Background

Giardia duodenalis (syns. G. lamblia and G. intestinalis) is a common zoonotic protozoan that inhabits the gastrointestinal tracts of animals and humans [1,2,3]. Giardiasis, caused by G. duodenalis, is an important diarrheal disease concerning both public and veterinary health worldwide [4, 5]. In recent decades, G. duodenalis infection has been widely reported in domestic (e.g. goats, pigs, cats, rabbits, dogs and calves) and wild animals (e.g. chipmunks, chinchillas, red foxes, lizards, aquatic birds, boars and rhesus macaque) in Asia, Europe, Australia, North America and Africa [6,7,8,13,14,15,16,17,18,19], and a wide distribution and high prevalence of up to 100% in some studies have been reported [20, 21]. Although the infection is commonly asymptomatic or self-limiting in immunocompetent hosts [23], watery diarrhea and other significant clinical impacts due to G. duodenalis infection could be observed in young, undernourished, or immunocomprised individuals [24, 25]. Furthermore, dormant and long-lasting infectious cysts excreted from hosts can pollute food, water and the environment, causing food-borne and water-borne outbreaks for humans and animals [26, 27].

The bamboo rat (Rhizomys sinensis), a rodent species belonging to the subfamily Rhizomyinae, is widely distributed in southern Asia, i.e. southern China, India, Myanmar, northern Vietnam and Thailand [28, 29]. Because of the high protein content, low fat and cholesterol of bamboo rat meat [30], the medicinal and edible values of this animal are favored by people [30]. Since the 1990s, the artificial breeding of bamboo rats began and was rapidly developed [36]. Furthermore, the transmission risk to people of some zoonotic pathogens (e.g. Cryptosporidium spp. [34], Penicilliosis marneffei [29], Escherichia coli [33], Trichinella spp.) in these animals should also not be neglected [37]. Although the prevalence of G. duodenalis in bamboo rats was, until now, unknown, the presence of this parasite in other rodents (e.g. rats, mice, rabbits, chipmunks, chinchillas, guinea pigs, beavers) has been reported in China, Romania, Laos, America, Germany, Austria, Denmark, Hungary, Finland, France, Italy, Luxembourg, the Netherlands, Norway, Poland, Portugal and Sweden, with a prevalence of 1.9–83.0% [13, 14, 36, 42]. Among them, high prevalence was detected in chinchillas (55.7%) from Romania [14], chinchillas (61.4%) from European countries [42] and mice (83%) from Germany [40]. Of eight assemblages (A-H) reported in animals and humans [43], assemblage G has been detected in rats and mice [36], and some animal-specific (C-H) and zoonotic assemblages (A and B) were reported in other rodents, i.e. chinchilla, beaver, muskrat and rabbit [44, 45].

To reveal the infection status of G. duodenalis in farmed bamboo rats, the prevalence and assemblages of this parasite in bamboo rats from six farms in four cities of Hunan Province, China, were determined, and the genetic diversity of G. duodenalis was also investigated by using the multilocus genoty** technique based on three gene loci [46,47,48], namely beta giardin (bg), triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh).

Methods

Collection of faecal specimens

From August to October 2017, 480 fresh faecal samples (Additional file 1: Table S1), were collected from seemingly healthy bamboo rats in six farms located in four cities (Wugang, Chenzhou, Huaihua and Jishou) of Hunan Province (Additional file 2: Figure S1), south-central China. Bamboo rats within four age groups (< 6 months; 6–12 months; > 12–24 months; and > 24 months) were investigated in the present study. Because of similar housing conditions in all examined sampling sites in this study, a fraction of animals were included (Additional file 1: Table S1). Approximately a third of bamboo rats representing each age group were examined in Farms 1–4. Since animals belonging to limited age groups (Additional file 1: Table S1) were raised on Farm 5 and Farm 6, a small number of samples within the existed age groups were collected. A faecal sample from each animal was collected immediately after defecation using disposable plastic bags, marked with the age, date, site and number, then transferred into a 15 ml centrifuge tube with 2.5% potassium dichromate solution and stored at 4 °C.

Extraction of genomic DNA from faecal samples

To remove potassium dichromate solution, all faecal specimens of bamboo rats were washed several times using distilled water under centrifugation at 13,000× rpm for 1 min. Then, the genomic DNA sample was extracted from approximately 300 mg of washed faeces using the commercial E.Z.N.A® Stool DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer’s protocol. The extracted DNA samples were stored at -20 °C for further analysis.

