Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea.

Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide. Data on the prevalence of Cryptosporidium spp. and its subtypes among calves in the Republic of Korea (KOR) are sparse. Hence, our study aimed to investigate the prevalence and association between the age of calf and the identified Cryptosporidium spp. and to determine the genotypes/subtypes of Cryptosporidium spp. in pre-weaned calves with diarrhea in the KOR. A total of 460 diarrheic fecal samples were collected from calves aged 1-60 days and screened for Cryptosporidium spp. by the 18S rRNA gene. Species identification was determined using the sequencing analysis of the 18S rRNA gene, and C. parvum-positive samples were subtyped via the sequence analysis of the 60-kDa glycoprotein (gp60) gene. Sequence analysis based on the 18S rRNA gene revealed the presence of three Cryptosporidium spp., namely, C. parvum (n = 72), C. ryanae (n = 12), and C. bovis (n = 2). Co-infection by these species was not observed. The infection rate was the highest in calves aged 11-20 days (26.1%, 95% CI 17.1-35.1), whereas the lowest rate was observed in calves aged 21-30 days (7.7%, 95% CI 0.0-16.1). The prevalence of C. parvum was detected exclusively in calves aged ≤20 days, and the highest infection rate of C. ryanae was seen in calves ≥31 days of age. The occurrence of C. parvum (χ2 = 25.300, P = 0.000) and C. ryanae (χ2 = 18.020, P = 0.001) was significantly associated with the age of the calves. Eleven different subtypes of the IIa family that belonging to C. parvum were recognized via the sequence analyses of the gp60 gene. Except for two (IIaA18G3R1 and IIaA15G2R1) subtypes, nine subtypes were first identified in calves with diarrhea in the KOR. IIaA18G3R1 was the most frequently detected subtype (72.2% of calves), followed by IIaA17G3R1 (5.6%), IIaA15G2R1 (4.2%), IIaA19G4R1 (4.2%), IIaA16G4R1 (2.8%), IIaA17G4R1 (2.8%), IIaA19G3R (2.8%), IIaA14G1R1 (1.4%), IIaA14G3R1 (1.4%), IIaA15G1R1 (1.4%), and IIaA19G1R1 (1.4%) These results suggest that the prevalence of Cryptosporidium spp. is significantly associated with calf age. Furthermore, the findings demonstrate the high genetic diversity of C. parvum and the widespread occurrence of zoonotic C. parvum in pre-weaned calves. Hence, calves are a potential source of zoonotic transmission with considerable public health implications.

Introduction

Cryptosporidium spp. are protozoan parasites that cause mild-to-severe diarrhea in humans and a wide range of animals [1]. Infections with these parasites occur via the fecal−oral route either by direct contact with infected animals or by the ingestion of infective oocysts from contaminated water or food [2-5]. To date, 40 Cryptosporidium spp. have been described [6], and among them, four species, namely, C. andersoni, C. bovis, C. parvum, and C. ryanae, have been identified in cattle. The distribution of these species is known to vary according to age [4, 7]. In particular, C. parvum is one of the most important pathogens causing diarrhea in neonatal calves worldwide and leads to severe economic losses owing to poor growth, decreased productivity, and even death [8]. Moreover, C. parvum is the major pathogenic species that affects humans [9, 10]. Unlike C. parvum, C. bovis, and C. ryanae usually infect post-weaned calves and yearlings without causing illness, and C. andersoni is mainly found in adult cattle [11-13]. The pathogenicity of C. bovis, and C. ryanae in post-weaned calves has not been established [9]. The oocysts of C. parvum, C. bovis, and C. ryanae are similar in size and shape. While C. ryanae is smaller than the others and requires molecular methods for its determination [14, 15], C. andersoni is larger in size and infects the abomasum [16].

According to the subtyping of C. parvum based on sequence analysis of the 60-kDa glycoprotein (gp60) gene, Ⅱa and Ⅱd subtypes have been detected in both humans and calves and can cause zoonotic cryptosporidiosis [17]. The Ⅱa subtype is mostly identified in calves, and IIaA15G2R1 is the predominant subtype [7] globally, including the Republic of Korea (KOR) [18]. The IId subtype is usually found in lambs and goat kids [4, 19] and has been described in calves in some countries such as Sweden, Turkey, Egypt, and China [20-23]. To date, most investigations of cryptosporidiosis in calves caused by C. parvum have focused on the IIa subtype in most countries. However, there are a few studies on C. parvum subtypes in calves in the KOR [18, 24].

