Molecular surveillance of viral pathogens associated with diarrhea in pre-weaned Korean native calves
Calf diarrhea causes severe economic losses in the cattle industry worldwide. This study investigated the prevalence of bovine coronavirus (BCoV), bovine norovirus (BNoV), bovine group A rotavirus (BoRVA), and bovine torovirus (BToV) in calves aged ≤ 60 days, regardless of diarrhea, across nine different regions in the Republic of Korea (ROK) and reported associations between these viruses and diarrhea. Fecal samples were collected rectally from 689 calves: normal (n = 360) and diarrheic (n = 329). BNoV (84/689, 12.2%) was the most prevalent regardless of diarrhea, followed by BCoV (37/689, 5.4%), BToV (15/689, 2.2%), and BoRVA (13/689, 1.9%). Although BCoV (P = 0.032) and BoRVA (P = 0.007) were significantly associated with diarrhea in pre-weaned calves, BNoV and BToV showed no association. Infection by the four pathogens had no relationship with calf age; BoRVA was detected only in calves aged < 30 days, but this finding was not statistically significant. Phylogenetic analysis revealed that BCoV isolates obtained in this study were distinct from the other known BCoVs, and all BNoV isolates belonged to GIII.2 genotype; genetic variations in BNoVs are present in the ROK. BoRVA isolates distributed in the ROK were assigned to G6P. Within the P genotype, our isolates were divided into two lineages: P-III and P-VIII. P- VIII lineage was dominant in pre-weaned Korean native calves. Our BToV isolates were more closely related to a European isolate, B145, than to Japanese isolates. Here, BNoV was frequently identified in calves, suggesting its non-significant contribution to calf diarrhea, whereas BCoV and BoRVA were responsible for calf diarrhea in the ROK. Consequently, our results highlight the importance of rapid diagnosis against these viruses in calf diarrhea.
KeywordsCalf diarrhea Bovine coronavirus Bovine norovirus Bovine group a rotavirus Bovine torovirus
Calf diarrhea (CD) leads to severe economic losses in livestock production worldwide owing to growth retardation and mortality of the affected young calves (Cho et al. 2013; Lyoo et al. 2018). CD can be attributed to multifactorial etiology, including various infectious agents as well as environmental and husbandry practices (Bendali et al. 1999; Meganck et al. 2015). Among numerous infectious agents causing diarrhea in calves, bovine coronavirus (BCoV) and bovine rotavirus (BoRVA) are recognized as the most important viral pathogens (Athanassious et al. 1994). Moreover, bovine norovirus (BNoV) and bovine torovirus (BToV) are frequently associated with acute diarrhea in calves (Gebregiorgis and Tessema 2016; Mohamed et al. 2017).
BCoV is the causative agent for severe diarrhea in neonatal calves, winter dysentery (WD) in adult cattle, and respiratory diseases in cattle (Clark 1993; Heckert et al. 1990; Saif 1990). Enteric BCoV replicates in the epithelial cells of the gut and destroys the villi, thereby causing severe and often bloody diarrhea in calves; this could be life-threatening owing to the loss of electrolytes and malnutrition (Clark 1993). Disease in calves usually occurs within the first month of life. BCoV infection has a high morbidity but a low mortality. BCoV has four major structural proteins: nucleocapsid (N), membrane (M), hemagglutinin esterase (HE), and spike (S) (Spaan et al. 1988). BCoV is divided into three genotype groups: groups 1, 2, and 3 depending on N protein gene. Variations in the host range and tissue tropism of coronaviruses are attributed to the spike (S) glycoprotein (Bidokhti et al. 2012).
Noroviruses are known to cause epidemic and sporadic gastroenteritis in humans and animals (Ferragut et al. 2016). Phylogenetically, noroviruses are classified into six genogroups (GI–GVI) based on RNA-dependent RNA polymerase (RdRp) and capsid gene (Pourasgari et al. 2018). GI, GII, and GIV infect humans, whereas GIII and GV infect ruminants and rodents, respectively (Thomas et al. 2014). GVI is responsible for infections in canine (Vinje 2015). BNoVs are divided into two distinct genotypes: GIII.1 and GIII.2. Both subtypes have increasingly been detected in cattle with enteric or respiratory diseases worldwide (Turan et al. 2018). BNoV was initially identified in diarrheic calves aged < 7 days (Ando et al. 2000; Gunther and Otto 1987), but BNoV was subsequently found in the feces of both diarrheic and healthy animals (Di Martino et al. 2014; van der Poel et al. 2003).
