**1. Introduction**

*Giardia duodenalis* (syn. *G. lamblia*, *G. intestinalis*) is a flagellate protozoan parasite recognized as a significant global contributor to diarrheal disease, affecting humans, domestic animals, and wildlife across the globe [1,2]. The majority of *G. duodenalis* infections are asymptomatic; however, in rare cases, some patients may experience severe gastrointestinal disturbances for several weeks [3]. As *G. duodenalis* utilizes the fecal–oral route for lifecycle maintenance, projections indicate that this parasite causes ~28.2 million foodborne disease cases [4,5]. Based on the above data, the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) in 2014 ranked *Giardia* 11th of 24 food-borne parasites [4].

The current wide use of genotyping tools has immensely improved our understanding of *G. duodenalis* transmission in humans and animals [5,6]. At least eight genotypes or assemblages have been described, including assemblages A and B containing zoonotic isolates potentially infecting humans and animals, and assemblages C–H, which exhibit specificity to particular animal hosts [7]. Moreover, several molecular markers (triosephosphate isomerase, *tpi*; glutamate dehydrogenase, *gdh* and beta giardin, *bg*) have been developed to

**Citation:** Cui, Z.; Wang, D.; Wang, W.; Zhang, Y.; Jing, B.; Xu, C.; Chen, Y.; Qi, M.; Zhang, L. Occurrence and Multi-Locus Analysis of *Giardia duodenalis* in Coypus (*Myocastor coypus*) in China. *Pathogens* **2021**, *10*, 179. https://doi.org/10.3390/ pathogens10020179

Academic Editors: David Carmena, David González-Barrio and Pamela Carolina Köster Received: 29 December 2020 Accepted: 4 February 2021 Published: 7 February 2021

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create a multi-locus genotyping (MLG) tool for subtyping assemblages A, B, and E and to explore the population genetic characterizations of *G. duodenalis* [6,8]. Subsequently, the MLG tool subdivided assemblage A into sub-assemblages AI, AII, and AIII and assemblage B into sub-assemblages BIII and BIV [9].

In recent years, studies on the epidemiology of *G. duodenalis* have been conducted for humans, non-human primates, ruminants, companion animals, domestic animals, wildlife, and in the environment in China [10]. However, limited information has been provided on the infection rate and genotype characteristics in rodents in China. The coypus (*Myocastor coypus*) is a large, amphibious rodent native to South America, which has become invasive in Europe and other parts of the world except Oceania and Antarctica [11]. Coypus were first introduced to China in 1956, then later widely reared in farms as important fur-bearing animals. The climate of China is very suitable for the growth of coypus, and 16 color-type strains have been bred. The number of the national stock reached more than 400,000 in 2000. To date, little is known about the genetic characteristics of *G. duodenalis* in coypus globally. Only two studies in Italy and the USA reported *Giardia* spp. prevalence in coypus, but neither identified the species [12,13]. Thus, the present study aimed to explore the distribution and genetic diversity of *G. duodenalis* in coypus in China and assess its zoonotic potential based on MLG analysis.

#### **2. Results**

#### *2.1. Occurrence of G. duodenalis*

All samples were initially tested using nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene. Of the 308 samples, *G. duodenalis* was present in 38 samples (12.3%, 95% Cl: 8.5–16.2%) (Table 1). Each examined farm had infected animals. Notably, the highest infection rate of *G. duodenalis* in coypus was detected in Baoding (28.6%, 10/35), followed by Ganzhou (25.7%, 9/35), Chengdu (15.0%, 6/40), Laibin (13.6%, 3/22), Yongzhou (13.0%, 3/23), Kaifeng (11.5%, 6/52), and Anyang (1.0%, 1/101) (Table 1).



N = number of positives for *G. duodenalis*; No. = number of samples; T = total analyzed samples.

By age, the highest infection rate was reported in the 3–6-month-old group (14.9%, 7/47), followed by >6-month-old group (14.8%, 29/196), and <3-month-old group (3.1%, 2/65) (Table 2). Furthermore, the correlation of age with the infection rates was evaluated based on the calculated ORs and 95% CI values (Table 2). There was a strong positive correlation between the infection rate and age, with an OR of 5.51 (95% CI: 1.09–27.87, *p* = 0.023) associated with the 3–6-month-old group and an OR of 5.47 (95% CI: 1.27–23.59, *p* = 0.011) associated with the >6-month-old group.

**Age (Month) N/T (%; 95% CI) Assemblage**


N = number of positives for *G. duodenalis*; OR: odds ratio; T = total of analyzed samples.

**Table 2.** Distribution of *G. duodenalis* assemblages in coypus of different ages.

#### *2.2. Assemblage A and B Subtypes*

Here, two genotypes, assemblages A (2) and B (35), were identified based on sequence analysis of the SSU rRNA, *tpi*, *gdh*, and *bg* loci (Table 1). Notably, assemblage B was the dominant genotype (92.1%, 35/38). Mixed infection was found in one sample. To reveal the genetic diversity of the *G. duodenalis*-positive samples, we sequenced the *tpi*, *gdh*, and *bg* genes, from which 25, 11, and 12 sequences were obtained, respectively (Table 3).

**Table 3.** Multi-locus characterization of *G. duodenalis* isolates in coypus in China based on *bg*, *gdh* and *tpi* genes.


\* New variants without heterogeneous positions. MLG: multi-locus genotypes; PN: PCR negative.

Of the *tpi* sequences, 3 and 22 belonged to assemblages A and B, respectively. Sequences from all isolates from assemblage A exhibited two single-nucleotide polymorphisms (G155T and G186A) relative to the MN174855 sequence. Within the assemblage B isolates, three subtypes were formed, which were designated as B1 (*n* = 17), B2 (*n* = 3), and

B3 (*n* = 2) for convenience. Of note, B1, B2, and B3 sequences were identical to MH644772, KM977653, and HM140711, respectively. At the *gdh* locus, 1 and 10 samples were found positive and identified as assemblages A and B, respectively. The one assemblage A sequence was identical to MN174853. Among the assemblage B sequences, B1 (*n* = 5), B2 (*n* = 3), and B3 (*n* = 2) sequences were identical to KM977648, MK952603, and MK982476, respectively. Sequence analysis demonstrated high genetic diversity in assemblage B at the *bg* locus. Ultimately, five subtypes (B1–B5) were formed in the 11 assemblage B sequences. The subtypes B1 (*n* = 4), B2 (*n* = 2), B3 (*n* = 3), B4 (*n* = 1), and B5 (*n* = 1) exhibited consistency with MT487587, MK982544, MN174847, MT487587, and KY696837, respectively. Moreover, one assemblage A sequence was identical to MN704938.
