., 2013G.-H. Zhao et al., , 2014) also detected in the QTP in the study.Some nematodes not only found in the QTP area but also out of original host and may have a wide range of infection.
Cylicocyclus nassatus (n = 9) and Strongylus edentatus (n = 7) infected horses out of China (Fabiani et al., 2016;Kooyman et al., 2016;McClure et al., 1994;Studzinska et al., 2012;van Doorn et al., 2014) but infect goats and cattle in the study as well.Cylicodontophorus bicoronatus usually live in the colon and cecum of equine animals (Silva et al., 1999) but detected in cattle in the QTP area.The latter three parasites were found all beyond original hosts and may have a wide range to infect.
In the process of this study, we were surprised to see a number

CONCLUSION
This study included whole typical area in different elevation distributed with domestic animals and provided useful and valuable information on the prevalence and species diversity of GINs in QTP of China.A total of 20 species of GINs were detected and identified from seven species of domestic animals, four of which were zoonotic species, such as T. axei, T. colubriformis, T. vitrinus and O. stephanostomum.Some parasites detected the presence in QTP area for the first time, such as T. The results indicated the severe situation that animal husbandry faced both in domestic livestock and wild animals.
/160) of the sheep (Tibetan and Mongolian sheep) in QTP identified in this study.Combining our result and others in the recent study, the infection of GINs is widely distributed in China, indicating the severe situation that animal husbandry faced both in domestic livestock and wild animals.
the M. marshalli in cattle, Tibetan sheep and goats, H. contortus in Tibetan sheep and goats and O. ostertagi in yaks.These results were F I G U R E 2 The phylogenetic relationship of the gastrointestinal nematodes (GINs) species detected in this study and those of related nematodes.Bootstrap values > 50% calculated from 1000 replicates are shown at the nodes.The black filled circles represent nematode species identified in the present study consistent with a few investigations on domestic animals, such as M.
of parasites normally parasitic to ruminants were detected in horse, as well as normally parasitic to equine animals but found in ruminant faeces.Therefore, we re-examined every process of sample collection, DNA extraction, PCR amplification, sequencing and alignment, reperformed the above experiments on the samples.Once again, the results above were confirmed, showing there was no fault such as sample and PCR contamination or the label confused.The reason of the results might be one possible way due to cross-species transmission and mixed infection of these parasites caused by the livestock overlap in raising plot at night in this area, and hence more investigation would be needed in the area, such as microscopic examination.Because of the serious harm to humans and impeding to livestock production and livestock, more investigations and studies for GINs are needed to accurately determine the species, pathogenicity, distribution and prevalence at different altitudes and regions in the QTP.
Detection and species distribution of gastrointestinal nematodes (GINs) in different kinds of livestock in the Qinghai-Tibetan Plateau (QTP) of China Prevalence of gastrointestinal nematodes (GINs) in different areas and altitudes in Qinghai-Tibetan Plateau (QTP) of China TA B L E 1 circumcincta, C. nassatus, S. edentatus, C. longibursatus, T. brevicauda, C.   catinatum, M. digitatus, C. punctata, C. bicoronatus, N. oiratianusand O. asperum.To our knowledge, this is the first report identifying the presence of O. stephanostomum in yak, cattle and Mongolian sheep, T. circumcincta in horse, C. nassatus in goat, S. edentatus and C. bicoronatus in cattle worldwide, suggesting that O. stephanostomum, T. circumcincta, C. nassatus, S. edentatus and C. bicoronatus may have a wide range of hosts.