Prevalence and Genetic Characterization of Giardia duodenalis and Blastocystis spp. in Black Goats in Shanxi Province, North China: From a Public Health Perspective

Simple Summary Blastocystis spp. and Giardia duodenalis are two prevalent intestinal parasites with a worldwide distribution that can infect humans and animals, resulting in significant public health concerns and economic losses. For goats, infection with the two parasites can cause symptoms such as diarrhea, which is not conducive to the development of stockbreeding. Shanxi Province is one of the largest goats breeding provinces in China. However, the prevalence of Blastocystis spp. and G. duodenalis in black goats in Shanxi Province remains unknown. Thus, 1200 fecal samples of black goats were collected in five representative geographical locations in Shanxi Province to examine the presence and genotypes of G. duodenalis and Blastocystis spp. by using a molecular approach. The results showed that the total infection rates of G. duodenalis and Blastocystis spp. were 7.5% and 3.5%, respectively. The presence and genotypes of G. duodenalis were determined based on three established loci (tpi, bg, and gdh). Among the detected assemblages B and E of G. duodenalis, the most prevalent assemblage was E in black goats in the five study areas. One novel MLG (MLG-E12) was identified by multilocus genotypes (MLGs) analysis. Through DNA sequence analysis, four subtypes of Blastocystis spp. were found in black goats, namely ST5, ST10, ST14, and ST30, among which ST10 was the dominant subtype in this study. This is the first report of Blastocystis spp. and G. duodenalis infection in black goats in Shanxi Province, which not only enhances our understanding of the genetic diversity of Blastocystis spp. and G. duodenalis in black goats in China but also provides essential baseline data for the prevention and control of Blastocystis spp. and G. duodenalis infection in black goats in the study areas. Abstract Blastocystis spp. and Giardia duodenalis are two prevalent zoonotic intestinal parasites that can cause severe diarrhea and intestinal diseases in humans and many animals. Black goat (Capra hircus) farming is increasingly important in China due to the remarkable adaptability, high reproductive performance, rapid growth rate, and significant economic value of black goats. A number of studies have indicated that black goats are the potential reservoir of multiple zoonotic protozoans in China; however, the prevalence and zoonotic status of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province is still unknown. Thus, a total of 1200 fecal samples of black goats were collected from several representative regions at different altitudes in Shanxi Province and were examined for the presence and genotypes of G. duodenallis and Blastocystis spp. by amplifying the beta-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) loci of G. duodenalis and SSU rRNA of Blastocystis spp. using PCR and sequence analysis methods, respectively. The overall prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province were 7.5% and 3.5%, respectively. Two assemblages (B and E) of G. duodenalis and four subtypes (ST5, ST10, ST14, and ST30) of Blastocystis spp. were identified, with assemblage E and ST10 as the prevalent genotype and subtype in black goats, respectively. One novel multilocus genotype (MLG) was identified in MLG-E and was designated as MLG-E12. For both G. duodenalis and Blastocystis spp., the prevalence was significantly related to the region and age groups (p < 0.05). This is the first report on the prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province. These results not only provide baseline data for the prevention and control of both parasites in black goats in Shanxi Province, but also enhance our understanding of the genetic composition and zoonotic potential of these two parasites.


