Occurrence and genetic characterization of Giardia duodenalis and Cryptosporidium spp. from adult goats in Sichuan Province, China

Giardia duodenalis and Cryptosporidium spp. are common gastrointestinal protozoa in mammals. Many studies have been conducted on the distribution of G. duodenalis and Cryptosporidium spp. genotypes in sheep and cattle. However, in China, information about molecular characterization and genetic analysis of G. duodenalis and Cryptosporidium spp. in goats is limited. In this study, 342 fecal samples from adult goats were collected from 12 farms in Sichuan Province, China. The occurrence of G. duodenalis and Cryptosporidium spp. in adult goats was 14.9% (51/342) and 4.7% (16/342), respectively. All G. duodenalis were identified as assemblage E, with two novel genotypes (assemblages E17 and E18) being detected at the beta-giardin (bg) locus. Based on three loci—beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh)—multilocus sequence typing revealed three novel multilocus genotypes (MLGs) of assemblage E (MLG-E1, E2, E3 (sc)). Small Subunit (SSU) rRNA-based PCR identified two Cryptosporidium species, namely C. xiaoi (11/16) and C. suis (5/16). This study is not only the first to report C. suis infection in adult goats in China but is also the first to use the MLG approach to identify G. duodenalis in adult goats.


Introduction
Giardia duodenalis and Cryptosporidium spp. are two genera of intestinal parasitic protozoa that infect humans and a broad range of animals, including livestock, companion animals, and wildlife [1][2][3]. Infection is acquired following ingestion of highly resilient, infective stages (cysts or oocysts) via the fecal-oral route [4]. Damage to animals is dependent on multiple factors, such as the strain of the parasite involved and the immunological and nutritional status of a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the host, and clinical manifestations vary from asymptomatic to acute or chronic diarrheal disease [5,6].
Giardia duodenalis and Cryptosporidium spp. are two of the most common identified parasitic protists in ruminants (i.e., cattle, goat, and sheep) and G. duodenalis is considered a multispecies complex with at least eight distinct assemblages (A-H). It has been reported that G. duodenalis assemblages A, B, and E are capable of infecting goats [7], and of these, assemblage E has been commonly detected in goats worldwide [8][9][10][11]. The zoonotic assemblages A and B have also been detected in goats, which suggests a potential threat to human beings [12][13][14][15][16].
In recent years, G. duodenalis has been identified in a wide variety of animals, including cattle (assemblages A, B, and E), sheep (assemblages A, B, and E), goats (assemblages A, B, and E), dogs (assemblages A, C and D), cats (assemblages A, F), pigs (assemblages A, B, D, E, and F), rabbits (assemblages B and E), rodents (assemblages A, B, and G), non-human primates (NHPs) (assemblages A, B, and E), and some other wild animals in China [17]. Of the abovementioned animals, information about production animals, including cattle and sheep, is more frequently reported, mostly in provinces with developed husbandry. In China, G. duodenalis infection in goats is limited with reports from only four provinces (Anhui, Henan, Shananxi, and Heilongjiang) [11,13,14]. Similarly, Cryptosporidium spp. has also been widely reported in various animals in China, especially for ruminants, such as cattle, sheep, and goats [18]. Several Cryptosporidium species (C. ubiquitum, C. xiaoi, C. parvum, C. hominis, C. andersoni, and rat genotype II) have been reported in goats worldwide with C. hominis and C. parvum frequently reported in humans, especially in developing countries including China, which suggests the potential occurrence of zoonotic transmission between goats and humans [18]. However, in China, only C. ubiquitum, C. xiaoi, C. parvum, and C. andersoni have been identified in goats, with C. ubiquitum and C. xiaoi being predominant [7,14,19,20]. The Cryptosporidium spp. genotypes that have the potential to infect goats are still not clear.
Recent studies have mostly focused on reservoirs of G. duodenalis and Cryptosporidium spp. in cattle, particularly calves, and only a limited number of reports have focused on goats as the reservoir for these two parasites [21]. According to the National Bureau of Statistics of the People's Republic of China, in 2016, the total population (~15.67 million heads) of goats in Sichuan Province was the fourth largest in China, which has the largest goat population in the world [5]. However, no information about G. duodenalis or Cryptosporidium spp. infection in goats is available in Sichuan Province. To gain a better understanding about the genetic diversity and frequency of G. duodenalis and Cryptosporidium species in adult goats, we conducted the first known molecular study on G. duodenalis and Cryptosporidium spp. infection for Sichuan Province, China. Here, we report for the first time the occurrence and genetic characterization of G. duodenalis infection in adult goats in Sichuan Province, China.

