Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli in Dairy Goat Kids with Diarrhea in Partial Regions of Shaanxi Province, China

Simple Summary Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli are major zoonotic pathogens causing diarrhea in humans and various animals. Knowledge of the distribution and genetic diversity of pathogens can shed a new light on the prevention and control of diseases. This study investigated the colonization frequency and genetic make-up of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in dairy goat kids with diarrhea in partial regions of Shaanxi Province. The frequent occurrence of zoonotic species/genotypes/subtypes/pathotypes of these four pathogens in the present study indicated the potential for zoonotic transmission between humans and animals. Abstract Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli are important diarrheal pathogens threatening the health of humans and various animals. Goats, especially pre-weaned goat kids, that carry these pathogens are important reservoirs related to human infection. In the present study, PCR-based sequencing techniques were applied to characterize Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in 202 fecal samples of diarrheal kids for Guanzhong dairy goats from five locations in Shaanxi Province. The positive rates of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli were 37.6% (76/202), 16.3% (33/202), 55.4% (112/202) and 78.7% (159/202) in these goat kids, respectively. Co-infection of two to four pathogens was found in 114 of 202 fecal samples. Significant differences (p < 0.001) in the positive rates of Cryptosporidium spp. and G. duodenalis were found among locations and age groups. Furthermore, two Cryptosporidium species (C. parvum and C. xiaoi), two G. duodenalis assemblages (E and A), nine E. bieneusi genotypes (CHG3, CHG1, BEB6, CHG5, CHG2, SX1, CHG28, COS-II and CD6) and two E. coli pathotypes (EPEC and EHEC) were identified. As for Cryptosporidium, two (IIdA19G1 and IIdA19G2) and two (XXIIIa and XXIIIg) subtypes were recognized in samples positive for C. parvum and C. xiaoi, respectively. A phylogenetic analysis based on the ITS locus of E. bieneusi indicated that all nine genotypes of E. bieneusi identified in this study belonged to the group 2. Four virulence factors (ehxA, eae, stx2 and stx1) of EPEC and EHEC were found in E. coli strains. Collectively, this study explored the colonization frequency of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats in Shaanxi Province and expanded our understanding of the genetic composition and zoonotic potential of these pathogens in goats.


Introduction
Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli are important zoonotic pathogens closely related with the occurrence of diarrhea, significantly endangering the health of humans and various animals [1][2][3][4][5].These pathogens can be transmitted between humans and animals via several routes, e.g., consumption of contaminated food/water and direct contact with infected persons/animals [6][7][8][9].Although Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli are usually reported as opportunistic pathogens, they have caused hundreds of outbreaks of diarrhea in humans and animals, resulting in considerable economic losses for human health and the animal-breeding industry [10][11][12][13][14].In addition to the public health significance, infections of these pathogens can also cause growth retardation in children and immune-compromised individuals [15][16][17][18].
Advances in the occurrence and molecular make-up of pathogens shed a novel light on the prevention and treatment of diseases.Recently, over 40 species and 100 genotypes of Cryptosporidium have been found in humans and various animals based on a molecular analysis targeting the small subunit ribosomal RNA gene (SSU rRNA) [19].Anywhere from one to four Cryptosporidium species/genotypes have been recognized in one host species, e.g., the occurrence of Cryptosporidium hominis and C. parvum in humans, C. canis in dogs, and C. parvum, C. bovis, C. ryanae and C. andersoni in cattle [20].Among those identified species and genotypes, some of them, e.g., C. parvum, C. hominis, C. meleagridis, C. felis, C. canis and C. ubiquitum, are zoonotic, threatening the health of both humans and animals [20].Further subtyping analyses targeting the 60-kDa glycoprotein gene (gp60) contribute to the understanding of transmission and tracing studies of Cryptosporidium spp.[10,[21][22][23].Based on the genetic diversity of the beta-giardin gene (bg), glutamate dehydrogenase gene (gdh) and triose phosphate isomerase gene (tpi), a total of eight assemblages (A-H) were identified in G. duodenalis [3].Of them, a wide host range was found for assemblages A and B, which can infect humans and various mammals.Assemblages C and D are mainly found in canines.Assemblage E is commonly recognized in artiodactyls.Assemblages F, G and H are frequently identified in felines, rodents and marine mammals, respectively [3,10,24].As for E. bieneusi, 11 genetic groups (groups 1-11) with divergent host specificity have been identified based on PCR sequencing of the internal transcribed spacer (ITS) gene [6,25].Most genotypes in the group 1, e.g., Type IV, EbpC, D and Peru6, are frequently identified in humans and various animals, indicating the zoonotic importance of this group [6].Although genotypes from the group 2 were reported to be ruminantspecific at the beginning, an expanding host range for more and more genotypes (e.g., BEB4, BEB6 and I) reflects increasing zoonotic potential within this group [12,26,27].Host specificities of genotypes are frequently found in the groups 3-11, indicated by the unique occurrence of PtEb VIII and WL6 in cats and rodents, respectively [6].Escherichia coli is one of the most common bacteria in animals and humans with important significance, especially for pathogenic E. coli.Pathogenic E. coli can produce toxins and is classified into two groups, namely intestinal (IPEC) and extra-intestinal (ExPEC) pathogenic E. coli, based on divergent virulence factors (e.g., toxins and adhesin) [28].Further, IPEC can be sub-classified into enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and enterohemorrhagic (EHEC) E. coli [29], causing diarrhea, inflammation or even death in infected children and young animals [30].
As one of the most important livestock in China, goats, which can carry Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli, are important reservoirs related to human infections, threatening the health of humans and development of the breeding industry [31][32][33][34].The Guanzhong dairy goat is a famous and excellent breed of dairy goat in China and is mainly distributed in Shaanxi Province.However, little is known of the occurrence of these pathogens in Guanzhong dairy goats.This study investigates the positive rates and genetic composition of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats, and the results expand our understanding of the distribution and zoonotic potential of these diarrhea-related pathogens in goats.