Cryptosporidium parvum infects the intestinal mucosa and accounts for over 90% of Cryptosporidium infections in neonatal calves [23]. In contrast, in pre-weaned calves, the prevalence of C. bovis and C. ryanae and their effects on causing diarrhea remain unclear. Several studies have reported that C. bovis and C. ryanae are present in pre-weaned calves [23, 25, 26] and that C. ryanae infections are particularly associated with moderate diarrhea in pre-weaned calves [23]. However, little is known about the association between C. bovis and diarrhea. In addition, a previous study has indicated the high prevalence of C. bovis and C. ryanae in hemorrhagic diarrhea in the KOR [24]. Nevertheless, the pathogenicity of these organisms is still unclear.

So far, for the identification of Cryptosporidium spp., a nested polymerase chain reaction (PCR) technique based on the SSU rRNA gene has been the most widely used method [27]. However, in the present study, a conventional PCR method using species-specific primers was used [24]. Although the amplification had a short fragment compared with a previous method, this PCR technique enabled the differentiation between C. bovis and C. ryanae. Therefore, this study aimed to investigate the prevalence of Cryptosporidium spp. using species-specific primers in pre-weaned calves with diarrhea and to evaluate the association between the age of calf and the identified Cryptosporidium spp. Furthermore, we intended to determine the genotype of Cryptosporidium spp. and subtyping of C. parvum in calves in the KOR and to assess the significance of calves as a source of human infections.

Materials and methods

Ethics statement

All animal procedures were conducted according to ethical guidelines for the use of animal samples, and were approved by the Jeonbuk National University (Institutional Animal Care and Use Committee Decision No. CBNU 2020–052). All procedures and possible consequences were explained to the managers of the surveyed farm, and written consent was obtained.

Sample collection

Between August 2019 and August 2020, fresh fecal samples were collected directly from the rectum of 460 diarrheic pre-weaned calves (up to 60 days of age) by an experienced veterinarian using sterile plastic gloves in 11 different farms located in the KOR. The samples were placed in labeled sterile plastic tubes and transported to the Animal Immunology Laboratory of Kyungpook National University in a cooler with ice packs. Upon arrival, sampling date, age, animal identification number, and fecal consistency (pasty, loose, watery, or hemorrhagic) were recorded for each animal. The collected feces were mostly pasty or loose. Prior to DNA extraction, all feces were stored at 4°C for no more than 2 days without the additional treatment of preservation. The fecal samples were divided according to age as follows; 1−10 days (n = 271), 11−20 days (n = 92), 21−30 days (n = 39), and ≥31 days (n = 58). No microscopic examination was performed for the detection of oocysts.

DNA extraction, molecular analysis, and sequencing

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 instructions. In brief, samples were suspended in lysis buffer, followed by boiling at 70°C for 5 min. Next, the inhibitors provided in the kit were added to the solution to remove substances that can degrade DNA and inhibit downstream enzymatic reactions. Supernatants were subsequently transferred into a tube containing proteinase K and then heated at 70°C for 10 min. A final volume of 200 μL of each DNA sample was then stored at −20°C until PCR amplification. The identification of Cryptosporidium spp. was first tested using the 18S rRNA gene [28]. Samples that yielded positive results for Cryptosporidium spp. via the sequence analysis were further screened to detect the four species using species-specific primers [24]. Positive samples for C. parvum were retested using the 60-kDa glycoprotein (gp60) gene to determine its subtype [4], whereas positive samples for C. bovis/C. ryanae were differentiated by sequence analysis. The subtypes of gp60 were named based on the repeated number of TCA (A), TCG (G), and ACATCA (R), as described previously [29]. All positive PCR products were purified using the AccuPower PCR Purification Kit (Bioneer, Daejeon, KOR) and employed for direct sequencing (Macrogen, Daejeon, KOR). The nucleotide sequences obtained in this study were analyzed using BioEdit (version 7.2.5) and compared with the reference sequences using the Basic Local Alignment Search Tool available at the National Center for Biotechnology Information database. As the sequences of C. bovis and C. ryanae are highly similar, all amplified samples were differentiated by comparing the sequences between the two species. To determine the subtype of C. parvum as well as the genotypes of C. bovis and C. ryanae, nucleotide sequences were aligned using ClustalX and then analyzed via direct comparison with reference sequences from GenBank. In this study, only samples showing a good sequencing result were considered positive for each Cryptosporidium spp. All nucleotide sequences generated in this study were deposited in the GenBank database with appropriate accession numbers (18S rRNA: MZ736386−MZ736399; gp60: MZ736314−MZ736385).

Statistical analysis

Statistical analysis was performed using SPSS Statistics 26 software package for Windows (SPSS Inc, Chicago, IL, USA). Chi-square test was used to determine the association between the prevalence of each species and age. Moreover, multinomial logistic regression analysis was used to determine any associations between the subtypes of C. parvum and age. A p-value of less than 0.05 was considered statistically significant.

