Introduction

Giardia duodenalis, also known as Giardia lamblia, or Giardia intestinalis, is a significant cause of diarrhoeal diseases globally and can infect a wide range of vertebrates, including humans (Ryan and Cacciò 2013; Rojas-López et al. 2022). In domestic animals, infection with G. duodenalis can lead to reduced reproductive performance in adults and developmental delays in young animals (O’Handley and Olson 2006). G. duodenalis is mainly transmitted through the faecal-oral route (Feng and Xiao 2011). The global prevalence of this disease is estimated to be between 2 and 7% in developed countries and up to 30% in low-income countries (Fantinatti et al. 2016). Giardiasis can cause diarrhoea and other intestinal symptoms. Additionally, it has been associated with arthritis and irritable bowel syndrome. The World Health Organisation (WHO) included giardiasis in its Neglected Diseases Initiative in 2006 (Painter et al. 2017; Nakao et al. 2017).

G. duodenalis has been classified into eight assemblages (A–H) based on host specificity and genetic differences. Assemblages A and B are considered typical zoonotic assemblages, while assemblages C–H have stricter host specificity (Heyworth 2016; Ryan et al. 2021). G. duodenalis assemblage E has been found to be the dominant assemblage in dairy cattle worldwide (Taghipour et al. 2022). Only a few studies have reported assemblage A/B as the dominant assemblage in dairy cattle (Coklin et al. 2007; Wang et al. 2014; Baroudi et al. 2017). Recently, multilocus genotyping (MLG) has been used to analyse the genetic diversity and zoonotic risk of G. duodenalis based on the bg*, gdh* and tpi* genes (Fu et al. 2022; Zhao et al. 2023b).

The National Bureau of Statistics reports a significant increase in the number of dairy cattle in Xinjiang, China, which is attributed to the implementation of scientifically informed large-scale breeding practices. However, large-scale animal husbandry can lead to the rapid spread of epidemics due to the close contact and similar living conditions of dairy cattle in high-density farming environments (Brooks-Pollock and Keeling 2009).

Compared to the data on Giardia duodenalis infections in cattle from central and eastern China, there is limited epidemiological data for G. duodenalis in large-scale dairy cattle farms in Xinjiang. The aim of this study was to investigate the infection rate and genetic characteristics of G. duodenalis in dairy cattle from large-scale farms in Xinjiang, China.

Materials and methods

Ethical standards

The protocol in the present study was not required to be reviewed and approved by the Animal Ethical Committee. Prior permission was obtained from the farm owner before collecting faecal samples. In the present study, faecal samples were collected from the rectum of each dairy cow without harm.

Sample collection

A total of 749 faecal samples were collected from dairy cattle between 2019 and 2020 from five large-scale farms (farm 1–farm 5) in Xinjiang. The farms were located in different positions in Alar city (farms 1–3), Hutubi city (farm 4), and Tiemenguan city (farm 5). The samples were collected from five age groups of dairy cattle. The study involved 153 pre-weaned calves (aged 0–3 months), 150 post-weaned calves (aged 3–6 months), 148 fattening dairy cattle (aged 6–12 months), 148 replacement heifers (aged 1–2 years and unproduced), and 150 adult heifers (over 2 years old and produced).

Faecal samples were collected directly from the rectums of the dairy cattle using disposable gloves. The amount of each samples ranged from 10 to 30 g. The samples were then placed in clean, self-sealing bags, labelled with the sampling information, and stored at 4 °C.

DNA extraction and PCR amplification

DNA was extracted from each faecal sample (approximately 200 mg) using an E.Z.N.A. Stool DNA Kit (Omega Bio-Tek Inc., Norcross/GA, USA) following the manufacturer’s suggested technique. The extracted DNA was stored at − 20 °C.

To determine the presence of G. duodenalis, the SSU rRNA* gene was targeted in all DNA samples (Appelbee et al. 2003). Each sample was analysed in duplicate using positive controls (dog-derived assemblage D DNA) and negative controls (sterile water). All samples positive for G. duodenalis were genotyped using multilocus analysis based on the bg* (Lalle et al. 2005), gdh* (Cacciò et al. 2008) and tpi* (Sulaiman et al. 2003) genes to determine G. duodenalis subtypes. The genotyping analysis included 291 bp for SSU rRNA*, 511 bp for bg*, 520 bp for gdh* and 530 bp for tpi*.

Sequence and phylogenetic analyses

The SSU rRNA*, tpi*, gdh* and bg* genes of positive secondary PCR products were sequenced by GENEWIZ (Suzhou, China). The obtained sequences were uploaded to SEQMAN in DNAStar (http://www.dnastar.com/) for DNA profile proofreading and were compared and identified using Clustal X2.1 (http://www.clustal.org/) and GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

To investigate the relationships between different isolates of G. duodenalis and reveal its genetic diversity, we constructed a phylogenetic tree based on the maximum likelihood neighbour-joining method using Mega7.0 software. The substitution rate calculation used the general time-reversible model, and we used 1000 replicates in the bootstrapping analysis.

