Giardia duodenalis in Hu sheep: occurrence and environmental contamination on large-scale housing farms

Giardia duodenalis is a common zoonotic intestinal parasitic protozoan and sheep are among its hosts; however, limited information is available on sheep kept in large-scale housing. The Hu sheep is a first-class protected local livestock breed in China. In this study, we investigated the seasonal dynamics of G. duodenalis infection in Hu sheep and the environmental contamination of large-scale sheep farms. We collected 474 fecal samples and 312 environmental samples from Hu sheep on a large-scale sheep farm in Henan, China. The prevalence of G. duodenalis was determined by nested PCR targeting the β‑giardin (bg) gene. The assemblages and multilocus genotypes (MLGs) were investigated based on analyses of three genetic loci, i.e. bg, glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi). To detect mixed infections of different assemblages, assemblage A/E-specific PCRs were performed to amplify the tpi gene. The prevalence of G. duodenalis infection in sheep was 17.9% (81/474) and the positivity rate in environmental samples was 0.96% (3/312). Genetic analysis revealed the presence of two assemblages (assemblages A and E), with assemblage E being detected in both fecal and environmental samples, and assemblage A detected only in fecal samples. A total of 23 MLGs were obtained in fecal and environmental samples, all of which belonged to assemblage E. These results indicate the seasonal dynamics of G. duodenalis infection in sheep and environmental contamination on large-scale housing sheep farms and provide an important reference for the prevention and control of G. duodenalis on large-scale housing sheep farms.


Introduction
Giardia duodenalis is a typical intestinal parasitic protozoan that is most often spread through polluted food and water. Most animals and humans are susceptible to infection by this parasite, which hinders the reproductive ability of adult animals and the growth of young animals. Giardiasis, which is usually asymptomatic but can cause symptoms such as bloating, diarrhea, nausea, vomiting, and indigestion, affects more than 28.5 million people in China each year and is the second most common cause of infectious diarrhea in humans after viruses [5,6,13,22]. The World Health Organization included giardiasis in the Neglected Diseases Initiative in 2006 [29].
Based on genetic analysis, G. duodenalis can currently be divided into eight assemblages (assemblages A-H) [15]. Most mammals can be infected by assemblages A and B, which are commonly categorized as zoonotic assemblages [16,17,26], while the other assemblages (C-H) are considered hostspecific [11,28]; however, this is a relative concept. Cats [24] and pigs [21] have been found with assemblage D, humans with assemblages C [31] and E [10], and pigs with assemblage F [1].
The dominant assemblage in sheep is assemblage E; assemblages A and B are also reported, of which the former is more common than the latter [18,19,25,35]. Previous research has shown that the infection rates for G. duodenalis ranged from 0% to 64.11% [4,22,34] in sheep in China, and the potentially high infection rates in sheep mean that they are considered potential transmission sources for human infection.
The Hu sheep is a first-class protected local livestock breed in China with characteristics like heat and moisture resistance, early maturity, and good meat quality. The breed was listed in the National Catalogue for Livestock and Poultry Genetic Resources of the Ministry of Agriculture in 2000 and 2006. However, the occurrence of G. duodenalis on large-scale sheep farms, including those housing Hu sheep, has not been thoroughly investigated.
Henan Province is located in the southern part of the North China Plain in the middle and lower reaches of the Yellow River, and most of the area is located in the warm temperate zone with four distinct seasons and simultaneous rain and heat. As an important grain growing base, and sheep farming is mostly intensive. The large breeding density leads to a favourable condition for the occurrence of G. duodenalis in sheep. In this work, the seasonal dynamics of G. duodenalis, based on environmental and fecal samples, were explored on a largescale housed Hu sheep farm in Henan Province. In addition, based on MLG analysis, we aimed to identify the genetic features of G. duodenalis infection and environmental contamination of Hu sheep on large-scale sheep farms, to evaluate the danger of zoonotic transmission, and to specify the importance of this protozoan parasite for public health.