Results

Prevalence of Cryptosporidium spp.

Among the 460 diarrheic fecal samples examined, 86 (18.7%) were positive for Cryptosporidium spp. on PCR analysis and sequencing based on the 18S rRNA gene. Three Cryptosporidium spp. were identified in pre-weaned Korean native calves (Table 1). No C. andersoni was detected in this study. Of these, C. parvum (15.7%, 72/460) was the most detected, followed by C. ryanae (2.6%, 12/460) and C. bovis (0.4%, 2/460). Co-infection of these species was not observed. The prevalence of the three Cryptosporidium spp. was compared according to the age groups. As shown in Table 1, the infection rate of Cryptosporidium spp. was highest in calves aged 11−20 days (26.1%, 95% CI 17.1−35.1), whereas the lowest infection rate was observed in calves aged 21−30 days (7.7%, 95% CI 0.0−16.1). All three Cryptosporidium spp. were detected only in calves aged 1−10 days (Table 1). The association between Cryptosporidium spp. and age-distribution was investigated. Interestingly, the identified Cryptosporidium spp. varied according to the age of the calves. C. parvum infection was detected exclusively in calves ≤20 days of age (Table 2). The prevalence peaked at the age of 11−20 days and decreased rapidly thereafter (Table 2). C. parvum infection was significantly associated with the age of the calves (χ2 = 25.300, P = 0.000). Unlike C. parvum, C. ryanae was found in all age groups, and the highest infection rate was observed at ≥31 days of age (Table 2). C. ryanae infection also had a significant age-related distribution (χ2 = 18.020, P = 0.001). In contrast, C. bovis was detected only in two calves aged 10 days and 35 days, and there was no statistical significance in the age-related distribution (P = 0.590).

Table 1

Prevalence and distribution of Cryptosporidium species according to age group in pre-weaned calves.

Age (days)	Sample size	No. of positive (%)	95% CI	Cryptosporidium species (No.)
				C. parvum	C. ryanae	C. bovis
1−10	271	53 (19.6%)	14.8–24.3	49	3	1
11−20	92	24 (26.1%)	17.1–35.1	23	1	0
21−30	39	3 (7.7%)	0.0–16.1	0	3	0
31−60	58	6 (10.3%)	2.5–18.2	0	5	1
Total	460	86 (18.7%)	15.1−22.3	72	12	2

Table 2

Distribution of Cryptosporidium species in pre-weaned Korean native calves according to age group.

Age (days)	Frequency of C. parvum positivity (%)	χ2 (P-value)	Frequency of C. ryanae positivity (%)	χ2 (P-value)	Frequency of C. bovis positivity (%)	χ2 (P-value)
1−10	49/271 (18.1%)	25.300 (0.000)	3/271 (1.1%)	16.020 (0.001)	1/271 (0.4%)	2.824 (0.419)
11−20	23/92 (25.0%)		1/92 (1.1%)		0
21−30	0		3/39 (7.7%)		0
31−60 (Ref.)	0		5/58 (8.6%)		1/58 (1.7%)

Distribution of Cryptosporidium spp. and C. parvum subtypes

All 72 C. parvum-positive samples were successfully amplified and subtyped by sequence analysis of the gp60 gene. A total of 11 different subtypes belonging to the family IIa were identified (Table 3). Subtype family IId was not detected. The distinction of each subtype within the IIa was in the number of trinucleotide region of TCA and TGA repeats (i.e., had one copy of sequence ACATCA immediately after the trinucleotide repeats). As shown in Table 3, in pre-weaned Korean native calves, the most frequently detected subtype was IIaA18G3R1 (72.2%), followed by IIaA17G3R1 (5.6%), and then IIaA15G2R1 (4.2%) and IIaA19G4R1 (4.2%). Other subtypes, namely, IIaA14G1R1 (1.4%), IIaA14G3R1 (1.4%), IIaA15G1R1 (1.4%), IIaA16G4R1 (2.8%), IIaA17G4R1 (2.8%), IIaA19G1R1 (1.4%), and IIaA19G3R1 (2.8%) were also identified. Except for the IIaA18G3R1, no statistical correlation was found between calf age and a specific subtype (Table 3). IIaA19G4R1 was observed only in calves aged 1−10 days, whereas IIaA17G3R1 was found exclusively in calves aged 11−20 days. Several more subtypes were found in calves aged 1−10 days (Table 3). The most predominant subtype, IIaA18G3R1, was seen in all ages.

