Giardia duodenalis (also known as Giardia lamblia and Giardia intestinalis) is the most prevalent protozoan pathogen, commonly found in the intestinal tract of humans and animals worldwide (Adam, 2021). Transmission of G. duodenalis infection occurs by several routes either directly (i.e., person-to-person, animal-to-animal, or zoonotic infection) or indirectly (i.e., water or food) (Dixon, 2021). Approximately 280 million people are considered to be infected with G. duodenalis worldwide, with infection rates at the range of 8.0–30.0% in developing countries and 0.4–7.5% in developed countries (Feng and Xiao, 2011; Ryan and Zahedi, 2019). Giardiasis is generally a self-limiting clinical illness in humans, whereas it can be threatening to infants, young children, the elderly, institutionalized individuals, travelers, and immunocompromised individuals (Cacciò et al., 2018; Cai et al., 2021). Nitroimidazoles (e.g., metronidazole and tinidazole) are the most commonly drugs used to treat giardiasis, although requiring multiple doses and being often associated with adverse effects (Argüello-García et al., 2020).

To date, according to the reservoir and genetic characteristics of the protozoan, eight Giardia species have been recognized (Ryan et al., 2019). However, most studies for both public and veterinary health have focused on the taxonomy, population genetics, and epidemiology of G. duodenalis (Li et al., 2017a; Xiao and Feng, 2017). Based on studies employing iso-enzymatic and nucleic acid polymorphisms, G. duodenalis is known as a multispecies complex, consisting of eight genetic assemblages (A–H) considering different host distribution (Cacciò et al., 2018); assemblages A and B are commonly found in humans and occasionally in other mammals; assemblages C and D are often found in canids; assemblage E mainly infects ungulates; assemblages F, G, and H are specific to cats, rodents and pinnipeds, respectively (Ryan and Cacciò, 2013; Cai et al., 2021). Furthermore, putative sub-assemblages have been identified within assemblage A (AI–III) and assemblage B (BIII and BIV) using a multilocus genotyping (MLG) approach (Capewell et al., 2021).

China has the largest wild and captive populations of alpine musk deer (Moschus chrysogaster) in the world, which are mainly distributed in the Qinghai Tibet Plateau, Sichuan and Gansu Province (Jiang et al., 2021). Musk, produced by adult male alpine musk deer, is an important raw material for preparations of the traditional Chinese medicine and the fragrance industry. However, illegal hunting, habitat fragmentation, and other human activities have decimated wild alpine musk deer populations in China (Cai et al., 2020). For these reasons, the alpine musk deer has been listed as an endangered species by the International Union for Conservation of Nature (IUCN) and as category I-protected wild animal in China. Although the Chinese government has encouraged enterprises to participate in programs of breeding of captive alpine musk deer, gastrointestinal infections are the most significant threats to population growth and breeding scale whose fatality rate is approximately 30% (Li et al., 2017b). Currently, little information is available on the prevalence and genetic characteristics of G. duodenalis in cervids in China.

Thus, the aim of the present study was to investigate the prevalence and genetic diversity of G. duodenalis in captive alpine musk deer. The findings discussed herein provide insights into the development of preventive measures against Giardia infection.

Six species of musk deer (Moschus spp.) have historically been bred in China, which include siberian musk deer (M. moschiferus), forest musk deer (M. berezovskii), black musk deer (M. fuscus), alpine musk deer (M. chrysogaster), himalayan musk deer (M. leucogaster), and anhui musk deer (M. anhuiensis) (Fan et al., 2018). In previous studies, deer have been considered as a major reservoir of viruses, bacteria, and parasites for humans and livestock (Böhm et al., 2007; Mehrpad et al., 2018). In particular, a recent study has suggested the potential emergence of a new reservoir of SARS-CoV-2 viruses in free-ranging white-tailed deer, which may open new pathways for evolution, transmission to other wildlife species, and potential spillback of novel variants to humans (Hale et al., 2022).

Giardia spp. infects a broad range of hosts including humans, livestock, companion animals, wildlife and birds (Ryan et al., 2021). However, information on the distribution, molecular characteristics and zoonotic potential of Giardia in cervids is scant. To date, G. duodenalis infections in cervids have been reported in several countries, including Australia, Bangladesh, Canada, Croatia, Italy, Japan, Netherlands, Norway, Poland, Spain, Sweden, USA and China, with the infection rates ranging from 0.6% to 24.0% (Table 4). Interestingly, the rodent-specific species Giardia microti has been isolated in roe deer (Capreolus capreolus) in Croatia (Beck et al., 2011). In the present study, the prevalence of G. duodenalis was 19.3% (39/202) in alpine musk deer, which is higher than that reported in sika deer (0.6% and 0.8%) and forest musk deer (2.2%) in China. The discrepancies in infection rates of G. duodenalis may be related to the differences in geographical location, sampling season, animal species, and sample size. To the best of our knowledge, this is the first study to isolate and characterize G. duodenalis from alpine musk deer in China using MLG.