Introduction
Giardiasis and blastocystosis are two common gastrointestinal diseases in both humans and most animals caused by protozoal parasites known as Giardia and Blastocystis spp., respectively [1,2].Healthy humans and animals with competent immunity are usually asymptomatic [3].However, immunocompromised individuals ingest the cysts of Giardia or Blastocyst, which are contained in the food or water via the fecal-oral route, resulting in stunted growth and gastrointestinal disturbances, such as diarrhea [4,5].
The Giardia genus is considered to have eight valid species, including Giardia microti, Giardia cricetidarum, Giardia muris, Giardia ardeae, Giardia psittaci, Giardia agilis, Giardia peramelis, and Giardia duodenalis.Of them, G. duodenalis (synonmy G. intestinalis and G. lamblia) is the only species of Giardia that infects humans [6][7][8][9].In early investigations, previously reported G. duodenalis were divided into eight assemblages/genotypes (A-H), in which A and B belonged to the zoonotic group [10,11], and C-H were considered as hostspecific assemblages: C and D for canines, E for artiodactyls, F for felines, G for rodents, and H for marine mammals [12].In the last decade, assemblages C, D, E, and F were also detected or isolated from human stool specimens; however, the zoonotic role of these assemblages in humans was still unknown [13,14].In recent years, multilocus genotyping (MLG) of the beta-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) loci has increasingly been used to characterize G. duodenalis infection in humans and animals [15].In developing countries, the prevalence of G. duodenalis infection ranges from 8.0% to 30.0% in children and from 1.5% to 42.2% in sheep and goats [16].In addition, assemblage E has been found in humans infected with G. duodenalis in Europe and Egypt [16].In 2022, G. duodenalis was detected in feces samples from western chimpanzees and individuals with close contact in Comoé National Park, Côte d'Ivoire, which indicates the risk of zoonosis [17].Additionally, assemblage E was the most frequently detected assemblage in goats in the world, followed by assemblages A and B [18].In China, MLG-E is often identified in goats; however, MLG-A has also been detected in Yunnan Province, southwestern China [19].
Regarding Blastocystis spp., a total of 28 distinct lineages have been identified and recognized based on nucleotide polymorphisms within the small subunit ribosomal RNA genes (SSU rRNA) of Blastocystis spp.and designated as subtypes ST1 to ST17, ST21, ST23-ST29, and ST30-ST32; with ST1-ST9 and ST12 commonly detected in humans and in other mammals [20][21][22].More evidence indicated that ST1 to ST4 contributed to more than 90.0% of human infection cases, in which ST1 and ST3 have the strongest pathogenicity, and ST3 was commonly detected in school children [23,24].Worldwide, the Blastocystis spp.prevalence in humans was documented in Europe (22.0% to 56.0%) and African and Asian countries (37.0% to 100.0%) [25,26].Moreover, the global prevalence of Blastocystis spp. in goats was 20.5%, with ST10 being the predominant subtype [22].
China is one of the most important agricultural countries in the world and has been the country with the largest number of goats since the late 1980s [27].Shanxi Province is located inland of the mid-latitude zone of North China and has a temperate semi-humid and semiarid continental monsoon climate.Black goats (Capra hircus), as an endemic goat breed, are extensively bred in many provinces of China, including Shanxi Province, due to their exceptional meat production performance and favorable reproductive capabilities [28,29].In China, the highest prevalence of G. duodenalis and Blastocystis spp. in black goats were reported in Guangdong Province (27.5%, 56/226) and Yunnan Province (41.3%, 375/907), respectively [29,30].Moreover, MLG was detected in black goats from Yunnan Province [31].So far, there have been no reports regarding the prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province.Thus, the objectives of the present study were to explore the prevalence and zoonotic potential of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province, aiming to provide the baseline data for executing the prevention and control strategy against these two parasites in black goats in Shanxi Province.

Ethics Approval
The experimental procedures of the study were reviewed and approved by the Experimental Animal Ethics Committee of Shanxi Agricultural University (Approval No. SXAU-EAW-2023G.PB.008011172, approved on 11 August 2023).The animals were handled in accordance with good animal practice as defined by the relevant Animal Ethics Procedures and Guidelines of the People's Republic of China.

