Molecular characterizations of Giardia duodenalis based on multilocus genotyping in sheep, goats, and beef cattle in Southwest Inner Mongolia, China

Giardia duodenalis is an important zoonotic parasite that causes economic losses to animal husbandry and threatens public health. In the present study, a total of 1466 fresh fecal samples were collected from sheep (n = 797), goats (n = 561) and beef cattle (n = 108) in Southwest Inner Mongolia, China. Giardia duodenalis was initially screened via nested polymerase chain reaction (PCR) targeting the β-giardin (bg) gene, and bg-positive samples were subjected to PCR amplification targeting the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. A total of 4.0% of samples (58/1466) were positive for G. duodenalis, with a prevalence of 3.4% in sheep, 3.7% in goats and 5.2% in beef cattle. Three G. duodenalis assemblages (A, B, and E) were identified, with E as the prevalent assemblage. Four and one novel assemblage E sequences were obtained for the gdh and tpi loci, respectively and four assemblage E multilocus genotypes (MLG) were obtained. This study demonstrates high genetic variations in G. duodenalis assemblage E, and provides baseline data for preventing and controlling G. duodenalis infection in livestock in Inner Mongolia.


Introduction
Giardia duodenalis (synonym G. intestinalis and G. lamblia) is one of the most common intestinal pathogens in both humans and animals [25]. The symptoms of Giardiasis are diarrhea, abdominal pain and weight loss [1,10,30]. Livestock has been reported as a common reservoir of G. duodenalis, with an individual prevalence ranging from 0 to 73% [9,17]. Although G. duodenalis infection is commonly asymptomatic, many reports of Giardiasis in calves, goats and lambs show decreased weight gain and impairment in feed efficiency, causing significant economic losses to the farm [1,12,29].
Thus far, eight assemblages (A-H) of G. duodenalis have been identified based on genetic analysis and specific hosts [19]. Assemblages A and B have low host specificity and can infect humans as well as several other vertebrates; there are three assemblage A subgroups (AI, AII and AIII) and subgroup AIII has only been found in wildlife. However, assemblages C-H seem to be host-adapted; of these, assemblages C and D are mainly found in canines, assemblage E in artiodactyls, assemblage F in felines, assemblage G in rodents, and assemblage H in seals and some aquatic mammals [5,24]. Previous studies have shown that artiodactyls are predominately infected by assemblages A and E, and a few reports have described assemblage B in artiodactyls [32,33].
Giardia duodenalis is widely distributed in sheep, goats, and cattle (including dairy cattle, beef cattle, and yaks) in China [17]. Inner Mongolia is the third largest province in China, and animal husbandry makes an important economic contribution to the area. In Inner Mongolia, there are only three reports of G. duodenalis, in sheep and Bactrian camels [6,34,37]. More investigations are needed to facilitate improved interventions and minimize the burden of G. duodenalis in livestock. The objectives of this study were to further investigate and expand the prevalence information on G. duodenalis in ruminants in Southwest Inner Mongolia, China.

Ethical standards
Following the Chinese Laboratory Animal Administration Act of 1988, the research protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University (Approval No. IRB-HENAU-20180914-01). Appropriate permission from farmers was obtained before collecting fecal samples, and no animals were harmed.

Sample collection
From October 2019 to July 2021, a total of 23 farms were chosen randomly in northwest Inner Mongolia, China (Fig. 1). A total of 1466 fresh fecal specimens were collected from sheep (n = 797), goats (n = 561), and beef cattle (n = 108), respectively (Table 1). Of these, 1083 were from more than 12-month-old livestock, and 383 were from 7-12 month-old livestock; 419 samples were collected in the summer, 289 in autumn and 758 in winter (Table 2). Fresh fecal samples were collected by rectal sampling from ruminants in pens, and samples were gathered from the top layer of feces when grazing livestock defecated on the ground to ensure that there was no contamination [27]. Samples were stored in clean bags and transported in foam containers under ice conditions. No abnormal fecal specimens were observed during sample collection.

