Introduction

Cryptosporidium and Giardia are important protozoan parasites that cause diarrhea and other clinical symptoms in humans and numerous animals, including livestock, companion animals, and wild mammals (Huang et al. 2014). Infections with these pathogens occur via the fecal-oral route, by either direct or indirect contact with infected individuals or by the ingestion of infective oocysts or cysts from contaminated water or food (Feng and Xiao 2011; Xiao 2010). Cryptosporidium and Giardia have attracted attention as the major cause of human infections (Maddox-Hyttel et al. 2006; Monis and Thompson 2003). Cattle, especially calves, may be considered the most important reservoir of zoonotic infections (Fan et al. 2017). Cryptosporidium and Giardia infections in these animals could lead to significant economic losses owing to high morbidity and mortality (Olson et al. 2004).

To date, four Cryptosporidium species, namely C. andersoni, C. bovis, C. parvum, and C. ryanae, have been identified as the most common and frequently occurring in cattle (Slapeta 2013), and the distribution of these species varies with age (Xiao 2010). Cryptosporidium parvum is usually found in pre-weaned calves and is the main pathogenic species that affects humans in certain countries (Snel et al. 2009; King et al. 2019); on the other hand, C. bovis and C. ryanae usually infect post-weaned calves and yearlings, and C. andersoni is mainly observed in adult cattle (Santin et al. 2004; Zhao et al. 2013). Based on sequence analysis using the 60-kDa glycoprotein (gp60) gene, IIa and IId subtypes of C. parvum have been found in both humans and calves and can cause zoonotic cryptosporidiosis (Avendano et al. 2018; Chalmers and Giles 2010). Of the IIa family members, IIaA15G2R1 is most commonly reported as the major zoonotic subtype in cattle (Ryan et al. 2014). Other IIa subtypes such as IIaA16G1R1 and IIaA18G3R1 have different distributions according to region (Xiao 2010).

Giardia duodenalisconsists of eight distinct assemblages, A–H(Feng and Xiao 2011). Among these, assemblages A and B can infect both humans and other animals including companion animals, livestock, and wild mammals, whereas the remaining assemblages (C–H) appear to be more host-specific, with assemblages C and D observed in dogs and other canids, assemblage E mainly observed in ungulates including cattle, sheep, and goats, assemblages F and G observed in cats and rodents, and assemblage H observed in marine mammals (Ryan and Caccio 2013). In particular, assemblages A, B, and E have been detected in cattle, with assemblage E being predominant in many countries (Feng and Xiao 2011; Li et al. 2016). Recently, several studies have also identified assemblage E in humans (Abdel-Moein and Saeed 2016; Zahedi et al. 2017). There is no difference in the distribution of G. duodenalis assemblage among cattle of different age groups (Naguib et al. 2018).

Despite the threat of cryptosporidiosis and giardiasis to humans and livestock, the importance of these diseases (particularly in cattle) is often overlooked, and information on these diseases is limited in the Republic of Korea (ROK). Recently, our group revealed the presence of assemblages A and E as well as G. duodenalis in pre-weaned Korean native calves. Moreover, assemblage A has been detected in calves with normal feces (Lee et al. 2018). Only one recent study on the molecular epidemiology of Cryptosporidium spp. in the ROK has identified the presence of C. parvum, C. bovis, and C. ryanae in young calves with diarrhea (Lee et al. 2016b). Thus far, little is known about the subtype and genotype of C. parvum and G. duodenalis among the calves in the ROK (Lee et al. 2016a, b, 2018). Therefore, this study aimed to investigate the prevalence of Cryptosporidium and Giardia in pre-weaned calves, analyze the association between these parasites and diarrhea, and identify the zoonotic potential of C. parvum and G. duodenalis subtypes/assemblages.

Materials and methods

Sample collection

Between January and October 2018, 315 fecal samples were collected directly from the rectum of pre-weaned Korean native calves aged 1–60 days from 10 different farms (Anseong, Geochang, Gimje, Gyeongju, Jeongup, Mungyeong, Naju, Sangju, Yecheon, and Yeongju) in the ROK. The collected samples were placed into a disposable plastic bag and immediately transported to the laboratory for subsequent DNA isolation and molecular testing. The fecal consistency of each calf was categorized as normal or diarrheic according to its physical characteristics. For each animal, the sampling date, age, sex, fecal consistency, farm location, and farm management system were recorded.