Ethical standards
This study was conducted in accordance with the Chinese Laboratory Animal Administration Act (1988) after it was reviewed and its protocol approved by the Research Ethics Committee of Henan Agricultural University. Appropriate permission was gained from the animal owners before the collection of fecal sample specimens from the animals. Sheep at different physiological stages were selected, including lactating lambs (1-20 days old), weaning lambs (1-3 months old), fattening lambs (4-6 months old), young lambs (7-12 months old), non-pregnant ewes, early pregnancy ewes (3 months gestation), late pregnancy ewes (1 week before parturition), lactating ewes, and breeding rams. Fresh feces from the animals were collected by rectal sampling, stored in clean containers, numbered, registered, and taken to the laboratory for storage at 4°C until the DNA was extracted.

Sample collection
Sheep housing soil samples (5-30 g) were randomly collected at the entrance, fecal leakage board, exit, and corridor of the sheep house, put in clean containers, numbered, and register, delivered to the laboratory, and stored at 4°C until the DNA was extracted.

DNA extraction and PCR amplification
For DNA extraction, 100 mg of each fecal or environmental sample was put into a 1.5-mL centrifuge tube, and an E.Z.N.A Stool DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) was used to extract genomic DNA, according to the manufacturer's instructions. The DNA was stored at À20°C before use.
To investigate the presence of G. duodenalis, all DNA samples were tested by nested PCR targeting the bg gene. All bg-positive samples were further subjected to gdh and tpi gene amplification to analyze the MLGs of G. duodenalis and record mixed infections. To further understand the characteristics of the mixed infections, specific primers targeting the tpi locus were selected to identify assemblages A and E [14,20,27,30,34]. The PCR products were subjected to 1% agarose gel electrophoresis, developed with DNA Green (Tiandz, Beijing, China), and analyzed on a Tanon 3500 Gel Image Analysis System (Tanon, Shanghai, China).

Sequencing and sequence analysis
All secondary positive PCR amplicons were purified and sequenced in both directions by a commercial company (SinoGenoMax Co. Ltd, Beijing, China). The obtained sequences were uploaded to seqman in DNAStar (http://www. dnastar.com/) to proofread the DNA chromatogram and compared and identified using Clustal X 2.0 (http://www.clustal. org/) and GenBank. Analysis of the amplification results for three genes (bg, gdh, tpi) allowed us to further conceptualize the genetic diversity of G. duodenalis.

Statistical analysis
The chi square test in SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used to calculate differences in the incidence of G. duodenalis among sheep of different physiological states, samples from different enclosure environments, and samples from different seasons. Differences were considered statistically significant when p < 0.05.

Phylogenetic analysis
To study the relationship between different isolates in more detail, phylogenetic analyses were performed using a concatenated dataset of bg, tpi, and gdh sequences with the MLGs of G. duodenalis.
The tandem sequence (bg-tpi-gdh) was used for phylogenetic reconstruction using Neighbor-Joining analysis, and the genetic distances were calculated in the Kimura 2-parameter model in MEGA10 (https://www.megasoftware.net/). Bootstrap analysis (1000 replicates) was used to evaluate the reliability of the phylogenetic tree.

Seasonal meteorological information
Information on the average temperature and precipitation data over the four seasons in Ruzhou was obtained through the Chinese Central Meteorological Station (http://www.nmc.cn/).

Nucleotide sequence accession numbers
The NCBI's GenBank database has received a deposit of representative nucleotide sequences from this study, and the accession numbers are OP142425-OP142427 for the bg gene, OP142428-OP142431 for the gdh gene, and OP156823-OP156824 for the tpi gene.
Among the fecal samples, the rate of parasite-positive samples was the highest in spring (30.37%) and the lowest in autumn (7.75%). The rates of positive samples in winter and summer were 19.23% (20/104) and 9.43% (10/106), respectively. The rate of positive samples differed among the seasons, and all differences between seasons were statistically significant (p < 0.05), except for between summer and autumn (p > 0.05). The differences between spring, summer, and autumn, and the difference between winter and autumn were highly significant (p < 0.01). In environmental samples, G. duodenalis was only detected in summer (all positive samples were taken from the fecal leakage board), and there were no significant differences between these environmental samples and those in other seasons (p > 0.05) ( Table 1).
On analyzing the presence of the parasites in Hu sheep of different physiological stages, we discovered that the highest infection rates of G. duodenalis occurred in weaning lambs (43.08%), and lactating ewes had the lowest infection rates (4.23%). The prevalence of G. duodenalis in late-pregnancy ewes (8.62%) was lower than that in early-pregnancy ewes (20.37%) and non-pregnant ewes (22.73%), while the prevalence of other groups ranged from 5.00% to 22.73%. The G. duodenalis infection rate of weaned lambs was significantly higher than that of Hu sheep of other physiological stages (p < 0.05) ( Table 2).