Samples
From February 2022 to May 2023, a total of 202 fecal samples were collected from diarrheal Guanzhong dairy goat kids in five locations of Shaanxi Province (Figure 1), containing 47, 102 and 53 fecal samples from goat kids aged < 2 weeks, 2-4 weeks and 4-12 weeks, respectively.All samples were directly collected from the rectum of goat kids using sterile cotton swabs (HUNAUT, Qingdao, Shandong, China), placed into separate 50 mL centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA), marked with sampling date, city name, location and age, and transported to the Parasitology Lab of Northwest A&F University for examination under cool conditions as soon as possible.
and genetic composition of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. col diarrheal kids of Guanzhong dairy goats, and the results expand our understanding the distribution and zoonotic potential of these diarrhea-related pathogens in goats.

Samples
From February 2022 to May 2023, a total of 202 fecal samples were collected fr diarrheal Guanzhong dairy goat kids in five locations of Shaanxi Province (Figure 1), c taining 47, 102 and 53 fecal samples from goat kids aged <2 weeks, 2-4 weeks and 4 weeks, respectively.All samples were directly collected from the rectum of goat kids us sterile cotton swabs (HUNAUT, Qingdao, Shandong, China), placed into separate 50 centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA), marked with sampl date, city name, location and age, and transported to the Parasitology Lab of Northw A&F University for examination under cool conditions as soon as possible.

Genomic DNA Extraction
Genomic DNA was isolated from fecal samples using an E.Z.N.A. Stool DNA kit (Omega, Norcross, GA, USA) as per the manufacturer's instructions.All gDNA samples were kept at −20 • C until use.

Detection, Genotyping and Subtyping of Cryptosporidium spp., G. duodenalis and E. bieneusi
A nested PCR-based sequencing technique targeting the SSU rRNA gene (~830 bp) was used to investigate the positive rates and species compositions of Cryptosporidium spp. in goat kids, as previously reported [35].Then, C. parvum and C. xiaoi identified in the present study were subtyped using two nested PCR-based sequencing techniques of the gp60 gene [21,36].
A nested PCR-based sequencing technique targeting the ITS gene locus (~392 bp) was used to determine the positive rates and genotypes of E. bieneusi in fecal samples, as previously reported [40].

Sequence Analysis
All positive secondary PCR products were sent to Sangon Biotech (Shanghai, China) for sequencing in both directions.The obtained sequences were assembled, edited and aligned using ChromasPro V1. 33  To assess the relationships of Cryptosporidium spp., G. duodenalis and E. bieneusi found in the present study, three phylogenetic trees were constructed with the maximum likelihood (ML) method, a general time-reversible model and a bootstrap evaluation of 1000 replicates using MEGA V6.0 [41].

Bacterial Isolation and Identification
Escherichia coli strains were isolated from fecal samples using screening on MacConkey agar (Solarbio, Beijing, China), as previously reported [42].Subsequently, the isolated strains were confirmed to be E. coli using a PCR-based sequencing assay targeting the 16S rRNA gene [43].The E. coli strains of each sample were suspended in 20% glycerol and stored at −80 • C for further analysis.