Group A rotavirus (RVA), a member of the Reoviridae family, is a major pathogen associated with severe, acute gastroenteritis in various host species worldwide (Manuja et al. 2008). RVAs have 11 genome segments of double-stranded RNA encoding six structural viral proteins (VP1–VP4, VP6, and VP7) and five or six non-structural proteins (NSP1–NSP6) (Estes and Cohen 1989; Pesavento et al. 2006). RVAs were classified into G (for glycoprotein) and P (for protease-sensitive) genotypes based on the two outer capsid proteins, VP7 and VP4, respectively (Matthijnssens et al. 2011). To date, based on genetic characterization, 32 G and 47 P genotypes have been identified in humans and animals (Dian et al. 2017). Among those, G6, G8, G10, P1, P5, and P11 were associated with most cases of diarrhea in cattle (Midgley et al. 2012).
Torovirus (ToV) is a genus belonging to the family Coronavidae, order Nidovirales (Ito et al. 2016). ToVs cause enteritis and respiratory diseases and have been identified in humans, horses, cattle, and swine worldwide. BToV, formerly called Breda virus, was first detected in the USA during an outbreak of diarrhea in calves (Woode et al. 1982). Since then, BToV has been established as a causative agent of diarrhea in cattle and was identified in diarrheic calves across several countries (Gulacti et al. 2014; Kirisawa et al. 2007; Park et al. 2008). The prevalence of BToV has been detected in 2.9–36.4% of fecal samples obtained from diarrheic calves (Duckmanton et al. 1998; Park et al. 2008).
Although several studies have been conducted on the detection of viral pathogens associated with CD in the Republic of Korea (ROK) (Park et al. 2018; Park et al. 2007a; Park et al. 2011; Park et al. 2007b; Park et al. 2008; Ryu and Choi 2019), there is little available information about the association between diarrhea and viral pathogens. The objective of the present study was to investigate the prevalence of BCoV, BNoV, BoRVA, and BToV in calves aged 1–60 days with or without diarrhea and the relationships between calf age and viral pathogens as well as to report any associations between these pathogens and diarrhea in calves.
Materials and methods
A total of 689 fecal samples were collected between March 2017 and October 2018 from pre-weaned Korean native calves for < 61 days. A veterinarian collected fecal samples rectally from individual calves. Depending on fecal condition, the obtained fecal samples were classified into normal (n = 360) and diarrheic (n = 329). Fecal samples were categorized according to age: 1–10 days (n = 140), 11–20 days (n = 197), 21–30 days (n = 129), 31–40 days (n = 89), 41–50 days (n = 79), and 51–60 days (n = 55). All samples were obtained from nine regions in the ROK. All fecal samples were placed in a 50-mL conical tube and subsequently transferred on ice in the Animal Immunology Laboratory at Kyungpook National University.
RT-PCR and sequencing
Information of primers used in this study
Amplicon size (bp)
Annealing temp (°C)
Tsunemitsu et al. (1999)
Smiley et al. (2003)
Isegawa et al. (1993)
Kirisawa et al. (2007)
The obtained sequence data were applied to a Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) database for homology analyses of BCoV, BNoV, BoRVA, and BToV genes. The sequence identities of all viruses were verified after comparing the sequences deposited in GenBank database using the BLAST software (https://blast.ncbi.nlm.nih.gov/Blast). The sequences were aligned using ClustalX (version 1.8) program. A phylogenetic tree was constructed using the neighbor-joining method based on nucleotide alignments. Bootstrap analysis was conducted with 1000 replicates using the MEGA version 6. Nucleotide sequences of BCoV, BNoV, BoRVA, and BToV obtained in this study were deposited in the GenBank database under the accession numbers MN650885–MN650894, MN156317–MN156366, MN650895–MN650906, and MN650907–MN650917, respectively.
The PCR results for each fecal sample were recorded either as positive or negative for each pathogen and categorized according to calf age (1–10, 11–20, 21–30, 31–40, 41–50, or 51–60 days) and fecal consistency (normal or diarrheic feces). The data were analyzed using the SPSS Statistics 25.0 software package for Windows (SPSS, Chicago, IL, USA). The associations between diarrhea and each pathogen were calculated for all ages together and for each age group separately using Pearson’s chi-squared test or Fisher’s exact test, as appropriate. P value of < 0.05 was considered to be statistically significant.