DNA extraction and PCR amplification

Genomic DNA was extracted from 200 mg of each fecal sample using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations and subsequently frozen at – 20 °C until use. Small subunit (SSU) rRNA gene was used to identify Cryptosporidium spp. (Xiao et al. 1999). C. parvum was subtyped by targeting the 60-kDa glycoprotein (gp60) gene using nested PCR (Alves et al. 2003). G. duodenalis assemblage types were analyzed using the triose phosphate isomerase (tpi) gene (Sulaiman et al. 2003), β-giardin (bg) gene (Caccio et al. 2002; Lalle et al. 2005), and glutamate dehydrogenase (gdh) gene (Read et al. 2004). In this study, only samples showing a good sequencing result were considered to be positive for C. parvum and G. duodenalis.

Nucleotide sequencing and phylogenetic analysis

All secondary PCR products were purified using the AccuPower PCR Purification Kit (Bioneer, Daejeon, ROK) and used for direct sequencing (Macrogen, Daejeon, ROK). The nucleotide sequences obtained in this study were analyzed using BioEdit (version 7.2.5) and the Basic Local Alignment Search Tool available from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov). To determine the subtype and genotype of C. parvum and G. duodenalis, nucleotide sequences were aligned using ClustalX and analyzed by performing a direct comparison with reference sequences from GenBank. A phylogenetic tree was constructed based on nucleotide alignments by employing the maximum-likelihood method. Bootstrap analysis was conducted with 1000 replicates using MEGA version 7 software (Kumar et al. 2016). Nucleotide sequences obtained in this study were submitted to the GenBank database under the accession numbers MK732491–MK732504 and MK720232–MK720271 for C. parvum and G. duodenalis, respectively.

Statistical analysis

Statistical analysis was performed using SPSS Statistics 25 software package for Windows (SPSS Inc., Chicago, IL, USA). Chi-square (χ2) and Fisher’s exact tests were used to compare the infection rates of C. parvum and G. duodenalis. In addition, the association between prevalence and specific variables (age and fecal consistency) was determined using binary univariate logistic regression models. Odds ratio (OR) with 95% confidence interval (CI) was calculated to assess the likelihood of association. A P value of ≤ 0.05 was considered to be statistically significant.

Results

Prevalence of C. parvum and G. duodenalis

The overall prevalence of C. parvum and G. duodenalis was 4.4% (14/315) and 12.7% (40/315), respectively. Co-infection with C. parvum and G. duodenalis was not detected in these calves. Among the 10 farms examined, C. parvum was detected in only three farms (Geochang, Gimje, and Gyeongju), whereas G. duodenalis was detected in all but two farms (Jeongup and Naju) (Table 1). Interestingly, a mixed infection with both assemblages A and E was found in two farms (Table 1). Infection with C. parvum (χ2 = 0.004, P = 0.952) and G. duodenalis (χ2 = 0.053, P = 0.817) was not associated with diarrhea in pre-weaned Korean native calves (Table 2). C. parvum– and G. duodenalis–positive samples were compared according to different age groups of the calves. As shown in Table 2, infection with C. parvum was observed only in calves aged ≤ 20 days. The prevalence of C. parvum was the highest in calves aged 11–20 days, but these differences were not significant (Table 2). On the contrary, the percentage of positive calves to G. duodenalis was significantly highest in calves aged 41–50 days (Table 2). Thus, the risk of being positive to G. duodenalis was 9.9- and 4.4-fold higher in calves aged 41–50 days (95% CI: 2.37–41.34; P = 0.00) and 31–40 days (95% CI: 0.70–7.77; P = 0.03) respectively, than in those aged 1–10 days (Table 2).