Giardia duodenalis assemblage distributions
In the nested PCR analysis of the bg gene, out of 81 G. duodenalis-positive fecal samples, six were zoonotic assemblage A and 75 were assemblage E, and all three positive environmental samples belonged to assemblage E.
When the 84 bg-positive samples were analyzed based on the gdh gene, 69 of the fecal samples and all 3 environmental samples belonged to assemblage E. For bg-positive samples, DNA sequence analysis of the tpi locus revealed the occurrence of assemblage A (15) and assemblage E (67) in fecal samples and assemblage E (3) in environmental samples, and assemblages A and E co-occurred in 12 fecal samples ( Table 1).

Subtypes of assemblages A and E
A total of 84, 72, and 85 sequences were obtained for the bg, gdh, and tpi loci, respectively, from Hu sheep and barn environment samples (Table 1).
Using GenBank sequence KJ027408 as a reference, six bg locus sequences were identified, three of subtype AI and three with two single-nucleotide polymorphisms, named A1. Similarly, the GenBank sequence FJ560569 was utilized as a reference, and the 15 sequences at the tpi locus were used to classify the parasites into three subtypes, including two known subtypes (A4 and A6) and a novel subtype A8 (Table 4).     (Table 5). Phylogenetic analysis showed that all assemblage E MLGs were significantly different from those reported previously [23,33] for G. duodenalis (Fig. 1).

Discussion
This paper reports, for the first time, the seasonal dynamics of G. duodenalis infections in Hu sheep and environmental contamination of large-scale housing sheep farms in China. The results showed that the incidence of G. duodenalis in Hu sheep was 17.09% (81/474), and in environmental samples, it was 0.96% (3/312). The prevalence of G. duodenalis in Hu sheep was higher than the previously reported prevalence of G. duodenalis infections in sheep in Henan Province (1.82-5.64%) [22]; however, it was far lower than that in Inner Mongolia (64.11%) [4]. This variation may be due to a range of factors, including the presence of other animal species on the farm, farming practices and size, inspection methods, study design, number of samples analyzed, timing of sample collection, and environmental conditions.
Giardia cysts can survive for months in high-humidity, low-temperature, low-sunlight-level, and low-salinity environments [9,11]. Ruzhou City, Henan Province, has a warm temperate semi-humid continental monsoon climate with four distinct seasons: it is warm in spring, hot in summer, cool in autumn, and cold in winter. This may explain why G. duodenalis infection rates are highest in spring (30.37%). Furthermore, the relatively cold winter temperatures, as well as the dry air in Ruzhou, may be the reason why the infection rate is lower in winter (19.23%) than spring. It is possible that the summer is not suitable for G. duodenalis transmission, resulting in lower infection rates (9.43%), and the influence of the summer results in lower infection rates in the autumn (7.75%).
A literature search shows that most reports describe a negative correlation between G. duodenalis infection rates and age [7,32,36], which is supported by our findings. Not all lambs had higher infection rates than adult sheep, but the rates of G. duodenalis were the highest in weaning lambs (43.08%, 28/65), which may be because weaning is a period of transition of feeding methods, and the lambs' immunity is low because of stress. Moreover, the parasite prevalence in lactating lambs (12.82%, 5/39) was lower than that in fattening lambs (17.65%, 9/51), probably because the lactating lambs were fed by fewer infected lactating ewes, reducing the probability of cross-infection. It was also found that the infection rate of late-gestation ewes was lower than that of early-gestation ewes and non-pregnant ewes. This phenomenon may be attributable to pregnant ewes being fed a more varied diet than their nonpregnant counterparts. Furthermore, pregnant ewes are typically better cared for and housed in more comfortable, quiet, and secure enclosures. Because of interactions among these factors, pregnant ewes may have had higher immunity and disease resistance, resulting in lower infection rates than non-pregnant ewes.
In this study, 312 environmental samples were collected. Although only three positive samples were found in the summer (all on the fecal leakage board), the results suggest that G. duodenalis was present in the environment. Because of the high density of livestock on large-scale sheep farms, G. duodenalis in the environment is likely to be consumed by sheep, allowing the transmission cycle to continue [2]. The wastewater used for cleaning fences is also at risk of entering the irrigation water used for vegetables, thus increasing the infection risk for humans and other animals [8,12].
Available studies suggest that assemblage E is the dominant assemblage of G. duodenalis in sheep, while assemblages A and B have also been occasionally reported [22]. The results Table 4. Intra-assemblage substitutions of assemblage A at the bg and tpi loci.

