Occurrence and multilocus genotyping of Giardia duodenalis in black-boned sheep and goats in southwestern China

Background Giardia duodenalis is an important intestinal protozoan infecting both humans and animals, causing significant public health concern and immeasurable economic losses to animal husbandry. Sheep and goats have been reported as common reservoirs of G. duodenalis, but only a limited amount of information is available for native breeds of these small ruminants in China. The present study investigated the prevalence and multilocus genotypes of G. duodenalis in black-boned sheep and goats, two important native breeds in Yunnan Province, southwestern China. Methods Fecal samples were collected from 336 black-boned goats and 325 black-boned sheep from five counties (Meishui, Shanshu, Shilin, Yongsheng and Nanping) of Yunnan Province and the genomic DNA was extracted from these feces. The prevalence of G. duodenalis was determined by the 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). Results Giardia duodenalis infection was detected in both black-boned sheep and goats, and the prevalence of G. duodenalis in black-boned sheep (21.8%, 71/325) was significantly higher (χ2 = 36.63, df = 1, P < 0.001) than that in black-boned goats (4.8%, 16/336). Significant differences in prevalence were also observed in goats and sheep from different counties (χ2 = 39.83, df = 4, P < 0.001) and age groups (χ2 = 97.33, df = 3, P < 0.001). Zoonotic assemblage A and animal-specific assemblage E were identified in both black-boned sheep and goats with the latter as the predominant assemblage. Based on sequences obtained from the three genetic loci (bg, gdh and tpi), 16 MLGs were obtained in black-boned sheep and goats, including 15 MLGs in assemblage E and one MLG in assemblage A. Conclusions Our results not only extended the host range of G. duodenalis, but also revealed high genetic variations in G. duodenalis assemblages. The findings of the present study also provide baseline data for preventing and controlling G. duodenalis infection in black-boned sheep and goats in Yunnan Province.


Background
Giardia duodenalis (syns Giardia lamblia, Giardia intestinalis) is one of the most common parasites that causes intestinal infections of humans and various animals worldwide [1][2][3], and about 280 million people have symptomatic infections annually [4,5]. The prevalence of G. duodenalis in humans in developing countries is much higher than that in developed countries [6,7]. In China, the average prevalence of G. duodenalis in humans was 0.85% from 2000 to 2017 (197/23,098) [3]; however, the infection rate is underestimated because people in many areas with poor medical care have not yet been assessed. Giardiasis, caused by G. duodenalis, is characterized by diarrhoea, abdominal pain, bloating, malabsorption and weight loss in symptomatic infections [1,8,9]. Moreover, diarrhoeal disease, causing about 800 000 fatalities worldwide per year, is the main cause of illness and death for children during the first five years of life [4,10]; G. duodenalis is the second-most common pathogen detected in diarrhoeal stools of children between 12-24 months of age [7]. However, due to drug resistance and known cases of treatment failure [11,12], it is essential to find the source of the infection for effective control of this parasitic disease.
Thus far, at least eight genetically distinct assemblages (A-H) of G. duodenalis have been identified using molecular biological analysis. Among them, assemblages A and B are believed to have zoonotic potential for their broad host range, and assemblage A is more frequently found in livestock than assemblage B, while the remaining assemblages (C-H) are animal-specific groups [1,13,14]. To better assess the zoonotic transmission of giardiasis and differentiate mixed infections of assemblages, highresolution multilocus genotyping analysis using more intra-assemblage variation genes, including β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes, has been widely used to characterize G. duodenalis isolates from humans and animals [1,15].
Sheep and goats are important for the animal husbandry economy and their populations are rising steadily in Asia. China has the largest population of sheep and goats around the world [16]. Thus far, a number of studies have reported the infection of G. duodenalis in sheep and goats in China, with prevalences of 1.82-13.11% [13,[17][18][19][20][21][22][23] and 0.95-27.78% [17,20,[24][25][26][27][28][29][30], respectively. Although asymptomatic infection of G. duodenalis commonly occurs in sheep and goats [31], an outbreak of giardiasis in a sheep farm located in central Italy had G. duodenalis-infected lambs showing malabsorption, weight loss, decrease of feed conversion ratio, malodorous and poorly formed greenish feces [32], causing significant economic loss to sheep on this farm. Assemblages A, B and E have been identified in sheep and goats in China, with assemblage E as the most frequently detected assemblage [3,13,30]. The identification of zoonotic assemblages A and B suggests that sheep and goats with G. duodenalis infections are likely to be the source of human giardiasis.
Black-boned sheep and black-boned goats, two important native breeds originated from Nanping county, Yunnan Province, southwestern China [33], are believed to have a medicinal function and nutritional value, as well as a health care value due to the great quantity of melanin in their tissues [33,34]. However, to date, there are no studies on G. duodenalis infection in black-boned sheep and goats. Therefore, the objectives of the present study were to investigate the prevalence and assemblage distribution of G. duodenalis in black-boned sheep and goats, and to evaluate the zoonotic potential of G. duodenalis in these two native breeds using a high-resolution multilocus genotyping tool targeting three genes (bg, gdh and tpi) useful for studying intra-assemblage variation.