Virulence Factor Determination of E. coli Strains
For each E. coli strain, a single colony of fresh bacterial culture on LB solid medium (Sangon Biotech, Shanghai, China) was selected and re-suspended in 150 µL ddH 2 O and then used as a DNA template in PCRs.Eight virulence genes for five pathotypes (EPEC, EHEC, ETEC, EAEC and EIEC) were detected in all the strains using two multiple PCRs, one for aggR, lt and st, and the other for ipaH, eaeA, ehxA, stx1 and stx2 [44][45][46].

Statistical Analysis
Differences in the positive rates of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in dairy goat kids with diarrhea among locations and age groups were analyzed using the χ 2 test in SPSS V18.0 (IBM, Armonk, NY, USA).Significant differences were identified if the p value was less than 0.05.

Nucleotide Sequence Accession Numbers
Representative nucleotide sequences generated in this study are available in GenBank TM with the accession numbers of OR220596-OR220604 for E. bieneusi, OR229417-OR229423 and OR232183-OR232196 for Cryptosporidium spp., OR230698-OR230699 for E. coli and OR237889-OR237906 for G. duodenalis.
Further phylogenetic analysis based on the ITS gene of E. bieneusi showed that all nine genotypes (CHG3, CHG1, BEB6, CHG2, CHG5, SX1, CHG28, COS-II and CD6) belonged to the group 2, with increasing zoonotic potential (Figure 4).Since there were no variations among the sequences of each genotype, one representative sequence of each genotype was included in the phylogenetic analysis in Figure 4.
Further phylogenetic analysis based on the ITS gene of E. bieneusi showed that a nine genotypes (CHG3, CHG1, BEB6, CHG2, CHG5, SX1, CHG28, COS-II and CD6) b longed to the group 2, with increasing zoonotic potential (Figure 4).Since there were n variations among the sequences of each genotype, one representative sequence of ea genotype was included in the phylogenetic analysis in Figure 4.

Occurrence of E. coli Pathotypes in Dairy Goat Kids with Diarrhea
The PCR-based sequencing analysis targeting the 16S rRNA gene indicated th 78.7% (159/202) of fecal samples were positive for E. coli (Table 4).Although the positi

Occurrence of E. coli Pathotypes in Dairy Goat Kids with Diarrhea
The PCR-based sequencing analysis targeting the 16S rRNA gene indicated that 78.7% (159/202) of fecal samples were positive for E. coli (Table 4).Although the positive rates of E. coli varied among locations (χ 2 = 5.499; df = 4; p = 0.240) and age groups (χ 2 = 0.164; df = 2; p = 0.921), no significant differences were identified.In addition, no significant differences in the positive rates of E. coli were identified among three farms in Fuping (χ 2 = 4.214; df = 2; p = 0.122).To further understand the pathotypes of E. coli in the present study, a total of eight virulence factors (eae, ehxA, stx2, stx1, lt, st, aggR and ipaH) representing EPEC, EHEC, ETEC, EAEC and EIEC E. coli were examined (Table 4).Four of eight virulence genes were identified in 159 strains in this study, namely eae (66.7%, 106/159), ehxA (36.5%, 58/159), stx2 (11.9%, 19/159) and stx1 (9.4%, 15/159).However, no strains were positive for the gene loci lt, st, aggR and ipaH (Table 4).Meanwhile, most strains carried anywhere from one to four virulence genes of EPEC and EHEC.Co-infection of pathogens was found in 114 of 202 (56.4%) fecal samples, with 69, 38 and 7 samples positive for two, three and four pathogens, respectively (Figure 5).As for co-infection of two pathogens, a total of five types were identified, with the co-infection of E. bieneusi and E. coli being the most common one.As for co-infection of three pathogens, four types were found, with the co-infection of Cryptosporidium spp., E. bieneusi and E. coli being the dominant one.