Methods based on sequence analysis of SSU rRNA, gdh, bg and tpi genes have currently been widely used for genotyping G. duodenalis isolates from human and animal samples in order to obtain high-sequencing resolution (Feng and Xiao, 2011). To date, molecular studies have identified G. duodenalis in fallow deer, forest musk deer, moose, red deer, reindeer, roe deer, sambar deer, spotted deer and white-tailed deer with a worldwide distribution (Table 4). In addition to zoonotic assemblages A and B, other G. duodenalis assemblages including E (mainly found in hoofed mammals) and D (mainly found in canines) have also been reported occasionally in these animal hosts (Beck et al., 2011; Huang et al., 2018), which suggests potential transmission routs of G. duodenalis assemblages between humans, livestock, companion animals and cervids. In the present study, both assemblage A and E were identified, and assemblage A was the predominant genotype. Mixed infections were observed in alpine musk deer at both assemblage and sub-assemblage levels, which may be a result of infection with Giardia parasites with different genetic profiles. Assemblages A is responsible for most giardiasis cases in humans, especially in South America and the Middle East (Xiao and Feng, 2017; Ryan et al., 2021). Interestingly, the host-adapted genotype assemblage E which was approximately 87% similar to assemblages A in genome, has been reported in at least 57 human giardiasis cases in Brazil, Egypt, Vietnam, Australia and New Zealand (Abdel-Moein and Saeed, 2016; Fantinatti et al., 2016; Zahedi et al., 2017; Garcia-R et al., 2021; Iwashita et al., 2021). Collectively, the presence of zoonotic assemblages of G. duodenalis in alpine musk deer in China suggests that these animals may potentially act as a reservoir of G. duodenalis for humans.

Moreover, in order to elucidate the genetic diversity of G. duodenalis in alpine musk deer, positive samples identified in the present were subjected for sub-genotyping by MLG. Moderate genetic variation was observed within assemblage A sequences, whereas no genetic variation was noticed within assemblage E sequences, which may be due to the low allelic sequence heterozygosity (ASH) in the genomes of assemblages A and E (Kooyman et al., 2019). In addition, the six assemblage A MLGs were composed of two novel MLG-subtypes which belonged to sub-assemblage AI. Among the three sub-assemblages within assemblage A, sub-assemblage AI is most commonly found in animals, whereas sub-assemblage AII is mostly found in humans; sub-assemblage AIII is rare and has been found in wild ruminants and two human giardiasis cases in Romania and New Zealand (Feng and Xiao, 2011; Cai et al., 2021; Ryan et al., 2021). In published studies, both sub-assemblage AI, AII and AIII were identified in various deer (Table 4). Further studies based on MLG analysis are necessary to gain a better understanding on the potential role of deer in the zoonotic transmission of G. duodenalis.

In conclusion, this is the first report of G. duodenalis in alpine musk deer with a high prevalence in China. Two G. duodenalis assemblages were identified, assemblage A and E. Moderate genetic diversity was observed within assemblage A sequences based on MLG analysis. Six assemblage A MLGs were identified which were composed of two novel MLG-subtypes belonging to sub-assemblage AI. Collectively, zoonotic assemblages of G. duodenalis identified in the present study point out that alpine musk deer may potentially act as reservoirs of this protozoan to humans.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

The animal study was reviewed and approved by Research Ethics Committee of Tarim University. Written informed consent was obtained from the owners for the participation of their animals in this study.

ZC, QW, XH, JB, BZ, BW, and XG performed the experiments. ZC drafted the manuscript. MQ and JL critically revised the manuscript. All authors contributed to the article and approved the submitted version.

The research was funded by the National Natural Science Foundation of China (32102689), Science and Technology Planning Project of Henan Province (222102110240), the Program for Young and Middle-aged Leading Science, Technology, and Innovation of Xinjiang Production & Construction Group (2018CB034), and Scientific research project of Xuchang University (2022GJPY009 and 2022YB034).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.