Table 3

Distribution of Cryptosporidium parvum subtype according to age group.

gp60 subtypes	Age groups (days)		No. of positive calves	P-value
	1−10	11−20
IIaA18G3R1	36	16	52 (72.2%)	0.000
IIaA17G3R1	1	3	4 (5.6%)	0.753
IIaA15G2R1	3	0	3 (4.2%)	0.785
IIaA19G4R1	3	0	3 (4.2%)	0.785
IIaA16G4R1	1	1	2 (2.8%)	0.823
IIaA17G4R1	1	1	2 (2.8%)	0.823
IIaA19G3R1	0	2	2 (2.8%)	0.677
IIaA14G1R1	1	0	1 (1.4%)	0.874
IIaA14G3R1	1	0	1 (1.4%)	0.874
IIaA15G1R1	1	0	1 (1.4%)	0.874
IIaA19G1R1	1	0	1 (1.4%)	0.874
Total	46	26	72

Based on the 18S rRNA gene, 14 (12 C. ryanae and 2 C. bovis) sequences were obtained and compared with the published literature. Twelve sequences of C. ryanae showed 95.1%−100% similarity with each other. The C. ryanae sequences shared 95.7%−100% identity with those found in Austria, China, India, Thailand, and Japan. Two sequences of C. bovis shared 94.1% similarity. These sequences demonstrated 95.5%−96.2% identity with those identified previously in the KOR and had 91.9%−96.2% homology with those from Austria, USA, Japan, and China. Interestingly, differences in nucleotides between C. ryanae and C. bovis were observed. As shown in Fig 1, the nucleotides in the six positions, i.e., 440, 460, 464−466, and 470, were different between the two species.

Fig 1

Sequence comparisons between C. bovis and C. ryanae for the partial18S rRNA gene from Korean sequences obtained in this study and reference strains.

Six nucleotide differences at 440, 460, 464−466, and 470 are shown. An asterisk indicates sequences obtained in this study.

Discussion

Cryptosporidium, along with rotavirus, has been well recognized as the main pathogen causing diarrhea in neonatal calves worldwide [30]. Our findings established the prevalence of Cryptosporidium spp. in pre-weaned diarrheic calves according to age, and the presence of various zoonotic subtypes of C. parvum in the KOR were identified. In the present study, the overall prevalence of Cryptosporidium spp. was found to be 18.7%, which is higher than that reported previously in the KOR [18, 24, 31]. These variations could be explained by the age of the animals, time of sample collection, and the differences in geographical location. However, the percentage of Cryptosporidium spp.-positive samples found in our study was lower than that reported in other countries such as Germany (88.9%), Japan (83.8%), China (38.4%), Italy (38.8%), Colombia (26.6%), Argentina (22.5%), and Estonia (22.6%) [25, 32–37].

In this study, the presence of three Cryptosporidium spp. in pre-weaned Korean native calves was ascertained: C. bovis, C. parvum, and C. ryanae. Of them, C. parvum was the most predominant species in the KOR. This finding agrees with the results observed in several other countries [7, 25, 33, 36, 38, 39]. Most studies have proven that C. parvum mainly infects calves up to 1 month of age [33, 40–43]. The results of the present study demonstrated that C. parvum was detected only in calves aged ≤20 days, and the infection rate was the highest in calves aged 11−20 days. This observation is consistent with a previous study performed by our group [18]. According to our findings, C. parvum was detected in calves aged ≤20 days. It is considered that calves in this age group are susceptible to C. parvum infection owing to their immature immune system [44]. In addition, it is well known that young calves can become infected with C. parvum and begin shedding the oocysts soon after birth [45-47]. This could be associated with cow-to-calf transmission. Several studies have reported that the possible source of infection in calves is transmission at birth from their mothers [48, 49]. However, at present, we do not have exact information on whether these calves were immediately removed from their mothers after birth, but the possibility of contamination via exposure to mother’s feces or the surroundings should be considered. Moreover, C. parvum is known to cause watery diarrhea [23, 30]. In this study, the number of animals with watery feces was small; hence, the association with diarrhea was not evaluated. Although we were not able to compare the occurrence of C. parvum with the diarrhea status, C. parvum was found to be the causative agent of diarrhea in young calves. Our results suggest that C. parvum infection is attributed to the significant age-related distribution (P = 0.000). Consequently, C. parvum was strongly associated with diarrhea in calves aged ≤20 days.