Discussion
Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli are common and important zoonotic diarrheal pathogens greatly endangering the health of humans and animals, especially children and young animals [1][2][3][4].The present study investigated the colonization frequency and genetic composition of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats, which could expand our

Discussion
Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli are common and important zoonotic diarrheal pathogens greatly endangering the health of humans and animals, especially children and young animals [1][2][3][4].The present study investigated the colonization frequency and genetic composition of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats, which could expand our knowledge on the occurrence and distribution of these four pathogens in goats.
Cryptosporidium infection in dairy goat kids was related to the age.In the present study, Cryptosporidium spp.were frequently found in all age groups, with the positive rates of 40.4% (19/47), 52.0% (53/102) and 7.5% (4/53) for goat kids aged <2, 2-4 and 4-12 weeks, respectively, indicating lower positive rates of animals aged >4 weeks compared with <4 weeks.Similar results have also been reported in sheep in Qinghai [54] and goats in Henan, Anhui and Qinghai [47], reflecting that immunity to Cryptosporidium in sheep and goats possibly increases with age.However, contrary results have been found in ewes and lambs in Inner Mongolia [55] and sheep in Henan, Anhui and Qinghai [47], reflected by higher positive rates in older animals compared with younger ones.
The sequences analysis indicated two Cryptosporidium species (C.parvum and C. xiaoi) in goat kids.Cryptosporidium parvum is a zoonotic species with a wide host range, including humans, cattle, sheep and goats, dogs, cats and mice [20].Although C. xiaoi is the most common species in sheep and goats in previous reports [20,32,55], the present study found that C. parvum contributed to over half of the cases, indicating that potentially zoonotic strains of Cryptosporidium circulated on the investigated farms.A further subtyping analysis showed the existence of subtypes IIdA19G1 and IIdA19G2 in C. parvum-positive samples, with the former being the dominant one, which was also found in previous studies [56].IIdA19G1 has also previously been reported in humans and animals [57][58][59][60], reflecting potential zoonotic transmission between humans and animals of this subtype.Cryptosporidium xiaoi was the other common species in the present study, which has also been widely reported in sheep and goats [31,32,53,54,56].A further subtyping analysis based on the gp60 gene locus found two potential goat-adapted subtypes (XXIIIa and XXIIIg) in C. xiaoi-positive samples, which was in accordance with a previous report [36].
Assemblages E and A were two G. duodenalis genotypes in goat kid samples in the present study, with the former being the major one, which is similar to studies in sheep and goats from Henan, Heilongjiang, Yunnan, Shaanxi and Qinghai [32,[61][62][63][64][65].Assemblage E has been widely reported in artiodactyls, such as cattle, sheep, pigs, goats and alpacas [24,66].For a period of time, assemblage E was recognized as one animal-adapted genotype, but a report of this genotype in humans indicated its zoonotic potential [67].Assemblage A is a zoonotic genotype found in humans, livestock, cats, dogs, beavers, guinea pigs and other primates and has also frequently been reported in sheep and goats [61,62,68,69].
A sequence analysis based on the ITS gene locus of 112 E. bieneusi isolates revealed nine known genotypes (CHG3, CHG1, BEB6, CHG5, CHG2, SX1, CHG28, COS-II and CD6) in goat kids in the present study, and these genotypes have been previously reported in sheep and goats [32,[70][71][72].Among the identified genotypes in this study, CHG3 was the most common genotype, which was also found in goats from Henan, Yunnan, Anhui, Chongqing and Shaanxi, as well as in sheep in Henan [71].BEB6 is a zoonotic genotype with a wide host range, including humans [26], non-human primates [73], bovines [74], deer [75], takins [76], alpacas [77], cats [78] and birds [79].Further phylogenetic analysis revealed that all nine genotypes belonged to the group 2.Although genotypes in the group 2 were reported to be ruminant-adapted at the beginning, more and more zoonotic genotypes identified in this group reflect an increasing risk of causing zoonotic infection between humans and animals [25,80].
A high positive rate of E. coli was commonly found in the present study, which is in accordance with previous reports in goats [42,81].Further pathotype analysis based on the eight virulence genes of these E. coli strains indicated the existence of EPEC and EHEC, with the former being the dominant one in goat kids, while no strains were identified to be positive for virulence genes of ETEC, EAEC and EIEC.However, divergent pathotypes were reported in sheep, reflected by the dominance of EAEC and EHEC [42].
Co-infection of these diarrhea-related pathogens was found in goats in the present study, which has also been reported in sheep and goats in previous studies [32,52,53].Previous studies have reported the co-infections of two pathogens (Cryptosporidium spp.and G. duodenalis, G. duodenalis and E. bieneusi, and Cryptosporidium spp.and E. bieneusi) and three pathogens (Cryptosporidium spp., G. duodenalis and E. bieneusi) in sheep and goats [32,52,53].Although the present study did not find the co-infection of Cryptosporidium spp.and G. duodenalis, the co-infection of E. coli with Cryptosporidium spp., G. duodenalis and E. bieneusi was identified for the first time in sheep and goats.