Table 1 Prevalence of Cryptosporidium parvum and Giardia duodenalis in pre-weaned Korean native calves
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Table 2 Prevalence of Cryptosporidium parvum and Giardia duodenalis considering the age of the animals and the fecal consistency
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Distribution of C. parvum subtypes

All Cryptosporidium-positive samples were identified as C. parvum using SSU rRNA sequence analysis. Based on gp60 sequence analysis, the C. parvum isolates were identified to belong to the IIa family and were further subtyped into IIaA15G2R1 (n = 13) and IIaA18G3R1 (n = 1) (Table 3). IIaA15G2R1 was the predominant subtype in pre-weaned Korean native calves and was detected in both diarrheic and normal feces, whereas IIaA18G3R1 was only found in the feces of a 14-day-old calf with diarrhea. Thirteen isolates of IIaA15G2R1 showed 89.4–99.5% similarity with each other and were closely related to a human isolate (EF576979) obtained from the Netherlands. Our isolate belonging to IIaA18G3R1 showed 100% identity with another Korean isolate (KX342059).

Table 3 Distribution of Cryptosporidium parvum subtypes in pre-weaned Korean native calves identified in this study
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Genotypes and subtypes of G. duodenalis

Sequence analysis of the bg gene revealed that two assemblages, A (n = 4) and E (n = 36), were identified (Fig. 1). All isolates belonging to assemblage A were identified as subtype A1 (Table 4) and detected in diarrheic feces. All nucleotide sequences were identical to those (MH104712 and MH104713) obtained from healthy calves previously identified by our group. Of the 36 assemblage E isolates, 17 and 19 isolates were identified from diarrheic and normal feces, respectively. Sequence analysis of the tpi, gdh, and bg genes revealed the genetic diversity within assemblage E. Among the 36 G. duodenalis bg-positive samples, the tpi and gdh loci were amplified and sequenced in 6 and 8 specimens, respectively. At the bg locus, the following six subtypes of assemblage E were observed: E1 (MK252651, n = 16), E2 (MK252652, n = 4), E3 (MK252653, n = 13), E5 (MK252649, n = 1), E8 (MK252650, n = 1), and E11 (KY769089, n = 1) (Table 4). Furthermore, at the tpi locus, the three subtypes, E1 (MF671900, n = 2), E3 (KT92259, n = 3), and E10 (KT710746, n = 1), were identified, whereas at the gdh locus, the three known subtypes, E1 (MK252654, n = 4), E3 (MK252657, n = 3), and E13 (MH621339, n = 1), were found. Altogether, two samples were successfully subtyped at all three genetic loci (Table 4). E1 was the most common subtype in pre-weaned Korean native calves (Table 4).

Fig. 1
figure 1

Phylogenetic analysis based on the β-giardin gene of Giardia duodenalis reference strains/isolates and Korean isolates identified in this study. The phylogenetic tree was constructed using MEGA7 software by employing the neighbor-joining method. The numbers over branches indicate bootstrap values as a percentage of 1000 replicates that support each phylogenetic branch. The boldface type indicates the sequences determined in this study

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Table 4 Multilocus sequence genotypes of Giardia duodenalis in pre-weaned Korean native calves identified in this study based on nucleotide sequence analysis of the beta-giardin (bg), triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh) genes
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Discussion