Sampling Collection
From October to November 2023, a total of 1200 fresh fecal specimens were collected from black goats without diarrhea in five representative regions of Shanxi Province, including Jinzhong City (n = 428), Lvliang City (n = 267), Datong City (n = 225), Changzhi City (n = 102), and Yuncheng City (n = 178) (Figure 1).Fresh stool samples were collected from the rectum of each animal and placed in a separate sterile polyethylene (PE) glove, isolated in time to prevent contamination.At the same time, detailed information (region, sex, and age) for each sample was recorded.As shown in Figure 1, all samples were divided into 2 age groups (older goats ≥3 years old and younger goats <3 years old), 2 sex groups (male and female), and 2 altitude groups (>1 km and <1 km).Subsequently, the collected specimens were transported to the Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, and stored at −20

DNA Extraction and PCR Amplification
Following the manufacturer's instructions, approximately 200 mg of each fecal sample was used to extract the genomic DNA using the E.Z.N.A. ® Stool DNA Kit (Omega, Bio-tek Inc., Norcross, GA, USA).According to the previously reported relevant primers (Table S1), the present study amplified the bg, tpi, and gdh loci of G. duodenalis and SSU The NOAA's National Center for Environmental Information (https://gis.ncdc.noaa.gov/maps/ncei/cdo/monthly, accessed on 15 March 2024) was used to extract relevant geoclimatic data (latitude, longitude, elevation, temperature, precipitation, humidity, and climate).

DNA Extraction and PCR Amplification
Following the manufacturer's instructions, approximately 200 mg of each fecal sample was used to extract the genomic DNA using the E.Z.N.A. ® Stool DNA Kit (Omega, Biotek Inc., Norcross, GA, USA).According to the previously reported relevant primers (Table S1), the present study amplified the bg, tpi, and gdh loci of G. duodenalis and SSU rRNA of Blastocystis spp.by PCR method and determined the occurrence and prevalence of both parasites [32,33].The PCR system consists of 2.5 µL of 10 × PCR Buffer (Mg 2+ free), 2 µL of dNTPs (25 mM MgCl 2 ), 1.25 U of Ex-Taq enzyme (Takara, Dalian, China), 1 µL of each primer (20 mM), 2 µL of DNA template, and double distilled water (ddH 2 O) was added to a final volume of 25 µL.PCR amplification procedures referred to previous reports [32,33], and the annealing temperatures are summarized in Table S1.Moreover, 2 µL negative (ddH 2 O) and positive (DNA of G. duodenalis or Blastocystis) controls were included in each PCR amplification round for detecting the contamination and reliability of results.All PCR products were visualized by 1.5% agarose gels containing ethidium bromide (EB) under UV light, and the positive products were sent to Sangon Biotech Co., Ltd.(Shanghai, China) for bidirectional sequencing.

Sequencing and Phylogenetic Analysis
Each sequence was obtained by overlapping the bidirectional peak map in Chromas V2.6 software.The genotypes and subtypes of G. duodenalis and Blastocystis spp.were determined by aligning with reported sequences available in the GenBank database using the Base Local Alignment Retrieval tool for nucleotide (BLASTn) on the NCBI website and novel genotypes and subtypes were identified according to the established nomenclature system [34].Additionally, multilocus genotypes (MLGs) of G. duodenalis were used to judge the potential correlation among three genetic markers using the multilocus sequence typing (MLST) tool [31].Subsequently, the phylogenetic evolutionary trees were separately constructed using identified genotypes or subtypes of G. duodenalis and Blastocystis spp.using the Neighbor-Joining (NJ) method with 1000 replicates of bootstrap value in Mega 7.0 to further explore the genetic characteristics.

Statistical Analysis
The chi-square (χ2) test was used to explore the correlation between risk factors (region, age, sex, and altitude) and prevalence of the two parasites by SPSS 26.0 software (SPSS Inc., Chicago, IL, USA).Odds ratios (ORs) and their 95% confidence intervals (95%CI) were calculated to assess the strength of the association between prevalence and risk factors.The correlation was considered statistically significant when the p-value was less than 0.05.