Cryptosporidium ryanae was the second most frequently detected species in pre-weaned Korean native calves. In general, C. ryanae is often found in post-weaned calves [15]. The results revealed that C. ryanae was detected in all age groups and that its occurrence increased with age. In particular, the infection rate of C. ryanae showed a low prevalence in calves aged <20 days, whereas it was rather high in calves aged ≥31 days (Table 2). The prevalence of C. ryanae found in this study was similar to that of a previous study performed in the KOR [24]. Our observation confirmed that C. ryanae has an age-associated distribution, similar to C. parvum. A recent study has reported that C. ryanae was common in pre-weaned as well as post-weaned calves and that the infection was associated with the occurrence of moderate diarrhea in pre-weaned calves [23]. In contrast, other studies have shown that C. ryanae was not associated with diarrhea [26, 39, 50]. So far, the pathogenicity of C. ryanae is controversial. A previous study conducted in the KOR demonstrated that although it is not a single infection, the prevalence of C. ryanae was significantly high in hemorrhagic diarrhea [24]. We could not arrive at a conclusion regarding the correlation with diarrhea since the number of C. ryanae-positive samples from diarrheic calves was small. Hence, C. ryanae infection may cause diarrhea in calves ≥21 days of age and should be considered as a causative agent of diarrhea in this age group. Further studies are necessary to clarify the pathogenicity of C. ryanae in pre-weaned calves.

We found that the prevalence of C. bovis was the lowest in pre-weaned Korean native calves. This observation is contradictory to the results reported by several studies in which C. bovis was the dominant species in pre-weaned calves [20, 47, 51–53]. In this study, C. bovis was detected only in two calves aged 10 and 35 days. Several studies have stated that C. bovis is common in 2−3-week-old calves [42, 50]. However, our result signified that C. bovis was not detected in this age (Table 1). Cai et al. mentioned that C. bovis usually appears after weaning and that the infection can last weeks or months and contribute to the small increase in Cryptosporidium infection rates soon after weaning [26]. This observation may also explain the low prevalence of C. bovis in the present study. To date, information on the prevalence and clinical signs of C. bovis infection in both pre-weaned and post-weaned calves is very limited in the KOR. C. bovis could have probably been considered to be less important than C. parvum and therefore overlooked as an etiological agent of diarrhea in calves. Moreover, the results revealed that infection by C. bovis, unlike the two other species, was not age-related. Most importantly, the involvement of C. bovis in diarrhea remains unclear. Unlike C. ryanae, many studies have suggested that C. bovis was associated with diarrhea [23, 26, 39, 54]. However, infection by C. bovis/C. ryanae may lead to clinical signs owing to the presence of C. parvum [33]. Therefore, the prevalence and pathogenicity of C. bovis in pre-weaned and post-weaned calves must be investigated through large-scale epidemiological surveys.

C. parvum IIa family is common in humans as well as calves and is considered potentially zoonotic. To date, three C. parvum subtypes have been detected in calves in the KOR [18, 24], whereas one subtype (IIaA16G3R1) was not found in this study. In addition to the two subtypes (IIaA15G2R1 and IIaA18G3R1) described above, nine other subtypes (IIaA14G1R1, IIaA14G3R1, IIaA15G1R1, IIaA16G4R1, IIaA17G3R1, IIaA17G4R1, IIaA19G1R1, IIaA19G3R1, and IIaA19G4R1) that have not previously been detected in the KOR were identified for the first time, showing the presence of high genetic diversity. Among them, IIaA18G3R1 was most commonly found in pre-weaned Korean native calves with diarrhea. This result is inconsistent with that of a previous study in which IIaA15G2R1 was shown as the predominant subtype [18]. This difference could be attributed to the fact that in the previous study, both normal and diarrheic feces were used and that IIaA15G2R1 was detected regardless of diarrhea [18]. Other variations are due to the differences in the season of sampling, regions, the number of samples, and herd management. IIaA15G2R1 has been known as the most prevalent C. parvum subtype infecting humans and cattle in many countries [7, 34, 55–59] and has also been detected in calves without diarrhea [18, 33, 60]. There seems to be no relationship between the subtype and diarrhea. In the present study, IIaA15G2R1 was detected only in three calves with diarrhea and was the third frequent subtype along with IIaA19G4R1.

Here, IIaA18G3R1 was the dominant subtype that accounted for 72.2% of C. parvum-infected pre-weaned Korean native calves and was the frequent cause of human cryptosporidiosis, besides being reported in calves and foals [61-66]. The second common subtype in the KOR, IIaA17G3R1, has been found in calves and humans in several countries [67-71]. IIaA19G4R1 was the third frequent subtype identified in the pre-weaned Korean native calves and was also detected in small ruminants and fish as well as humans and calves [61, 70, 72–74]. Interestingly, all sequences belonging to the IIaA19G4R1 subtype were identical to those reported from other countries previously. These subtypes are considered to be the most common ones in calves in the KOR.