Conclusions
This study explored the colonization frequency and genetic make-up of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats from partial regions of Shaanxi Province.The findings in the present study indicated high positive rates and zoonotic species/genotypes/subtypes/pathotypes of these four diarrhearelated pathogens in goat kids.Considering the zoonotic potential of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in goat kids in this study, interventions are needed to prevent the cross-transmission of these diarrhea-related pathogens between animals and humans.

Figure 1 .
Figure 1.Geographical distribution of sampling sites of dairy goat kids with diarrhea in partial gions of Shaanxi Province in the present study.

Figure 1 .
Figure 1.Geographical distribution of sampling sites of dairy goat kids with diarrhea in partial regions of Shaanxi Province in the present study.

Figure 2 .
Figure 2. Phylogenetic relationships of Cryptosporidium spp.from sheep and goats based on the SSU rRNA gene by maximum likelihood analysis using general time-reversible model.Red-filled circles before the bold sample names represent species identified in the present study.Bootstrap values over 50 are presented at the nodes.Eimeria tenella (AF026388.1) is used as the outgroup.Representative sequences of each sequence type in this study are included in the phylogenetic analysis.

Figure 2 .
Figure 2. Phylogenetic relationships of Cryptosporidium spp.from sheep and goats based on the SSU rRNA gene by maximum likelihood analysis using general time-reversible model.Red-filled circles before the bold sample names represent species identified in the present study.Bootstrap values over 50 are presented at the nodes.Eimeria tenella (AF026388.1) is used as the outgroup.Representative sequences of each sequence type in this study are included in the phylogenetic analysis.

Figure 3 .
Figure 3. Phylogenetic relationships of Giardia duodenalis based on the tpi gene by maximum likelihood analysis using general time-reversible model.Red-filled circles before the bold sample names represent assemblages identified in the present study.Bootstrap values over 50 are presented at the nodes.Representative sequences of each sequence type in this study are included in the phylogenetic analysis.

Figure 4 .
Figure 4. Phylogenetic relationships of representative sequences for the ITS genotypes of E. bieneu identified in this study with reference sequences by maximum likelihood analysis using gener time-reversible model.Red-filled circles before the bold sample names represent genotypes iden fied in the present study.Bootstrap values over 50 are presented at the nodes.Genotype SW3 fro stormwater (KF591679.1) is used as the outgroup.

Figure 4 .
Figure 4. Phylogenetic relationships of representative sequences for the ITS genotypes of E. bieneusi identified in this study with reference sequences by maximum likelihood analysis using general time-reversible model.Red-filled circles before the bold sample names represent genotypes identified in the present study.Bootstrap values over 50 are presented at the nodes.Genotype SW3 from stormwater (KF591679.1) is used as the outgroup.

Table 1 .
Occurrence and distribution of Cryptosporidium species and subtypes in dairy goat kids with diarrhea in partial regions of Shaanxi Province.Cryptosporidium species in goat kids in the present study, namely C. parvum (n = 41) and C. xiaoi (n = 35) (Table1and Figure2).Both C. parvum and C. xiaoi were found in goat kids in Fuping and Yangling, while only one species was recognized in Yanliang (C.parvum) and Jingyang (C.xiaoi).Meanwhile, both C. parvum and C. xiaoi were found in goat kids in all three age groups (Table1).Further sequence analysis based on the gp60 gene showed subtype diversity within C. parvum and C. xiaoi in goat kids in the present study.As for C. parvum, 38 positive samples were successfully subtyped, with IIdA19G1 (n = 37) being the dominant one, followed by IIdA19G2 (n = 1).Meanwhile, two subtypes, namely XXIIIa (n = 22) and XXIIIg (n = 9), were found in 35 C. xiaoi-positive samples, with the mixed infection of XXIIIa and XXIIIg in two samples.
NA: not available.A sequence analysis of the SSU rRNA gene indicated the existence of two

Table 2 .
Occurrence of Giardia duodenalis assemblages in dairy goat kids with diarrhea in partial regions of Shaanxi Province.

Table 2 .
Occurrence of Giardia duodenalis assemblages in dairy goat kids with diarrhea in part regions of Shaanxi Province.

Table 3 .
Occurrence of Enterocytozoon bieneusi genotypes in dairy goat kids with diarrhea in partial regions of Shaanxi Province.

Table 4 .
Occurrence of Escherichia coli pathotypes in dairy goat kids with diarrhea in partial regions of Shaanxi Province.
3.5.Co-Infection of Pathogens for Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in Dairy Goat Kids with Diarrhea