The Prevalence of G. duodenalis and Blastocystis spp.
In this study, 90 out of 1200 fecal samples were detected as G. duodenalis-positive with an overall G. duodenalis prevalence of 7.5%.As shown in Table 1, the presence of G. duodenalis was observed in all five sampling sites, with the highest prevalence of G. duodenalis in black goats detected in Changzhi City (24.5%, 95% CI: 16.2-32.9).Statistical analysis revealed that significant differences in the prevalence of G. duodenalis were observed among region groups (p < 0.001), age groups (p = 0.005), and altitude groups (p = 0.023), but not found in sex groups.The overall prevalence of Blastocystis spp. in the examined black goats in Shanxi Province was 3.5% (42/1200) (Table 1).The highest prevalence of Blastocystis spp. was detected in Changzhi City (22.6%, 95% CI: 14.4-30.7),which was significantly higher than that in the other four sampling regions (p < 0.001).Likewise, the prevalence of Blastocystis spp. between younger goats (4.7%, 30/641) and elder goats (2.2%, 12/559) was statistically significantly different (p = 0.017).Notably, the prevalence of Blastocystis spp. was not statistically significantly different in relation to altitude and sex factors (p > 0.05).
For all the fecal samples investigated in this study, the results showed that both G. duodenalis and Blastocystis spp.infections were more likely to be found in black goats who were under three years, female, and living in areas with altitude above 1 km.Notably, black goats in Changzhi City had the highest prevalence of the two parasites, significantly higher than that in the other four study areas (Table 1).

Population Genetic Analyses of G. duodenalis
In order to investigate the population genetic structure of G. duodenalis in black goats in five study regions, out of 90 G. duodenalis-positive samples, 70, 7, and 32 sequences were obtained by further sequencing and analyses at the bg, tpi, and gdh loci, respectively.As shown in Table 2, among the 70 obtained sequences at the bg locus, 38 were assemblage B, and 32 belonged to assemblage E. Regarding tpi and gdh loci, all 7 and 32 sequences were identified as assemblage E. Interestingly, assemblage B was detected in four regions, except Yuncheng City where the altitude was less than 1000 m.In addition, assemblage E was also not detected in black goats in Datong City.As shown in Table 3, out of the obtained 70 sequences at the bg locus, 38 belonged to sub-assemblage BIII and 32 represented assemblage E (including 27 known assemblage E35, four known assemblage E1, and one novel assemblage E41).Single nucleotide polymorphisms (SNPs) analysis of assemblage B at the bg locus of G. duodenalis indicated that 76.3% (29/38) of sequences showed 98.8% similarity to the reported assemblage BIII (LC184000) with 4 SNPs, and another 9 sequences exhibited 98.5% to 98.8% similarity with the reference sequence (LC184000) with 4-6 SNPs.Meanwhile, the known sub-assemblage E1 and E35 sequences showed 100.0%similarity to the sequences of assemblage E detected in Tan sheep in the Ningxia Hui Autonomous Region of China with accession number MK610387 (E35) and MK610388 (E1), respectively.Moreover, a novel sub-assemblage E41 was identified in this study with one SNP when referred to the reported assemblage E35 (MK610387).At the gdh locus, 30 out of 32 sequences were identical to the known assemblage E34 (MK645786); two sequences showed a 99.8% similarity and were designated as novel sub-assemblage E53 (Table S2).In addition, seven sequences detected at the tpi locus showed a 100.0%similarity to the reported assemblage E (EU189333), designated as E33 (Table S3).The representative sequences obtained in the present study were deposited in the GenBank database with the following accession numbers: PP754423 to PP754425, PP754427 to PP754435 for the bg gene, PP754419 to PP754420 for the gdh gene, and PP754421 for the tpi gene.In this study, six samples were successfully sequenced at all three intra-assemblage variation genetic loci, forming one novel MLG-E12 within the assemblage E (Table S4).This novel MLG was only found in black goats in Changzhi City.
A phylogenetic tree was conducted to explore the relevance of detected assemblages and sub-assemblages in this study.As shown in Figure 2, the tree revealed that the assemblage E detected in this study consistently clustered with the reported assemblage E and had a close relationship with reported assemblage E in cattle and goats.It is noteworthy that the tree also demonstrated that the novel assemblage B was closely related to the reported assemblage B, which was detected in humans.In this study, six samples were successfully sequenced at all three intra-assemblage variation genetic loci, forming one novel MLG-E12 within the assemblage E (Table S4).This novel MLG was only found in black goats in Changzhi City.
A phylogenetic tree was conducted to explore the relevance of detected assemblages and sub-assemblages in this study.As shown in Figure 2, the tree revealed that the assemblage E detected in this study consistently clustered with the reported assemblage E and had a close relationship with reported assemblage E in cattle and goats.It is noteworthy that the tree also demonstrated that the novel assemblage B was closely related to the reported assemblage B, which was detected in humans.

