Prevalence and Genotype Distribution of Giardia duodenalis in Rabbits in Shandong Province, Eastern China

Giardia duodenalis is a zoonotic enteric parasite that can infect humans and a number of animal species including rabbits with a worldwide distribution. Infection with G. duodenalis can cause serious public health problems and significant economic losses to animal husbandry. So accurate understanding of the prevalence and genotype distribution of G. duodenalis in rabbits is necessary. In the present study, a total of 616 fecal samples were collected from rabbits in Shandong province, eastern China, and examined in G. duodenalis prevalence and genotypes by nested PCR amplification of β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) gene loci of G. duodenalis. Sixty-nine (11.2%) of the examined rabbit fecal samples were G. duodenalis-positive. Of them, the prevalence of G. duodenalis is 8.4% (41/490) in Rizhao city and 22.2% (28/126) in Weihai city. Breeds, region, and feeding modes were highly correlated with G. duodenalis infection in rabbits. Moreover, three genotypes (assemblages A, B, and E) were identified in rabbits at three gene loci, and the assemblage E was the dominant genotype, while the assemblage A was reported in rabbits in China for the first time. It is noticeable that two rabbits were found to be infected with two different G. duodenalis assemblages (assemblages A and E, assemblages B and E, respectively). These findings enrich the genotype distribution of G. duodenalis in rabbits and provide baseline data for preventing and controlling G. duodenalis infection in rabbits in eastern China.


Introduction
Giardia duodenalis (syn. Giardia intestinalis, Giardia lamblia) is a common gastrointestinal protozoon that causes enteric disease in a variety of animal species and humans [1][2][3][4]. More than 40 species of animals have been reported to be infected with G. duodenalis over the world [2,5]. Humans can be infected through ingesting water and food contaminated with G. duodenalis cysts [6]. e clinical symptoms of giardiasis are diarrhea, dehydration, abdominal pain, nausea, vomiting, and weight loss [2,6,7]. Giardiasis caused by G. duodenalis also has been recognized as an important zoonotic disease for both public and animal health [2,7,8].
Eight assemblages have been identified in G. duodenalis, including assemblages A-H [2,5]. e structure of eight assemblages is similar, but the genotypes are distinct [8]. Among assemblages A-H, both of the assemblages A and B are usually identified in various mammals, including humans [9], while the remaining assemblages mainly occur in the relatively specific groups of animals [10,11].
Rabbits are one of the most important economic animals in China, and the consumption of meat and fur accounts for a large part of China's economy [12]. However, rabbits are susceptible to many pathogens [5,13,14], including G. duodenalis [15][16][17][18][19][20], which can cause significant economic losses to the rabbit breeding industry.
ere are limited reports about G. duodenalis prevalence in rabbits in China [15,[18][19][20]; therefore, the objective of the present study was to investigate the prevalence and genotype distribution of G. duodenalis in rabbits in Shandong province, eastern China.

Collection of Fecal Samples.
A total of 616 rabbit fecal samples (490 from Rizhao city, 126 from Weihai city) were collected from Shandong province, eastern China. e fecal samples were composed of 207 New Zealand white rabbits, 188 long-haired rabbits, and 221 Tolai hares. Each fresh fecal sample was collected with gloves and placed into a box with ice, then marked with the breed, age, and region, respectively, and transported to the laboratory. All of the fecal samples were stored at − 20°C for further DNA extraction.

Genomic DNA Extraction.
Each rabbit fecal sample was washed with distilled water, uniformly stirred, and the residue was filtered through a 60-mesh sieve (0.23 × 0.23 mm). e filtrate was centrifuged at 4000g for 5 min in a centrifuge, the filtrate was discarded, and the precipitate was retained; then approximately 300 mg of each precipitate of fecal sample was used to extract genomic DNA using the commercial E.Z.N.A.R ® Stool DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA), following the manufacturer's recommendations. e extracted genomic DNA was stored at − 20°C for further PCR amplification.