The other seven subtypes were also identified in pre-weaned Korean native calves with diarrhea, but their prevalence was relatively low. Subtypes IIaA14G1R1, IIaA14G3R1, and IIaA15G1R1 were each detected in one calf. IIaA14G1R1 was identified in calves, goat kids, and humans [7, 12, 17, 19, 25, 34, 57, 58]. IIaA14G3R1 was found in humans, calf, lambs, and fresh molluscan shellfish [19, 25, 75, 76]. IIaA15G1R1 has been reported in humans [29, 57, 58, 77, 78] as well as in cattle and goat kids [22, 79–81]. Subtypes IIaA16G4R1 and IIaA17G4R1 were each found in two calves in the current study. Unlike the other subtypes, IIaA16G4R1 has so far been noted only in neonatal calf with diarrhea [82], which is consistent with our findings. Subtype IIaA16G4R1 has not yet been detected in humans; however, the possibility that this may represent a significant health risk cannot be excluded. The IIaA17G4R1 subtype has been identified in humans, cattle, and goats [32, 34, 65, 76, 82, 83] and has also been detected in diarrheic calves [32]. Finally, subtypes IIaA19G1R1 and IIaA19G3R1 have each been identified in one calf. IIaA19G1R1 has been reported in humans, cattle, and sheep [36, 58, 69, 84–86]. IIaA19G3R1 has been identified in humans, cattle, and deer [66, 87–90]. To the best of our knowledge, this is the first study to report the presence of various subtypes in pre-weaned calves in the KOR.

To detect C. bovis and C. ryanae, 18S rRNA and heat-shock protein 70 genes are generally used [15]. According to sequence analysis of the 18S rRNA gene, C. bovis and C. ryanae showed ≥99% identity, and it is not always possible to differentiate between them by PCR [91, 92]. However, in this study, we used only the 18S rRNA gene. Even without phylogenetic analysis, the difference between the two species could be confirmed via sequence analysis. At the six nucleotide positions of 440, 460, 464−466, and 470, C. bovis had C, T, A, T, C, and A, whereas C. ryanae had T, C, G, C, T, and G, respectively. These positions are representative markers that distinguish C. ryanae from C. bovis. Our results suggest that these two species can be discerned using the 18S rRNA gene.

Conclusion

Our results confirm the presence of three Cryptosporidium spp. in pre-weaned calves with diarrhea: C. bovis, C. parvum, and C. ryanae. C. parvum was found to be the dominant species in young calves in the KOR. The occurrence of C. ryanae and C. parvum, but not C. bovis, in pre-weaned Korean native calves was significantly related to age; the prevalence of C. parvum decreased with age, whereas that of C. ryanae increased with age. The most frequently detected subtype in calves with diarrhea was IIaA18G3R1, which was responsible for zoonotic transmission. This is the first report to identify nine potentially zoonotic subtypes belonging to the family IIa, which have not previously been reported in cattle in the KOR. This study establishes the high genetic diversity of C. parvum in diarrheic calves and the widespread distribution of zoonotic C. parvum in the KOR. Therefore, the results emphasize that young calves may be a potential source of infection and may serve as an important zoonotic reservoir for human cryptosporidiosis [47, 49].

6 Oct 2021

PONE-D-21-26307Prevalence and distribution of Cryptosporidium spp. among diarrheic calves in the Republic of KoreaPLOS ONE

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Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Author,

I can say that I really like the subject of your article.

However, there is some confusion. What is the method by which you specify subtypes? According to which literature did you identify these subtypes?

I have not seen such a method in the literature number 24 that you use on this subject.

You should provide information about this in your article.

Kind regards

Reviewer #2: The current MS presented Cryptospordiosis distribution among calves in ROK. The main issue of this work is the methods in which the authors described it in a briefly way. As PLOS ONE has broad base of readers, so, authors should be mentioned the methods used in something of details. Extraction of DNA from fecal samples needs specific precautions, authors need to mention how they manage these samples, add preservative or not, what about the inhibitors from feces? Also, I can't understand how the authors differentiate between C. ryanae and C. parvum? How did the authors decide that this is C. ryanae and other one is C. bovis then they did sequence and then differentiate between them?

-The introduction needs a paragraph about importance and improvements that were done in the molecular identifications of Cryptosporidium.

- In the discussion the authors need to discusses why the age below 20 days calves are more susceptible to C. parvum?

Reviewer #3: Dear authors,

Thank you for this interesting work.

The manuscript is well written.

Here you are my comments/ remarks for your consideration.