Population Genetic Analyses of Blastocystis spp.
As shown in Table 4, sequence analyses indicated that the obtained 42 Blastocystispositive sequences from the examined black goats were divided into four known subtypes, including ST10 (n = 26), ST14 (n = 13), ST5 (n = 2), and ST30 (n = 1).Notably, the prevalent ST10 was detected in black goats in four study areas but not detected in Yuncheng City, and the ST14 was widely observed in Lvliang City, Yuncheng City, Changzhi City, and Datong City, and was the only ST detected in Yuncheng City.In addition, ST30 was only detected in black goats in Jinzhong City.
Moreover, the obtained sequences of Blastocystis spp.detected in this study were used to conduct a phylogenetic tree with 21 reported sequences, which were deposited into the GenBank database.As shown in Figure 3, the sequences detected in this study were closely related to and clustered with reported animal-derived STs, and other reported STs were separately positioned in distinct branches (Figure 3).The representative sequences obtained in the present study were deposited in the GenBank database with the following accession numbers: PP626090 to PP626111.

Co-Infection of G. duodenalis and Blastocystis spp. in Black Goats
In this study, among the detected 90 G. duodenalis-positive samples and 42 Blastocystis-positive samples, nine samples showed the co-infection of both parasites in black goats in Shanxi Province, in which eight samples were collected in Changzhi City (Table 5).Interestingly, among the nine co-infected black goat samples, five of them were successfully identified as G. duodenalis-positive at all three loci and formed a novel MLG-E12.

Co-Infection of G. duodenalis and Blastocystis spp. in Black Goats
In this study, among the detected 90 G. duodenalis-positive samples and 42 Blastocystispositive samples, nine samples showed the co-infection of both parasites in black goats in Shanxi Province, in which eight samples were collected in Changzhi City (Table 5).Interestingly, among the nine co-infected black goat samples, five of them were successfully identified as G. duodenalis-positive at all three loci and formed a novel MLG-E12.
Samples successfully amplified at three loci and forming a novel MLG-E12 are shown in bold.