Sample collection
A total of 661 fecal samples were collected from five counties (Meishui, Shanshu, Shilin, Yongsheng and Nanping) in Yunnan Province, southwestern China in August 2017; of these, 325 fecal specimens were collected from black-boned sheep and 336 were collected from blackboned goats. Both male and female animals' feces were collected, and these animals were divided into four age groups: 0-2 months; 3-6 months; 7-12 months; and > 12 months. Each fresh fecal specimen was randomly obtained from the rectum of each apparently healthy animal using a sterile disposal plastic glove. Approximately 5-20 g of feces were collected from each animal. The feces were marked with the sampling site, sex, age and breed immediately after collection, and kept on ice packs during transportation. All specimens were preserved in 2.5% potassium dichromate at 4 °C until genomic DNA extraction.

Genomic DNA extraction
To remove the potassium dichromate, approximately 300 mg of each fecal specimen was washed by centrifugation at 13,000 × g with sterilized distilled water until the supernatant was clear before genomic DNA extraction. Then, the genomic DNA of each washed fecal specimen was extracted using E.Z.N.A ® Stool DNA kit (Omega Bio-tek Inc., Norcross, GA, USA), following the manufacturer's instructions. The extracts of genomic DNA were stored at -20 °C for further PCR analysis.

Nested PCR amplification
The prevalence of G. duodenalis in black-boned sheep and goats was determined by nested PCR targeting the bg gene using previously described primers and procedures [9,35,36]. Then, the bg-positive samples were analyzed by nested PCR at the gdh and tpi loci using previously described primers and procedures [9,37]. Positive and negative controls were included in each PCR reaction. The secondary PCR products were screened by electrophoresis with 1% (w/v) agarose gels containing ethidium bromide.

Sequencing and sequence analysis
All G. duodenalis bg-, gdh-and tpi-positive secondary PCR products were sent to Xi'an Sangon Biotech (Shanghai) Co., Ltd. for direct sequencing. Sequences obtained were first proofread with their DNA peakform graph using Chromas v.2.6. Then, the checked sequences were amended by aligning with reference sequences downloaded from GenBank database using the software Clustal X v.1.83 [38] to identify G. duodenalis assemblages. Samples simultaneously successfully amplified at all three genetic loci (bg, gdh and tpi) were used to form MLGs to further reveal the genetic diversity. The nomenclature of novel sequence subtypes identified in the present study and those undesignated subtype sequences previously deposited in GenBank followed that of the former studies [13,23].

Statistical analysis
The differences in prevalence between different breeds, sexes, age groups and locations were calculated using a Chi-square test in SPSS 22.0 (SPSS Inc., Chicago, IL, USA). The differences were considered statistically significant when P < 0.05.
Significant differences in prevalence of G. duodenalis were found among different age groups of both blackboned sheep (χ 2 = 46.11, df = 3, P < 0.001) and goats (χ 2 = 32.51, df = 3, P < 0.001). Of the 325 fecal samples collected from the four age groups of black-boned sheep, the highest prevalence of G. duodenalis was found for the 0-2-month group (59.4%, 19/32); however, G. duodenalis was not found in this age group of black-boned goats (0/3) due to the number of samples being too small. The highest prevalence of G. duodenalis was found in 3-6 month black-boned goats (24.1%, 7/29). In black-boned sheep, the lowest prevalence was found in sheep aged more than 12 months (5.7%, 7/123), while only one of 200 (0.5%) black-boned goats in this age group was found with G. duodenalis infection ( Table 2).

Distribution of G. duodenalis assemblages
Sequence analysis of the bg gene indicated two assemblages (A and E) of G. duodenalis in both black-boned sheep and goats. Of the 71 bg-positive black-boned sheep samples, 70 isolates from three counties and four age groups belonged to the animal-specific assemblage E, while only one isolate from a 3-month sheep in Shilin county was identified as the zoonotic assemblage A. Ten isolates of assemblage E were detected in 16 bg-positive black-boned goat samples and all 6 assemblage A isolates were detected in Meishui County (Tables 1 and 2).

Subtypes of assemblages A and E
Among the 325 samples of black-boned sheep examined, a total of 35 and 43 sequences were obtained by amplifying 71 bg-positive black-boned sheep samples at the gdh and tpi loci, respectively (Table 1). At the bg locus, 14 subtypes were generated by comparison of the 71 amended sequences obtained from black-boned sheep, with one and 13 identified to be assemblages A and E, respectively, including nine known subtypes (A5, E2, E3, E5, E6, E8, E9, E13 and E15) and five novel subtypes (named as E16, E17, E18, E20 and E22) ( Table 3). Four subtypes were yielded by analysis of 35 amended sequences at the gdh locus. All of them represented assemblage E and three of them had been previously published with no subtype names; here we named them as E31, E32 and E33, respectively, while only one was identified as a novel subtype (named as E34) ( Table 4).
Sequence analysis of the 43 tpi-positive samples revealed one subtype of assemblage A and four subtypes of assemblage E in black-boned sheep. Among them, three subtypes had been previously reported, named as A4, E3 and E5 (Table 5). One sequence had been previously published without a subtype name; here we named it as E29, while the remaining novel subtype was named as E30 (Table 5).
In black-boned goats, one subtype of assemblage A and six subtypes of assemblage E were identified at the bg locus, including four known subtypes (named as A5, E2, E5 and E13) and three novel subtypes (named as E16, E19 and E21) ( Table 3). At the gdh locus, one known subtype (designated as A1), two previously published sequence with no subtype name (named as E32 and E33) were identified (Table 4). At the tpi locus two known assemblage A subtypes (A1 and A4), one known assemblage E subtype (E3) and one previously published sequence with no subtype name (named as E28) were identified (Table 5).