Title

Line2

Should be changed: “Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea”

Abstract

L24: Add Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide

L42: Illustrate that family IIa belongs to C. parvum

L45, 46: add the percentage of the rest subtypes that you found .

Key words

L52 Add : spp. to Cryptosporidium,

Materials and Methods

L93-L131 The Materials and Methods should be written in more details.

L110: Why did not you collect an equal number of samples in different age groups ?

Thus you can get more accurate prevalence and actual association between age and Cryptosporidium spp. distribution.

Results and discussion

Results and discussion were written in a good details.

Reviewer #4: Dear Author

This is a generally well written manuscript with minor grammatical errors. Just a few comments or corrections:

1. Correlations are mentioned in the results (line 170) but not described in the statistical analysis.

2. Consider moving the sentence in line 174 to the discussion as it is not a description of the results.

3. Line 216 statement on prevalence of C. ryanae in calves is ambiguous please revise.

4. Line 296 change "sequencing analysis" to "sequence analyses".

5. Line 297 add the word "respectively after the G at the end of the sentence. Line 313 add a recommendation.

Thank you and well done on this work.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Submitted filename: PONE-D-21-26307_reviewer.pdf

Click here for additional data file.

20 Oct 2021

Reviewer #1: Dear Author,

I can say that I really like the subject of your article.

However, there is some confusion. What is the method by which you specify subtypes? According to which literature did you identify these subtypes?

I have not seen such a method in the literature number 24 that you use on this subject.

You should provide information about this in your article.

Kind regards

Response: We are sorry for the confusion and agree with this comment. As the reviewer’s comment, we have provided them. Please see lines 131-133 and 466-468.

Reviewer #2: The current MS presented Cryptospordiosis distribution among calves in ROK. The main issue of this work is the methods in which the authors described it in a briefly way. As PLOS ONE has broad base of readers, so, authors should be mentioned the methods used in something of details. Extraction of DNA from fecal samples needs specific precautions, authors need to mention how they manage these samples, add preservative or not, what about the inhibitors from feces? Also, I can't understand how the authors differentiate between C. ryanae and C. parvum? How did the authors decide that this is C. ryanae and other one is C. bovis then they did sequence and then differentiate between them?

Response: We agree with this comment and understand the reviewer’s concern. Unfortunately, we did not examine oocysts from each feces, because we are not expert in this field. However, we used PCR method to detect Cryptosporidium species. All feces were stored at 4�C without additional treatment of preservation and were used for DNA extraction. We extracted directly DNA from feces using the QIAamp Fast DNA Stool Mini Kit (Qiagen) according to the manufacturer's instructions. We have provided the methods as the reviewer’s suggestion. Please see lines 121-126.

In addition, to detect Cryptosporidium species, we first used the 18S rRNA gene and sequenced all positive samples. By sequencing analysis, samples that yielded positive results for Cryptosporidium spp. were further screened to identify the species using species-specific primers. Positive samples for C. parvum was re-tested using the 60-kDa glycoprotein (gp60) gene to determine its subtype. To differentiate between C. bovis and C. ryanae, amplified sequences were compared and confirmed. According to the results of sequence, two species were different from each other. Please see lines 127-131, and 138-139.

-The introduction needs a paragraph about importance and improvements that were done in the molecular identifications of Cryptosporidium.

Response: In this study, we utilized the partial 18S rRNA gene, not SSU rRNA gene. In general, SSU rRNA gene is based on a nest PCR method. As you know, nested PCR has some problems such as contamination risks owing to multiple rounds of DNA amplification and concomitant DNA manipulation steps. To exclude these problems, we used conventional PCR method which have a short fragment. This method can easily be used to detect Cryptosporidium spp. We have provided them. Please see lines 87-91.

- In the discussion the authors need to discusses why the age below 20 days calves are more susceptible to C. parvum?

Response: We agree with this comment. At this point, we cannot make a conclusion about this, but it could be related to immune system of calves. In addition, it has been reported that C. parvum can be transmitted from cow to calf. Please see lines 219-227.

Reviewer #3: Dear authors,

Thank you for this interesting work.

The manuscript is well written.

Here you are my comments/ remarks for your consideration.

Title

Line2

Should be changed: “Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea”

Response: We have changed as the reviewer’s suggestion. Please see lines 2-3.

Abstract

L24: Add Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide

Response: We have modified as the reviewer’s suggestion. Please see lines 22-23.

L42: Illustrate that family IIa belongs to C. parvum

Response: We have modified as the reviewer’s suggestion. Please see lines 39-40.

L45, 46: add the percentage of the rest subtypes that you found .

Response: We have provided as the reviewer’s suggestion. Please see lines 43-45.