Discussion
G. duodenalis and Blastocystis spp.are two common intestinal protozoans that infect humans and many animals, including goats, which can cause diarrhea, weight loss, and reduced feeding efficiency [35].Blastocystis spp.has been reported in over half of the countries across the Asian continent [36].Furthermore, more than 28.5 million people in China are affected annually by G. duodenalis, positioning it as the second most prevalent causative agent for human infectious diarrhea following viral pathogens [37].To reveal the prevalence and genetic characterization of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province, the present study detected and characterized both parasites by PCR amplification and sequence analyses.
Overall, the prevalence detected in black goats in Shanxi Province was 7.5% for G. duodenalis, with assemblage E being the predominant genotype in the study areas.In the world, G. duodenalis was less well studied in goats, with the reported prevalence ranging from 4.0% to 43.5% [13].Moreover, the G. duodenalis prevalence in goats in China ranged from 0 to 46.2% (Table S5).In this study, the prevalence of G. duodenalis in black goats in Shanxi Province was higher than that in goats in some countries [38,39] and some provinces of China, such as Yunnan province [19,30,34,[40][41][42], but lower than that in goats in some other countries, e.g., Greece and India [43][44][45][46][47][48] and some other provinces of China [29,35,[49][50][51][52] (Table S5).These results indicate the importance of goats as hosts for G. duodenalis and their potential role in transmitting giardiasis caused by this parasite.Additionally, prevalence differences may be related to various factors, including geographical location, age distribution, sex composition, feeding and management practices, ecological conditions, animal immune statuses, and other contributing factors [7].
Statistical analysis revealed that significant differences in the prevalence of G. duodenalis in black goats were observed among region groups, age groups, and altitude groups.The highest G. duodenalis prevalence was detected in black goats in Changzhi City and was significantly higher than that in the other four study areas (Table 1).Changzhi City, situated in the southeastern of Shanxi Province, has a warm temperate sub-humid continental monsoon climate characterized by high temperatures and humidity levels.We speculated that the favorable climate and environment may contribute to the prevalence and spread of G. duodenalis.Additionally, the prevalence of G. duodenalis in black goats decreased as age increased, consistent with previous studies in goats [30].Generally, younger animals are more likely to acquire the parasites than adults, which have stronger immune systems [41].At altitude groups, 93.3% of G. duodenalis-positive samples were detected in areas above an altitude of 1000 m, which indicated that the altitude factor might play an important role in the transmission of G. duodenalis.However, there is no sufficient evidence to explain the correlation between the prevalence of G. duodenalis and the altitude.
In this study, G. duodenalis assemblages E and B were identified in black goat fecal samples; the former was the main genotype (57.8%, 52/90).The results of this study are similar to those reported in sheep and goats from Henan, Sichuan, Yunnan, Shaanxi, Anhui, and Guangdong Provinces and the Inner Mongolia Autonomous Region of China (Table S5).Numerous reports documented that the close association between humans and animals serves as a basis for the transmission of animal-specific subtypes to humans [53].Assemblage E has been widely reported in artiodactyls, such as cattle, sheep, pigs, goats, and alpacas, with strong host specificity [35].Notably, assemblage B was only amplified at bg loci, and 79.0% assemblage B (30/38) was detected in Jinzhong City, which is close to the provincial capital city, Taiyuan, where has the largest transportation hub and population in Shanxi Province.High population density and public transportation may be related to the fact that the majority of assemblage B occurred in black goats in Jinzhong City.Up to now, among the five studies reporting the presence of assemblage B in goats, assemblage B was always detected in SSU rRNA and the tpi locus, including China [34], India [46], Malaysia [39], and Tanzania [54].However, in Nigeria, assemblage BI was successfully identified at three loci (bg, gdh, and tpi) from fecal samples of goats for the first time, whereas assemblage BIII was only detected at gdh and tpi loci [55].Sub-assemblage BIII was a valid zoonotic assemblage of G. duodenalis, which has been reported in humans and many animals, including wild animals, domestic animals, and companion animals [13].G. duodenalis was detected in human fecal samples in Heilongjiang Province, and assemblages A and B were distributed in all ages [41].
The multilocus genotyping (MLG) employed in this study has been adopted worldwide as a more reliable method of designating Giardia (sub)-assemblages to isolates than single-locus genotyping [32,56].In this study, three loci of G. duodenalis were successfully amplified in six samples and designated as a novel MLG-E12.Previous reports highlighted that assemblage E was detected in humans from different countries, suggesting that assemblage E was a potential zoonotic assemblage [57,58].However, further epidemiological investigations involving larger sample sizes and diverse geographical regions are necessary to gain deeper insights into the G. duodenalis transmission dynamics between animals and humans [12,59].
The prevalence of Blastocystis spp. in black goats in Shanxi Province was lower than that in Guangdong Province (33.6%) [29], the Tibet Autonomous Region (8.5%) [60], Shaanxi Province (58.1%) [61] of China, Malaysia (30.9%) [62], Portugal (12.7%) [63], and Libya (10.5%) [64].In this study, mostly Blastocystis-positive samples in black goats were detected from Changzhi City, which exhibited a significant difference compared with the other four investigated regions (p < 0.001).Many factors could contribute to these variations, e.g., unique climate conditions and poor feeding and management practices [33].The prevalence of Blastocystis spp. in black goats was observed to be negatively correlated with altitude in the other four areas, except in Changzhi City.With regards to the age groups, there was a statistically significant difference in Blastocystis spp.prevalence in the examined black goats (p < 0.05).According to a previous study, the age factor is also considered as one of the pivotal factors influencing the transmission of Blastocystis spp.among animals, but this point is still controversial [33].
In this study, four subtypes (ST5, ST10, ST14, and ST30) of Blastocystis spp.were identified in black goats in Shanxi Province, with the predominant subtype being ST10, consistent with previous studies reported in Asia [36].ST10 and ST14 were the most prevalent subtypes of Blastocystis spp. in sheep and goats in Asia, and have been reported in some provinces of China, such as Guangdong Province [29].ST10 and ST14 were detected in children in Senegal, suggesting their zoonotic potential and threatening public health [33,65].Importantly, the present study first reported the occurrence of Blastocystis ST30 in black goats in China.A previous report detected ST30 in sheep in China [25]; however, the transmission dynamics and pathogenicity of ST30 are still unknown.ST5, a valid zoonotic subtype of Blastocystis spp., has been detected in humans, livestock, and wild animals [20], and was detected in the present study in only two fecal samples.
In this study, a total of nine samples were found to be co-infected with G. duodenalis and Blastocystis spp.(0.8%, 9/1200).Among these positive samples, eight samples were collected from female black goats aged less than 3 years old in Changzhi City.In fact, poor environmental conditions and chaotic management were observed in farms in Changzhi City, which may be the primary reasons for the occurrence of the highest prevalences of G. duodenalis and Blastocystis spp. in black goats.Therefore, some sanitation measures are necessary to be taken to control the transmission of two parasites in Changzhi City, including regular cleaning and deworming.