Ethics statement
This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-S20174604. The field studies did not involve any endangered or protected animal species. Prior to the study, permission was granted by the owners of 12 farms. No other specific permits were required for the described field studies. In this study, all fecal samples were carefully collected cautiously from the rectum of each goat without causing discomfort.

Sample collection
Sichuan Province, located in southwestern China, has a subtropical monsoon climate, where the breeding industry is still in the initial development stage although it has a long history of traditional husbandry. From June to August 2017, a total of 342 fecal samples were collected from goats at 12 different farms in Sichuan Province, China, with a history of animals with diarrhea, including Qingchuan (Fig 1). The city-level map was provided by the National Geomatics Centre of China (National Geomatics Centre of China, Beijing, China, http://ngcc.sbsm.gov.cn/). The 12 farms are distributed throughout Sichuan Province, except the western region where animal husbandry is relatively undeveloped. Of the 12 farms, six (Qingchuan, Dalin, Beichuan, Jianyang, Fushun, and Jingjiu) practiced intensive feeding, while the other six farms are free-range. All goats included in the study were more than 1 year old. Feces were collected directly from the rectum of each goat into a 50-mL centrifuge tube, which was immediately capped, labeled, and placed in a box with ice. Fecal samples were transported to the Sichuan Agricultural University and stored in 2.5% potassium dichromate at 4˚C. All samples were processed within 24 h after collection.

DNA extraction and PCR amplification
Before DNA extraction, feces were washed with distilled water to remove potassium dichromate. DNA was extracted from the feces using a PowerSoil 1 DNA isolation kit (MoBio, Carlsbad CA, USA) according to the manufacturer's instructions and stored at −20˚C in 100 μL of the solution buffer provided in the kit [22].
For detection of Giardia spp., DNA samples were subjected to nested PCR amplifications based on the detection of beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh) as previously described [23]. Nested PCR of Cryptosporidium spp. was performed targeting the small subunit (SSU) rRNA gene [24]. The primers and amplification conditions used in this study (Table 1) have been previously described [23,24]. The PCR reaction was composed of 12.5 μL 2× Taq PCR Master Mix (KT201-02, Tiangen, Beijing, China), 8.5 μL deionized water (Tiangen, Beijing, China), 2 μL DNA, and 1 μL each of set primers, for a total volume of 25 μL. Positive and negative controls were included in each test. Secondary PCR products were visualized by 1% agarose gel electrophoresis and staining with Golden View.

Sequencing and phylogenetic analysis
The secondary PCR products were sent to Invitrogen (Shanghai, China) and sequenced in both directions. To determine the species/assemblages of Giardia and Cryptosporidium species/ genotypes, sequences were aligned with reference sequences from GenBank using BLAST (http://blast.ncbi.nlm.nih.gov) and Clustal X. For the phylogenetic analysis, sequences at the three loci obtained in this study were concatenated to form one multilocus sequence for each isolate. A neighbor-joining tree was constructed using Mega 5 (http://www.megasoftware.net/)

Nucleotide sequence accession numbers
Representative nucleotide sequences were deposited into the GenBank database under the following accession numbers: MG602956-MG602963. GenBank accession numbers of reference sequences are listed as follows: E5 (KY769092) and E8 (KY633465) for the bg loci; E2 (KT92262) and E3 (KT92259) for the tpi loci; and E3 (KF843925) and E4 (KF843926) for the gdh loci.