Statistical analysis

Differences in infection rates between regions and physiological states were analysed by the χ2 test using the statistical software SPSS Statistics 26 (SPSS Inc., Chicago, IL, USA), and differences were considered statistically significant when P < 0.05.

Nucleotide sequence accession numbers

The nucleotide sequences obtained in this study have been deposited in NCBI’s GenBank database under the following accession numbers: OR538008–OR538012 for the bg* gene, OR538013–OR538016 for the gdh* gene and OR538017–OR538020 for the tpi* gene.

Results

Giardia duodenalis infection rate and distribution based on age

The study analysed 749 faecal DNA samples from dairy cattle using the SSU rRNA* gene of G. duodenalis. Of these, 180 samples (24.0%, 95% CI 21.0–27.1%) were identified as G. duodenalis-positive (Table 1). The lowest infection rate was found on farm 2 (0.7%), which was significantly lower than the rates on the other farms (farms 1 and 3) in Alar, as well as farms 4 and 5 (P < 0.05) (Table 1).

Table 1 Prevalence of G. duodenalis and assemblages determined by sequence analysis of the SSU rRNA*, bg*, gdh* and tpi* genes
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The study analysed the infection rate of G. duodenalis in dairy cattle across different age groups. The highest infection rate was observed in pre-weaned calves (45.1%, 69/153), which was significantly higher than the other age groups (P < 0.05). Adult heifers had the lowest infection rate (12.0%, 18/150). The infection rates of G. duodenalis in post-weaned calves, fattening dairy cattle and replacement heifers were 22.0% (33/150), 18.9% (38/148) and 21.6% (32/148), respectively (Table 2).

Table 2 Prevalence and assemblage distribution of G. duodenalis by physiologic stage
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Molecular identification and distribution of assemblages

In this study, 180 amplified sequences of G. duodenalis were analysed for the SSU rRNA* gene. Out of these, 176 sequences belonged to G. duodenalis assemblage E (97.8%, 176/180), three sequences belonged to G. duodenalis assemblage A (1.7%, 3/180) and one sequence belonged to G. duodenalis assemblage B (0.6%, 1/180). In farm 1, three samples tested positive for assemblage A, while in farm 4, one sample tested positive for assemblage B. Assemblage E was the only assemblage detected in farms 2, 3 and 5, and it was the dominant assemblage on all five farms (refer to Table 1).

In each of the five age groups defined in this study, one case of assemblage A was found in post-weaned calves, replacement heifers and fattening cattle. Only one case of assemblage B was found in fattening cattle, and both pre-weaned calves and adult cattle. Assemblage E was the dominant assemblage in all age groups.

Polymorphisms of Giardia duodenalis isolates

Out of the 180 positive samples, 69 sequences were obtained for the bg* gene, with 68 belonging to assemblage E and 1 to assemblage A. For the gdh* gene, 67 sequences were obtained, with 66 belonging to assemblage E and 1 to assemblage A. Lastly, 49 sequences were obtained for the tpi* gene, with 48 belonging to assemblage E and 1 to assemblage A (refer to Table 1). The present study obtained 68 assemblage E sequences obtained for the bg* gene, which were classified into 13 subtypes, including 8 known subtypes and 5 novel subtypes, by comparison with MN833266 in GenBank (accession numbers OR538008–OR538012) (Table S1). Additionaliy, 66 assemblage E sequences were obtained for the gdh* gene and classified into 14 isoforms, including 10 known isoforms and 4 novel isoforms (accession numbers OR538013–OR538016) (Table S2) using the reference sequence MK645797. Using MG820468 as a reference sequence, 48 sequences of the tpi* gene were obtained and categorized into 15 subtypes, including 11 known subtypes and 4 novel subtypes (accession numbers OR538017–538020) (refer to Table S3).

For assemblage A, one sequence was obtained for each of the three genes, which belong to different samples. The sequence obtained for the bg* gene showed 100% similarity to MK610391, while the gdh* gene sequence was 100% similar to MN047217, and the tpi* gene sequence was 100% similar to MK639171.

Multilocus genotyping

A total of 32 samples were identified as belonging to assemblage E based on being positive at all four genes (SSU rRNA*- bg*- gdh*- tpi*), forming 26 MLGs (named MLG-E1 to MLG-E26) (Table S4). Phylogenetic analyses of bg*- tpi*- gdh* tandem sequences showed that the MLGs obtained in this study were genetically distinct from the MLGs previously found in other regions (Fig. 1). However, they were similar to the MLGs found in Xinjiang, indicating a clear geographical segregation.