Subtypes
Nucleotide at position of this survey are the same as those of other surveys. Based on nested PCR amplification of bg, gdh, and tpi, assemblage E was still the dominant cluster of G. duodenalis in Hu sheep, followed by assemblage A, while assemblage B was not found. A total of 18 samples were detected with mixed infections of assemblages A and E using specific primers for the tpi locus of these assemblages. Because commonly used primers for the tpi locus were unable to detect two or more assemblages in the same sample in the past, it can be inferred that the identification of assemblage A and mixed infections in previous investigations were relatively low because of the specificity of the primers [30]. The public health impact of assemblage A, a common human-animal G. duodenalis assemblage, and the risk of sheep acting as potential G. duodenalis conservator hosts have been simultaneously underestimated.
To further reveal the genetic variations in assemblages A and E, another two genetic loci (gdh and tpi) of 84 bg-positive samples were analyzed with high resolution via the multilocus genotyping tool. In the fecal samples from Hu sheep, seven assemblage E subtypes were identified using the gdh locus, and four assemblage E subtypes as well as three assemblages A subtypes were identified using the tpi locus. In the environmental samples, three assemblage E subtypes were identified using the gdh locus, and two assemblage E subtypes were identified using the tpi locus (Tables 3 and 4). In addition, 14 samples contained different assemblages at the three loci (Table 5). Twenty-three MLGs at all three loci of assemblage E were successfully amplified from 57 samples with the same assemblage genotype. These findings suggest that there is high subtype diversity and genetic variation within assemblage E.

Conclusions
This dynamic survey of G. duodenalis in fecal and environmental samples collected in different seasons from a large-scale sheep farm in Henan Province revealed the prevalence of G. duodenalis in Hu sheep and environmental contamination. The prevalence of G. duodenalis in Hu sheep was 17.09% (81/474) and the rate of G. duodenalis in the environment was 0.96% (3/312), indicating that the prevalence of the protozoa in Hu sheep is high and it can be transmitted through the environment. Large-scale farms with a high density of sheep should take measures to avoid horizontal transmission of G. duodenalis. In addition, mixed infections of assemblages A and E in Hu sheep were more severe than reported by previous studies, suggesting that the presence of assemblage A has been underestimated. Therefore, the significance of G. duodenalis in sheep for public health may need to be re-examined. Figure 1. Phylogenetic relationships among G. duodenalis MLGs. The phylogenetic tree was constructed using a concatenated dataset of the bg, tpi, and gdh gene sequences and Neighbor-Joining analysis with the Tamura-Nei model. MLGs with unambiguous sequences identified in this study (Table 5) were analyzed with reference isolates. Bootstrap values greater than 50% from 1000 replicates are shown at the nodes. HN: Henan; SX: Shaanxi; JX: Jiangxi; HS: Hu sheep; EN: Environment.