Multilocus genotypes
In total, 33 samples were simultaneously amplified at all three intra-assemblage variation genetic loci, including 26 samples from black-boned sheep and seven from black-boned goats. Of the 26 black-boned sheep samples, sequences obtained from 24 samples belonged to assemblage E, forming 14 novel assemblage E MLGs (named as MLG-E1 to MLG-E8 and MLG-E10 to MLG-E15), and two samples were mixed infection with assemblages E and A (Table 6). In black-boned goats, three distinct assemblage E MLGs (named as MLG-E1, MLG-E2 and MLG-E9) were yielded from three samples and one assemblage A MLG (named as MLG-A1) was identified from another three samples; the remaining sample was a mixed infection with assemblages E and A (Table 6).
In black-boned sheep, MLG-E1 was found in four samples from two counties. MLG-E4, MLG-E6 and MLG-E7 were each found in two samples from one county. MLG-E5 and MLG-E10 were each found in three samples from one county. The remaining eight assemblage E MLGs were observed in only one sample (Table 7). Most MLGs were found in Shilin county, three MLGs were seen in Yongsheng county and only one MLG was observed in Nanping county (Table 7). Most MLGs were observed in black-boned sheep of 7-12 months (7/15) and 0-2 months (6/15) ( Table 8).
In black-boned goats, both assemblage E MLGs and assemblage A MLGs were found. MLG-E1 and MLG-E2 were detected in 9-month-old and 2-year-old   black-boned goats from Nanping county, respectively. MLG-E9 was found in a 3-month-old black-boned goat from Meishui county. All three samples of MLG-A1 were from Meishui county, two of them were collected from black-boned goats of 3-6 months and one was from a black-boned goat of the 7-12 months group (Tables 7  and 8).
Assemblage E was the most prevalent genotype in both black-boned sheep (70/71) and goats (10/16) in our study. Assemblage E was also reported as the predominate genotype in sheep and goats in other studies [13, 17, 23, 24, 29-31, 43, 45, 46, 50-54, 56, 57, 59, 61, 62]. Although assemblage E was generally believed to be animal-specific [65], sporadic reports from some areas with poor conditions in Egypt, Australia and Brazil suggest that it can also be found in humans [66][67][68][69][70], indicating its zoonotic transmission. In some studies, both zoonotic assemblages A and B were observed in sheep and goats [17,30,44,52,[61][62][63]. However, in our study, only one black-boned sheep and six black-boned goats were infected with G. duodenalis assemblage A. Interestingly, genomic analysis of assemblages A, B and E showed that the nucleotide similarity between assemblages A and E is much higher than that between assemblages A and B [71,72]. These results suggest that infected black-boned sheep and goats have the opportunity to transmit G. duodenalis to humans.
To further reveal the genetic variations in assemblages A and E, another two genetic loci (gdh and tpi) of 87 bg-positive samples were analyzed by the multilocus genotyping tool with high resolution [9]. A total of four assemblage E subtypes at the gdh locus, four assemblage E subtypes and one assemblage A subtype at the tpi locus were identified in black-boned sheep. Two assemblage E subtypes and one assemblage A subtype at the gdh locus, and two assemblage E subtypes and two assemblage A subtypes at the tpi locus were observed in black-boned goats (Tables 4 and 5). In addition, three samples had different assemblages at the three loci (Table 6). Fifteen MLGs of assemblage E and one MLG of assemblage A were yielded in 30 samples amplified successfully at all three loci with the same assemblage genotype. These findings suggest that there is a high subtype diversity and genetic variation in assemblages A and E. In addition, a higher prevalence of G. duodenalis assemblage A in black-boned goats (62.5%, 10/16) and the presence of the assemblage A MLGs suggest that the black-boned goats has higher potential in transmitting zoonotic G. duodenalis than the black-boned sheep.

Conclusions
The present study revealed the presence and identified assemblages of G. duodenalis in black-boned sheep and goats in Yunnan Province, southwestern China. Assemblages A and E were found in both black-boned sheep and goats, with assemblage E being the most prevalent in these two native species. The presence of zoonotic assemblage A in black-boned sheep and goats suggests their zoonotic potential. Multilocus genotyping at bg, tpi and gdh loci formed 14 novel assemblage E MLGs in blackboned sheep and four novel MLGs (three assemblage E MLGs and one assemblage A MLG) in black-boned goats. These results provide baseline data for preventing and controlling G. duodenalis infection in black-boned sheep and goats as well as humans.