Fig. 1
figure 1

Geographic map of the sampling locations of Xinjiang, China. The authors originally designed the figure using ArcGIS 10.2 software. The original vector diagram imported into ArcGIS was adapted from Natural Earth (http://www.naturalearthdata.com)

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Discussion

The study revealed that the infection rate of G. duodenalis in dairy cattle in certain areas of Xinjiang was 24.0% (180/149), which is higher than the 9.4% (52/556) reported in Jiangxi (Qi et al. 2016) and 19.8% (31/156) reported in Taiwan (Lam et al. 2021), but lower than of the 27.5% (144/524) reported in Yunnan (Heng et al. 2022) and 29.5% (149/505) reported in Inner Mongolia (Zhao et al. 2023a). The worldwide infection rate of G. duodenalis in dairy cattle ranges from 0 to 76.5%. The infection rate of G. duodenalis in dairy cattle based on molecular techniques was 21.8% (5845/26,767) (Taghipour et al. 2022), which is similar to the results of the present study. Variations in reported infection rates may be attributed to several factors, such as the testing method (microscopic or molecular), farming type (free-range or intensive), farm size, number of samples analysed, time of sample collection and environmental conditions.

Previous studies have demonstrated that the infection rate of G. duodenalis is higher in pre-weaned dairy cattle than in post-weaned dairy cattle (Liu et al. 2015; Wang et al. 2019; Heng et al. 2022; Taghipour et al. 2022). These findings are consistent with the results of the present study. Table 2 shows that the infection rate of G. duodenalis was significantly higher in pre-weaned calves compared to dairy cattle of other ages. This may be due to the incomplete development of the immune system in pre-weaned calves making them more susceptible to pathogens. Most reports indicate that G. duodenalis infection is negatively correlated with the age of the animals (Santín et al. 2009; Wang et al. 2014; Huang et al. 2014). The results of the present study are consistent with this pattern, but the infection rate was higher in large-breed dairy cattle (21.6%) than in small-breed dairy cattle (18.9%). This may be due to the fact that large-breed dairy cattle enter the stage of social maturation and have more contact with other individuals. In large and crowded breeding environments, contact between animals can lead to the transmission of G. duodenalis, which increases the risk of infection.

G. duodenalis is classified into eight assemblages (A–H). Studies have consistently shown that assemblage E is the most prevalent in cows, followed by assemblages A, B and D (Taghipour et al. 2022; Meng et al. 2023). This study, assemblage E, A and B were detected using the SSU rRNA* gene, with assemblage E being the most dominant. Assemblage A and assemblage B are considered zoonotic assemblages in the traditional sense due to their wide range of hosts (Heyworth 2016). Assemblage E, on the other hand, is commonly found in hoofed animals such as dairy cattle and sheep (Taghipour et al. 2022; Geng et al. 2023) and is not typically considered zoonotic. However, it has been documented that assemblage E has caused human infections in various countries such as Brazil (Fantinatti et al. 2016), Australia (Zahedi et al. 2017) and New Zealand (Abdel-Moein and Saeed 2016). A study conducted in Egypt found that assemblage E was present in up to 62.5% of human samples (Abdel-Moein and Saeed 2016). Moreover, a growing body of research has demonstrated that G. duodenalis not only infects livestock in large-scale farms but also contaminates the surrounding environment(Tram et al. 2022; Zhao et al. 2023b). This contamination poses a risk not only to the health of other animals within the farms but also to humans who come into contact with these farms and may potentially be exposed to G. duodenalis (Robertson 2009; Berrilli et al. 2012). Therefore, further research is necessary to eatablish the public health implications of G. duodenalis in dairy cattle.

Twenty-six assemblage E MLG genotypes were identified in this study. The genetic differences of assemblage E were confirmed in various regions, with limited MLG intersection among samples. Each MLG was found in at most three dairy cattle, and only MLG-E1 was present simultaneously in both Alar and Tiemenguan (see Table S4). This relationship could be attributed to the substantial genetic diversity of G. duodenalis observed in Xinjiang’s dairy cattle. Figure 2 shows that the occurrence of E MLGs in the Tiemenguan and Alar areas was clustered on farms, while no substantial farm-based grouping was observed in Hutubi. This may be due to the low infection rate on Hutubi farms and the limited discovery of only two E MLGs, making the trial outcomes unrepresentative. Previous studies have found that G. duodenalis experiences geographical isolation (Cui et al. 2018). The E MLGs of G. duodenalis identified in this study were highly aggregated with those previously found in Xinjiang. However, most of them were significantly distinct from the E MLGs of G. duodenalis detected in dairy cows from Inner Mongolia (Zhao et al. 2023a), Guangdong (Cui et al. 2018), Sichuan (Dan et al. 2019), Yunnan (Heng et al. 2022), Jiangsu (Wang et al. 2019) and Shanghai (Wang et al. 2017). The study showed notable distinctions between the geographical distribution and genetic makeup of the E assemblage.

Fig. 2
figure 2

Phylogenetic evolutionary tree of G. duodenalis assemblages analysed by the maximum likelihood method based on bg*, tpi* and gdh* gene tandem sequences (bg*-tpi*-gdh*), with substitution rates calculated using a general time-reversible model. Bootstrap values greater than 50% from 1000 replicates are shown at the nodes. MLGs marked by the black triangles were the sequences obtained in this study

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Conclusion

The study findings show a high infection rate of G. duodenalis in dairy cattle in Xinjiang, China, with a predominant infection of G. duodenalis assemblage E. The varying distribution of MLGs of G. duodenalis assemblage E in dairy cattle may indicate geographic isolation.