In the present study, we assessed the prevalence of Cryptosporidium spp. and G. duodenalis among different age groups as well as the association with diarrhea in pre-weaned Korean native calves aged ≤ 60 days. The prevalence of C. parvum infection was lower than that of G. duodenalis infection. Recent studies have reported infection rates of 6.1% for C. parvum and 10–13.1% for G. duodenalis in young calves (< 3 months) in the ROK (Lee et al. 2016a, b, 2018). In this study, the prevalence of C. parvum was lower than that reported in a previous study (Lee et al. 2016b). The differences between the two groups can be explained by the number of analyzed specimens, farm location, and season at the time of specimen collection. The percentage of C. parvum positive samples found in the present study was lower than that reported in several investigations (Smith et al. 2014; Ichikawa-Seki et al. 2015; Lee et al. 2016b; Naguib et al. 2018; Ouakli et al. 2018; Santoro et al. 2019). This may be due to low oocyst shedding when sampling, since oocyst excretion in calves is intermittent (Rieux et al. 2013; Santoro et al. 2019); so, resampling would allow us obtaining more accurate results. Moreover, we did not concentrate oocysts and did not apply freeze-thaw cycles before DNA extraction, resulting in lower DNA recovery and consequently decreasing PCR sensitivity. In this regard, previous studies have also reported that PCR analysis is less sensitive if the number of oocysts is less or if oocysts are not completely broken (Fayer et al. 2007; Mueller-Doblies et al. 2008). Additionally, co-infection with C. parvum and G. duodenalis was not detected; this might be attributed to the low prevalence of C. parvum in these calves. Of the four major Cryptosporidium species (C. andersoni, C. bovis, C. parvum, and C. ryanae) identified in cattle, only C. parvum was detected in pre-weaned Korean native calves (< 1 month). These results agree with previous studies revealing highest C. parvum prevalence in caves aged 1 month (Santin et al. 2004; Fayer et al. 2007; Thompson et al. 2007; Rieux et al. 2013; Ouakli et al. 2018). In this study, C. parvum was only identified in animals younger than 21 days, especially in 11–20-day-old calves; the absence of C. parvum ≥ 21-day-old calves could be attributed to the lower number of samples collected in these animals when compared with calves aged 1–20 days. Interestingly, C. bovis was not found in calves in the ROK, although several studies have shown that it is the most common species in pre-weaned dairy calves, especially healthy animals older than 4 weeks (Murakoshi et al. 2012; Rieux et al. 2013; Bjorkman et al. 2015; Feng et al. 2019). Thus, a recent study showed that C. bovis was mainly identified in calves older than 15 days (Ouakli et al. 2018). Nevertheless, it was also reported that the Cryptosporidium species detected in cattle farms could differ depending on the sampling year (Rieux et al. 2013). Therefore, further studies are warranted to investigate the prevalence of various Cryptosporidium species across different age groups and fecal consistency.

Based on our results, the prevalence of G. duodenalis was different according to the age of calves, indicating that G. duodenalis infection is related to age. The prevalence of G. duodenalis was significantly higher in calves aged 31–50 days, especially in those aged between 41 and 50 days (P = 0.00; Table 2). The results suggest that age could affect the occurrence of G. duodenalis in calves. The presence of G. duodenalis in the 80% of the studied farms indicates that this protozoan is endemic in cattle farms in the ROK; its infection may be explained by environmental contamination with cysts. According to recent studies, the highest occurrence of G. duodenalis was found in 2-month-old calves (Naguib et al. 2018; Feng et al. 2019). However, in the present study, G. duodenalis infection was not detected in calves aged 51-60 days. It is presumed that either the number of samples analyzed in this age group was too low or the infection rate of G. duodenalis was considerably low; consequently, G. duodenalis could not be detected by PCR. Similar to the approach adopted for C. parvum, we did not investigate the intensity of cyst shedding of G. duodenalis in calves. It is speculated that DNA was extracted directly from the feces without confirming the presence of cysts, which may affect the accurate diagnosis of G. duodenalis. These results suggest that microscopic examination should be performed for more accurate results. Therefore, it is necessary to conduct large-scale epidemiological studies in the future to determine the association between age and G. duodenalis infection.

Our study showed that C. parvum and G. duodenalis were not associated with diarrhea. According to the present results, C. parvum was found only in diarrheic feces of 1-10-day-old calves, whereas it was commonly found in the normal feces of 11-20-day-old calves, indicating that C. parvum was present regardless of diarrhea status. Cryptosporidium parvum reportedly causes diarrhea in younger animals; however, our result is inconsistent with that reported in previous studies (Geurden et al. 2010; Blanchard 2012). In addition, it remains unclear whether G. duodenalis causes diarrhea in calves because its prevalence was found to increase only when the calves turned 31–50 days old and usually in calves of this age group, G. duodenalis is detected regardless of their diarrhea status. The detection of C. parvum and G. duodenalis in normal feces cannot rule out the possibility of contamination with oocysts/cysts in an entire herd. Although the number of these oocysts/cysts present in feces is not sufficient to cause diarrhea, calves in which the passive transfer of colostral antibodies fails to occur usually have poor health condition and might develop diarrhea more easily (Lora et al. 2018). Moreover, if the location where a calf is raised is not properly cleaned and has piled-up feces, the risk of diarrhea increases (Maddox-Hyttel et al. 2006). These calves may become reservoirs, and care for these animals should not be neglected. Further studies are necessary to investigate the importance of C. parvum and G. duodenalis as the primary diarrhea-causing pathogens in pre-weaned Korean native calves.