Nested PCR Amplification.
e prevalence and genotypes of G. duodenalis were examined by nested PCR amplification of the bg, gdh, and tpi gene loci as described previously [2,15]. e primers and corresponding annealing temperature are shown in Table 1. Positive and negative controls were included in each PCR reaction. e secondary PCR products were electrophoresed in 1% (w/v) agarose gels containing ethidium bromide.

Sequence Analysis and Phylogeny.
All of the positive secondary PCR products were sent to Tsingke Biotechnology Technology Company (Xi'an city, China) for two-directional sequencing.

Statistical Analysis.
e relationship between the G. duodenalis prevalence in rabbits with different variables such as breed, region, age, and feeding mode was analyzed by the Chi-square (χ 2 ) test. e 95% confidence intervals (CIs) and odds ratios (ORs) were estimated by SPSS version 24.0 (SPSS Inc., Chicago, IL, USA). If the statistical resultP < 0.05, the difference was considered statistically significant.

Molecular Characterization of G. duodenalis Isolates.
Two G. duodenalis assemblages (assemblages B and E) have been reported in rabbits in China [15,[18][19][20]. In the present study, 616 rabbit fecal samples were used to identify G. duodenalis genotypes by nested PCR targeting the bg, gdh, and tpi genes. e results showed that 53 bg-positive samples and 39 gdh-positive samples were identified as assemblage E; two (2.9%, 2/69) tpi-positive samples and one (1.5%, 1/69) tpi-positive sample were identified as assemblage B and assemblage A, respectively. Interestingly, the assemblage E (98.6%, 68/69) was the dominant genotype in  rabbits in the present study, which was different from the previous studies [18,19]. Moreover, two mixed G. duodenalis assemblages (assemblages A and E, assemblages B and E) were identified in rabbits, and the assemblage A was firstly detected in rabbits in China in the present study. e G. duodenalis assemblage A was also reported in humans [9,22].
is finding suggests that the rabbits may be a potential source of human infection with G. duodenalis.

Phylogenetic Analysis of G. duodenalis Isolates in Rabbits.
To further elucidate the genetic relationship of G. duodenalis assemblages in rabbits, we aligned the obtained sequences with reference sequences in GenBank by Clustal X 1.83, which were used for phylogenetic analyses (Figures 1 and 2). e phylogenetic analyses showed that the G. duodenalis assemblage E in rabbits and dairy cattle was distributed on one branch, representing a closer genetic relationship ( Figure 2). ese findings indicated a possibility of spreading G. duodenalis between rabbits and dairy cattle. Moreover, the single nucleotide polymorphisms (SNPs) existed in bg sequences and gdh sequences in this study (Table 3) by comparing the obtained G. duodenalis sequences in the present study with corresponding sequences in the GenBank database. ese findings indicated that various G. duodenalis types are distributed in rabbits. ese findings enriched the genetic diversity of G. duodenalis in rabbits and other animals.

Conclusions
e present study revealed a higher (11.2%) G. duodenalis prevalence in rabbits in Shandong province, eastern China. Different feeding methods, breeds, and regions were highly correlated with G. duodenalis prevalence in rabbits (P < 0.05). ree G. duodenalis assemblages (assemblage A, assemblage B, and assemblage E) were identified in rabbits, assemblage A was reported in rabbits in China for the first time, while assemblage E was the dominant assemblage. ese findings not only enriched the genotype distribution of G. duodenalis in rabbits but also have an implication for better control of G. duodenalis in rabbits. Further studies are necessary to examine the infection status and genotype distribution of G. duodenalis in rabbits in other areas of the country.
Data Availability e G. duodenalis prevalence data used to support the findings of this study are included in the article.

Ethical Approval
All rabbits were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People's Republic of China, and the study was approved by the Animal Administration and Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Conflicts of Interest
e authors declare that they have no conflicts of interest.