Key words

L52 Add : spp. to Cryptosporidium,

Response: We have modified as the reviewer’s suggestion. Please see line 51.

Materials and Methods

L93-L131 The Materials and Methods should be written in more details.

Response: We agree with this comment. As the reviewer’s comment, we have provided them. Please see lines 114-115, 117, 121-133, and 138-140.

L110: Why did not you collect an equal number of samples in different age groups ?

Thus you can get more accurate prevalence and actual association between age and Cryptosporidium spp. distribution.

Response: We understand the reviewer’s concern and agree with this comment. Our lab is focused on calf diarrhea, especially neonates. As you know, calf diarrhea occurs mainly less than 1 month old. Because most of the samples requested to be tested by our lab are under 30-day-old and feces were less in the age group over 30 days of age. For this reason, currently, it is difficult to compare the association between age group and distribution of Cryptosporidium spp. We are going to try to compare the prevalence and association between age and Cryptosporidium spp., if the number of samples is similar in all age groups. Please understand our situation. Thank you for your comment.

Results and discussion

Results and discussion were written in a good details.

Reviewer #4: Dear Author

This is a generally well written manuscript with minor grammatical errors. Just a few comments or corrections:

1. Correlations are mentioned in the results (line 170) but not described in the statistical analysis.

Response: We agree with this comment. We have provided. Please see lines 186-187 and Table 3.

2. Consider moving the sentence in line 174 to the discussion as it is not a description of the results.

Response: We agree with this comment. We have removed it and added in discussion. Please see lines 311-312.

3. Line 216 statement on prevalence of C. ryanae in calves is ambiguous please revise.

Response: We are sorry for the confusion and have revised. Please see lines 236-238.

4. Line 296 change "sequencing analysis" to "sequence analyses".

Response: We have changed as the reviewer’s suggestion. Please see lines 317-318.

5. Line 297 add the word "respectively after the G at the end of the sentence. Line 313 add a recommendation.

Response: We have modified as the reviewer’s suggestion and added some references. Please see line 319 and 335.

Thank you and well done on this work.

Reviewer comment

1. Line 111. No microscopic examination was performed. How did you know for sure that the stools could be infective? Was it a coincidence?

Response: We agree with this comment and understand the reviewer’s concern. We were not able to examine the oocytes because we are not expert in this field. So, we tested all fecal samples for detection of Cryptosporidium infection.

2. Line 114. DNA was extracted from 200 mg. Specify what pretreatment was done to break up the oocyst wall.

Response: We understand the reviewer’s concern. Because we did not test the oocysts and did not perform the step to break up the oocysts wall. To extract DNA, we directly used feces according to the manufacturer’s instructions. Please see lines 114-115, 117, and 121-126.

3. Line 119. As far as I know, C.bovis/ryanae cannot be differentiated with this gene region. Clarification on the primers used should be clarified.

Response: We agree with this comment. We have provided the reference of species-specific primers which we use. Please see line 129.

4. Line 126. Since the base of sequences of C. bovis and C. ryanae are similar, all positive samples of the 18S rRNA were separated by comparing the sequences. Which method or according to which technique was this process done?

Response: We agree with this comment and understand the reviewer’s concern. We first used the 18S rRNA gene to detect Cryptosporidium species and positive samples for Cryptosporidium species by sequencing analysis were screened to identify the four species using species-specific primers. Finally, positive samples for C. bovis/C. ryanae were differentiated by sequence analysis and species was confirmed based on the sequence analysis. According to our results, these two species had differences in nucleotide sequences.

5. Line 142-143. If C. andersoni was not found, please specify.

Response: We have provided. Please see lines 158-159.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Oct 2021

Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea

PONE-D-21-26307R1

Dear Dr. Kyoung-Seong Choi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Saeed El-Ashram

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you very much for all of your hard work and dedication. I, the academic editor, welcome your future submissions on behalf of the PLoS ONE editorial members.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Accept

It can be published in this form

best wishes

Accept

It can be published in this form

best wishes

Reviewer #2: The authors addressed all comments and corrections that recommended by the reviewer. The manuscript could be accepted in this form

Reviewer #3: Dear authors,

Thank you for performing all the modifications i requested in the manuscript.

Well done and good effort.

With my best wishes

Reviewer #4: Dear Authors

Thanks for the thorough revision of the article and addressing all the reviewer comments. The manuscript is reading well and I recommended it to be accepted for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Shawky M Aboelhadid

Reviewer #3: No

Reviewer #4: No

5 Nov 2021

PONE-D-21-26307R1

Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea

Dear Dr. Choi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Saeed El-Ashram

Academic Editor

PLOS ONE