Nested PCR analysis and sequencing

The prevalence of G. duodenalis in bamboo rats was determined by using a nested PCR targeting the bg gene using primers reported previously [7, 49,50,51]. The samples positive for the bg gene were further analyzed with nested PCRs of gene loci tpi and gdh, respectively [7]. A 25 μl PCR mixture was used for PCR amplification, containing 1 μl genomic DNA (for the primary PCR) or primary amplification product (for the secondary PCR) as the template, 2.5 μl 10× Ex Taq buffer (Mg2+ free), 2 mM MgCl2, 0.2 mM dNTP mixture, 0.625 U TaKaRa Ex Taq (TaKaRa Shuzo Co., Ltd, Dalian, China) and 0.4 μM of each primer. Negative and positive controls were included in each PCR reaction and negative samples were spiked with positive material to investigate whether sample material had been associated with inhibition. The secondary PCR amplicons were analyzed by using 1% agarose gels electrophoresis with ethidium bromide. The positive secondary amplicons were sent to Sangon Biotech Co. Ltd. (Shanghai, China) for direct sequencing using the secondary PCR primers.

Sequence analysis

The nucleotide sequences obtained were aligned and edited by using the software Clustal X 1.83 [52]. Then, the corrected sequences were aligned with the reference sequences of G. duodenalis within GenBank by using Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) to identify the assemblages and subtypes.

Statistical analysis

The differences in prevalence among bamboo rats of different age groups and farms were calculated by using the Chi-square test within the software SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA), and the difference was considered statistically significant when P < 0.05.

Results and discussion

Giardia duodenalis infection has been reported in humans and a large number of animal hosts worldwide [8, 15, 47]. The prevalence has been reported as 1.09–72.4% in farmed animals (e.g. rabbits, goats, cattle, pigs, dogs, cats, Tibetan sheep and calves), and 3.2–83.0% in wild rodents (e.g. Apodemus spp., Microtus spp., Myodes spp., house mice, brown rats, Asian house rats, beavers, roof rats and deer mice) [7, 36, 54,55,56,57]. In the present study, this parasite was detected in bamboo rats from Hunan Province of China, and 52 (10.8%) of 480 samples were positive for G. duodenalis infection based on the nested PCR amplification targeting the bg gene. The prevalence of G. duodenalis in bamboo rats in this study was well in the range of previous studies in farmed animals and wild rodents. However, significant differences in prevalence (χ2 = 59.031, df = 5, P < 0.01) were detected among six farms from four sampling cities, ranging from 0 to 38.2%. The highest prevalence was found on Farm 3 of Wugang city (38.2%), while no infections were detected on Farm 4 of Chenzhou city and Farm 6 of Jishou city (Table 1). Furthermore, the prevalence was also significantly different (χ2 = 28.876, df = 2, P < 0.01) among three farms in Wugang city, with the highest on Farm 3 and lowest on Farm 1 (Table 1). The differences in prevalence among these farms could be due to different feeding and management levels. For example, bamboo rats in Farm 4 from Chenzhou city were mainly fed with concentrate (e.g. corn, wheat) and kept in multilayer vertical cages, which reduced the risks of pathogenic infections. On the other hand, in farms of Wugang city, both fresh bamboo and concentrate were fed to the animals, all of which were reared in wooden fences. Furthermore, different sampling practices may also have contributed to the differences between farms. Samples varying in number were collected from different farms (Table 1), ranging from 15 (Farm 6) to 207 (Farm 1). Different age groups and sample numbers within the same age groups tested (Additional file 1: Table S1) are also possible explanations for differences in prevalence. Additionally, detection procedures and targets are also possible factors affecting the rates of G. duodenalis infection. Due to its multiple-copy nature [58], the small subunit ribosomal RNA (SSU rRNA) gene was commonly used as a good marker to detect G. duodenalis infection in animals and humans [44] and rabbits [65,36, 42, 45, 71,72] and has a broad host range, including livestock, companion animals, fish, and marine and wild mammals [13, 18, 21, 26, 73, 74]. Furthermore, assemblage B was also detected in some water bodies [45]. Of the two assemblages (A and B) commonly found in humans, assemblage B was identified in over 50% of Giardia-infected children in some studies [44], an isolate (KY696836) from Hylobatidae in China [71], and an isolate (KM190805) from humans in Canada [45], respectively, while 99% identities of three novel subtypes Ba4, Ba5 and Ba6 were found with the reference sequence KM977640 [44]. Additionally, six subtypes (Bc1-Bc6) and four subtypes (named as Bb1-Bb4) were also identified in 27 gdh-positive samples and 12 tpi-positive samples (Table 2).

Table 2 Substitutions of intra-assemblage B in bg, tpi and gdh sequences
Full size table

Conclusions

Giardia duodenalis occurs in bamboo rats from Hunan Province of China and the infection was significantly associated with age and farms. Although only one assemblage (B) was identified in bamboo rats in the present study, zoonotic potential and genetic diversity were found. To our knowledge, this is the first report on G. duodenalis infection in bamboo rats.