Conclusions
This study revealed the prevalence and genotypes of Blastocystis spp.and G. duodenalis in black goats in Shanxi Province for the first time.Subtypes (ST5, ST10, ST14, and ST30) of Blastocystis spp.and two assemblages (B and E) of G. duodenalis were identified in this study, which suggests the risk of zoonotic transmission.These results provide baseline data for the execution of intervention strategies and measures against Blastocystis spp.and G. duodenalis in black goats in Shanxi Province.

Figure 1 .
Figure 1.Sampling sites of black goat feces in Shanxi Province, North China.

Figure 2 .Figure 2 .
Figure 2. Phylogenetic tree of the obtained sequences (marked with a black circle) in this study and previously reported sequences of G. duodenalis based on bg loci.The Kimura 2-parameter model Figure 2. Phylogenetic tree of the obtained sequences (marked with a black circle) in this study and previously reported sequences of G. duodenalis based on bg loci.The Kimura 2-parameter model method was used with bootstrap evaluation of 1000 replicates.Bootstrap values are shown when >50%.

Animals 2024 , 15 Figure 3 .
Figure 3. Phylogenetic tree of the obtained sequences in this study (marked with a black circle) and previously reported sequences of Blastocystis spp.based on SSU rRNA.The Kimura 2-parameter model method was used with bootstrap evaluation of 1000 replicates.Bootstrap values are shown when >50%.

Figure 3 .
Figure 3. Phylogenetic tree of the obtained sequences in this study (marked with a black circle) and previously reported sequences of Blastocystis spp.based on SSU rRNA.The Kimura 2-parameter model method was used with bootstrap evaluation of 1000 replicates.Bootstrap values are shown when >50%.

Table 1 .
Factors associated with the prevalence of Giardia duodenalis and Blastocystis spp. in black goats in Shanxi Province, North China.

Table 2 .
Occurrence of Giardia duodenalis genotypes (assemblages B and E) in this study in relation to different factors.

Table 3 .
Analysis of single nucleotide polymorphisms in G. duodenalis sequences at bg locus.

Nucleotide at Position of Reference Sequence No. of Sequences
Nucleotide substitutions are in bold and in capital letters; dots indicate identical to the reference sequences.
Nucleotide substitutions are in bold and in capital letters; dots indicate identical to the reference sequences.

Table 4 .
Distribution of Blastocystis spp.subtypes in black goats in Shanxi Province.

Table 5 .
Distribution of genotypes/subtypes of G. duodenalis and Blastocystis spp. in co-infected black goats in Shanxi Province.