Giardia duodenalis
Giardia duodenalis infection in goats has been reported from many countries such as Ghana, Malaysia, India, Greece, Tanzania, Iran, Spain, the Netherlands, and China [8][9][10]12,13,15,16,25,26]. However, in China, information regarding G. duodenalis infection in goats is limited, with only three reports available [11,13,14]. Our study, for the first time, reports the occurrence and genetic characterization of G. duodenalis infection in adult goats in Sichuan Province, China.
In the present study, goats from 10 of the 12 farms showed presence of G. duodenalis, with the positive rate ranging from 5-37.5% ( Table 2). The highest occurrence (37.5%, 15/40) was found in Linshui Farm which is free-ranging and relatively poorly managed. Giardia duodenalis was detected in 51 of 324 (14.9%) fecal samples from adult goats, which was higher than that observed in Shaanxi (10.8%) [14], Anhui (6.3%) [11], or Heilingjiang (2.9%) [13], but lower than that in Henan (17.3%) [14]. Moreover, only two studies related to goat kids are available to date, but the infection rates of G. duodenalis in kids in Greece (40.4%) [9] and the Canary Islands (42.2%) in Spain [15] are significantly higher than what has been reported in any study for adult goats. This suggests that G. duodenalis-infection may be associated with age, which had been previously proved in sheep and cattle [27,28]. However, whether this hypothesis is reliable still requires confirmation with a more extensive epidemiological investigation of goats of various ages. The genetic diversity of G. duodenalis was analyzed by sequencing the bg, tpi, and gdh loci with 51, 37, and 33 sequences were obtained, respectively (Table 3). Sequence analysis of 51 G. duodenalis positive samples showed mono-infection with the ruminant-specific assemblage E at the three loci, which is similar to the results of previous investigations on adult goats [8,10,11,29]. According to previous studies, the zoonotic genotypes A and B have been detected in goats, which suggests a zoonotic threat to human beings [12]. In the present study, our findings indicated that adult goats may not be a potential reservoir for zoonotic genotypes of G. duodenalis in Sichuan Province. However, recently, scientists concluded that assemblage E should also be considered potentially zoonotic [7,30]. More epidemiological research is needed regarding the potentially zoonotic genotypes of assemblage E.
In short, our study, for the first time, reports the occurrence and genetic characterization of G. duodenalis infection in adult goats in Sichuan Province, China. This first-time observation requires a more extensive epidemiological investigation in goats of various ages.
Among the 16 positive samples, two Cryptosporidium species (C. xiaoi and C. suis) were identified. C. xiaoi was the most predominant genotype detected in three farms, while C. suis was only found in Naxi. Todate, this is the first molecular identification of C. suis infection in adult goats. C. xiaoi was the predominant species found in this study (68.8%, 11/16), which is consistent with that reported in previous surveys in China [14,19]. However, it is different from studies in Henan and Chongqing [20], which revealed that C. andersoni and C. ubiquitum are predominant species, respectively. Other studies found that C. parvum was the dominant species in goats [34][35][36]. In this study, C. parvum, C. andersoni, and C. ubiquitum, which were considered zoonotic subtypes, were not detected. The difference in genotype between our survey and previous studies may be related to host age, raising density, geographical ecological conditions, and examination methods [7,14,19,20].

Conclusions
This is the first known report on the occurrence and genetic characterizations of G. duodenalis and Cryptosporidium spp. in adult goats in Sichuan Province, China. We found that assemblage E of G. duodenalis was dominant in adult goats in this region. For the first time, we used an MLG approach to identify G. duodenalis in adult goats, and we detect three novel assemblage E MLGs. For Cryptosporidium spp., two Cryptosporidium species (C. xiaoi and C. suis) were observed, which is, notably, the first time C. suis infection has been identified in adult goats. For a better understanding of the epidemiology and genotypes of G. duodenalis and Cryptosporidium spp. in goats, further investigation with a larger sample of goats of different ages is needed.