DNA extraction and PCR amplification
The genomic DNA of each fecal sample was extracted using a commercial E.Z.N.A Stool DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA), strictly following the specifications of the manufacturer. All the extracted DNA samples were stored at À20°C.
Giardia duodenalis was initially screened via nested PCR amplification targeting the bg [7] gene, and then studied by a MLG analysis based on the gdh [4] and tpi [28] genes. After amplification, the DNA fragments were separated by agarose gel electrophoresis (1% agarose) stained with DNA Green (TIANDZ, Beijing, China) and observed using a Tanon 3500 Gel Image Analysis System (TANON, Shanghai, China). Amplified samples with the target band were selected as positive PCR production (bg is 511 bp, gdh is 520 bp, tpi is 530 bp).

Sequence analysis
Positive PCR amplicons with the target band were sequenced by SinoGenoMax (Beijing, China). Bidirectional sequencing was chosen to ensure the veracity of sequences. The sequences in this study aligned with reference sequences from GenBank using ClustalX 2.1 (http://www.clustal.org/). Samples were amplified at the bg, gdh and tpi loci to form MLGs to further reveal genetic diversity. The same nomenclature system as in previous reports was used in naming G. duodenalis assemblage E subtypes at each genetic locus. Undesignated subtype sequences previously published and novel subtype sequences identified in this study were named accordingly as E36-E40 at the bg locus, E45-E52 at the gdh locus, and E32 at the tpi locus [6,7,22] (Table 3).
Phylogenetic analysis was conducted using the maximum composite likelihood model, and bootstrap values were calculated by analyzing 1000 replicates and the other cho-

Statistical analysis
A Chi-square test was performed, and 95% confidence intervals (CIs) were calculated using Crosstab in SPSS, version 24.0 (SPSS Inc., Chicago, IL, USA). A Pearson's chi-squared test was used for comparisons between two groups, and p < 0.05 was considered statistically significant.

Nucleotide sequence accession numbers
The representative nucleotide sequences were submitted to the GenBank at the National Center for Biotechnology Information under accession numbers: OL456202, OL456203, OL456204 and OL456206 for the gdh gene, and OL456207 for the tpi gene.
Among the positive samples in sheep, 11 were from pastoral sheep and 16 were from captive sheep, and there

Sequence and subtype analysis
A total of 58 bg sequences, 17 gdh sequences and 6 tpi sequences were obtained. Three kinds of assemblages were identified, including G. duodenalis assemblage A (n = 1), assemblage E (n = 56), and a mix of assemblages B and E (n = 1). Additionally, 4 samples were simultaneously amplified at all three intra-assemblage variation genetic loci (bg, gdh, tpi), forming 4 novel assemblage E MLGs (MLG-E1 to MLG-E4). The MLG-E2 and MLG-E4 sequences were obtained from sheep; the MLG-E1 sequences were obtained from goats, and the MLG-E3 sequences were obtained from beef cattle ( Table 4).

Phylogenetic analysis
Based on the G. duodenalis bg-sequences, gdh-sequences and tpi-sequences, three phylogenetic trees were constructed to evaluate the genetic relationships of the G. duodenalis isolates. The results showed that G. duodenalis isolates from this study were clustered within the G. duodenalis assemblage E, and high genetic diversity was observed in the assemblage E subtypes (Figs. 2-4).