Two C. parvum subtypes (IIaA15G2R1 and IIaA18G3R1) belonging to the zoonotic subtype family IIa were identified based on sequence analysis of the gp60 gene. This is the first report of the IIaA15G2R1 subtype in pre-weaned calves in the ROK. The other IIaA18G3R1 subtype has previously been reported in calves in the ROK (Lee et al. 2016b), and it was identified in only one diarrheic calf in the present study. Furthermore, this subtype is geographically restricted and mostly found in the UK and Australia (Xiao 2010). This may explain its minor role in the dissemination of cryptosporidiosis in animals and humans. In the present study, IIaA15G2R1 was identified as the predominant subtype (92.9%) in the ROK and was found in calves with both diarrheic and normal feces. In industrialized nations, the IIaA15G2R1 subtype is a major zoonotic subtype found in calves and humans (Trotz-Williams et al. 2006; Xiao 2010; Rieux et al. 2014; Hijjawi et al. 2016); however, no Cryptosporidium sequences have been obtained from humans in the ROK. To date, there is limited information regarding the subtypes of C. parvum isolated from calves in the ROK (Lee et al. 2016b), and this can be attributed to the lack of information or interest in C. parvum despite its high zoonotic potential. The results of that study suggested that calves could be a potential source of zoonotic infections. Further molecular epidemiological studies are necessary to identify the genetic variability and zoonotic potential of C. parvum in both cattle and humans.

Here, G. duodenalis assemblages A and E were detected. This result is in agreement with that of previous studies conducted in other countries (Abdel-Moein and Saeed 2016; Asher et al. 2016; Wang et al. 2017). A previous study reported that G. duodenalis assemblage E may cause intestinal lesions, leading to calf scours (Barigye et al. 2008). In this study, the genetic diversity of assemblage E was observed in pre-weaned Korean native calves with the detection of 6, 3, and 3 subtypes at the bg, gdh, and tpi loci, respectively. The reason underlying the diversity of assemblage E remains unclear. Moreover, the presence of multiple subtypes in a single farm was observed. This occurrence may be the result of intra-assemblage genetic recombination. In contrast to a previous study (Lee et al. 2018), G. duodenalis assemblage A was identified in four calves with diarrhea. The four sequences were identical to each other and to the sequences of assemblage A isolates reported in our previous study as well as in studies conducted in other countries. Previous studies have reported the detection of assemblages A and E in humans (Abdel-Moein and Saeed 2016; Zahedi et al. 2017); however, assemblage E had not been detected in humans in the ROK. Our findings suggest that calves play a potential role in the transmission of human G. duodenalis infection. Further studies are necessary to monitor its zoonotic transmission.

In conclusion, the present study demonstrated that the prevalence of G. duodenalis was higher than that of C. parvum in pre-weaned Korean native calves and that the occurrence of C. parvum and G. duodenalis was not associated with diarrhea. The most common C. parvum and G. duodenalissubtype/assemblage in pre-weaned Korean native calves were the IIaA15G2R1 subtype and assemblage E, respectively. This is the first report on the IIaA15G2R1 subtype of C. parvum in calves in the ROK. These findings suggest that calves may be an important source of zoonotic C. parvum and G. duodenalis infections. Because cryptosporidiosis and giardiasis prevention is important for maintaining good health of calves and humans, the risk of diseases caused by these parasites should be reduced by minimizing the infection pressure resulting from contamination of environment with C. parvum and G. duodenalisoocysts/cysts and by improving the immunity of calves. Additional epidemiological studies are warranted to better understand the transmission and public health significance of C. parvum and G. duodenalis in the ROK.