Discussion
This study presented G. duodenalis distribution in sheep, goats and beef cattle in Southwest Inner Mongolia. Giardia duodenalis in this study were detected by bg locus, and the total infection rate was 4.0%. In previous reports using the same method, there was a higher G. duodenalis infection rate in Tan sheep in northwestern China (10.95%) [22], cattle in Turkey (30.2%) [21], beef cattle in Scotland (10.1%) [3], Tibetan sheep (13.1%) and yaks (10.4%) in Qinghai province, China [14]. However, there was a similar infection rate in healthy adult domestic ruminants in central Iran (5.2%) [15], and sheep in Inner Mongolia, China (4.3%) [34], which were detected by the tpi locus. Based on the SSU rRNA gene, G. duodenalis was detected in livestock in the United Kingdom (34.3%) and sheep in Inner Mongolia, China (64.1%) [6,18].
The SSU rRNA, bg and tpi loci have frequently been used to detect G. duodenalis. In this study, G. duodenalis in fecal samples was detected by nested-PCR of the bg locus, and only 29.3% and 10.3% bg-positive samples were amplified based on the gdh and tpi loci, which were similar to previous studies [3,14,21,22]. The difference between the G. duodenalis infection rate in this study and that in other studies which used the bg locus may be partially attributed to the Table 3. Intra-assemblage substitutions in bg, gdh, and tpi sequences within G. duodenalis assemblage E.

Serial number of samples Host
Genotype or subtype MLGs (bg-gdh-tpi) Beef All samples in this study were collected from non-diarrhea livestock in the age groups of seven months and older. The G. duodenalis infection rate was significantly different between different age groups of beef cattle (p < 0.001). Previous studies showed a higher prevalence in sheep, goats and cattle before weaning, and G. duodenalis infection is inversely associated with animal age [8,17,35]. The G. duodenalis infection rates in winter were significantly higher than in summer and autumn (p < 0.01), and the same phenomenon was reported in dairy calves in Norway and pigs in Denmark [13,23]; however, the season was not significantly associated with giardiasis infection of yaks in Qinghai, China [26].
Giardia duodenalis assemblages A, B and E were identified, and G. duodenalis assemblage E was the dominant assemblage found in this study, which is consistent with previous reports [6,7,25]. Giardia duodenalis assemblages A and E were identified as the two most common assemblages in sheep, goats and cattle, with assemblage B reported occasionally [11,25,35,36]. A few studies have reported assemblage C and assemblage D in livestock, but it is unknown whether this was an actual infection or mechanical transmission [15,18,20,31]. The G. duodenalis assemblages in this study were also reported in humans, companion animals and wildlife [24], and more research is needed to verify the potential impact on public health safety.
High genetic diversity was observed in the assemblage E subtypes. At the bg locus, eight published assemblage E subtypes were found in sheep, goats and beef cattle, and the bgpositive samples were analyzed by the multilocus genotyping tool with high resolution (gdh and tpi) to further reveal the genetic variations in G. duodenalis. A total of four and one novel assemblage E subtypes were found at the gdh and tpi loci, respectively and the analysis yielded four novel MLGs of assemblage E. A high degree of genetic diversity in G. duodenalis assemblage E has been reported in livestock, which was probably a cause of the high occurrence rate of G. duodenalis in Tibetan sheep and yaks [14,32]. In this study, the same G. duodenalis assemblage E subtypes (E1, E35 at the bg locus Figure 2. Phylogenetic relationships of beta-giardin (bg) nucleotide sequences of G. duodenalis assemblages (A-G) and assemblage E subtypes, using the maximum composite likelihood model. Percent bootstrap values greater than 50% from 1000 replicates are shown next to the branches. The hollow triangles represent published isolates in this study. and E45 at the gdh locus) were found in sheep, goats and beef cattle simultaneously, which may indicate a potential occurrence of cross-species transmission. Cross-species transmission of G. duodenalis assemblage E subtypes was also found in Tibetan sheep and yaks [14], black-boned sheep and blackboned goats [7].

Conclusion
The results of this study show that G. duodenalis is a common parasite in sheep, goats and beef cattle in Inner Mongolia, and the infection rate is related to the season, and age of beef cattle. Based on molecular analysis, three G. duodenalis assemblages (A, B and E) were found and assemblage E was predominant. Novel subtypes found in this study show further genetic diversity of G. duodenalis assemblage E. This study provides baseline data for preventing and controlling G. duodenalis infection in livestock.