Prevalence of viral pathogens in pre-weaned Korean native calves
Detection rate of viral pathogens in 689 fecal samples collected from nine different regions
Anseong (n = 39)
Asan (n = 95)
Geochang (n = 37)
Gimje (n = 106)
Gyeongju (n = 6)
Mungyeong (n = 112)
Samrye (n = 80)
Sangju (n = 8)
Yeoungju (n = 206)
Detection rate of viral pathogens according to fecal consistency
No. (%) of positives among diarrheic calves (n = 329)
No. (%) of positives among normal calves (n = 360)
Total (n = 689)
Co-infections with two viral pathogens in 689 fecal samples
BCoV + BNoV
BCoV + BToV
BNoV + BToV
Prevalence of viral pathogens depending on calf age
Prevalence of BCoV, BNoV, BoRVA, and BToV infections according to age group of calves
1–10 (n = 140)
11–20 (n = 197)
21–30 days (n = 129)
31–40 days (n = 89)
41–50 days (n = 79)
51–60 days (n = 55)
Phylogenetic analysis of BCoV, BNoV, BoRVA, and BToV
In this study, we investigated the prevalence of BCoV, BNoV, BoRVA, and BToV infections regardless of diarrhea in pre-weaned Korean native calves from nine different regions in the ROK and performed genetic characterization of the identified viral pathogens. In our samples, BNoV was the most frequently detected pathogen; while BNoV and BToV were detected regardless of diarrhea, BCoV and BoRVA were mainly detected in diarrheic feces, suggesting an association with diarrhea. Moreover, the identified viral pathogens had no relationship with calf age. Our present finding that BCoV infection was associated with diarrhea was consistent with the result of a previous study conducted by our group (Park et al. 2018). However, the results of the present study did not reveal whether BCoV infection causes diarrhea in calves of only a certain age. Additionally, a possibility of the presence of other pathogens, including other bacteria, viruses, and protozoa, could not be ruled out in the fecal samples in which the abovementioned viral pathogens were not detected.
BCoV is considered a major viral pathogen causing CD (Torres-Medina et al. 1985). Here, BCoV was the second most prevalent viral pathogen detected in 5.4% of all fecal samples. BCoV infections were found in eight of the surveyed regions, reflecting a broad distribution of BCoV among pre-weaned Korean native calves. Although BCoV is more prevalent in calves aged 1–10 days compared with the calves of other age groups, our results demonstrated no association between BCoV infection and calf age. Several studies have reported no association between BCoV infection and CD (Bartels et al. 2010; Uhde et al. 2008). However, the present study showed that BCoV infections were more commonly identified in diarrheic feces than in normal feces, indicating that BCoV was associated with diarrhea. A phylogenetic analysis based on the S gene revealed that BCoV isolates detected in this study diverged from CD and WD isolates reported previously in the ROK and were clustered into a separate branch. Interestingly, our isolates were more closely related to the isolates from wild animals. This suggests that genetic variations exist within BCoV isolates prevalent in the ROK. Therefore, the relationship between genetic variations and pathogenicity should be established in future studies.
In this study, BNoV was the most prevalent viral pathogen in pre-weaned Korean native calves. BNoV was detected in eight farms, barring one (Sangju). BNoV alone was identified in Anseong farm, which was cleaner and more spacious than the other farms (Table 2). It is speculated that norovirus is a zoonotic pathogen; thus, it is transmitted to calves through humans. According to our results, BNoV had an infection rate of ≥ 10%, except in the Gimje farm (Table 2). Our study reported a BNoV prevalence rate of 12.2%, which was higher than that reported previously in the ROK (Park et al. 2007a). A previous study reported that BNoV infection is endemic in diarrheic calves in the ROK (Park et al. 2007a), whereas our results revealed that BNoV was found regardless of diarrhea and that BNoV infection had no association with diarrhea. Consequently, we cannot make a definite conclusion that BNoV infection is a direct cause of CD. Moreover, BNoV prevalence was higher in calves aged 1–10 days and gradually decreased with age (Table 5). We found no correlation between BCoV and calf age. BNoV occurred as a co-infection with BCoV or BToV, but not with BoRVA. Owing to few co-infection samples, it was not possible to demonstrate that those viruses were associated with diarrhea. Based on the partial RdRp region, 50 BNoV isolates were classified into GIII.2 genotype and were divergent from the previously reported Korean isolates (Fig. 2). The results suggest that GIII.2 is the dominant genotype of BNoV circulating in the ROK, with genetic variations occurring in this strain. Further studies are warranted to investigate the pathogenicity and pathogenesis of BNoV.
BoRVA is the primary cause of acute diarrhea in neonatal calves (Reynolds et al. 1986; Snodgrass 1986; Uhde et al. 2008). Here, the prevalence of BoRVA was considerably higher in diarrheic feces than that in normal feces. The present study revealed that BoRVA infection was significantly associated with diarrhea. BoRVA infection was observed only in calves aged < 30 days, indicating that BoRVA causes diarrhea in young animals. Here, the prevalence of BoRVA was very low, whether the infection rate of BoRVA is originally low or whether it varies according to different groups of rotaviruses, such as group B and group C, remains unclear to date. A previous study revealed that group C rotavirus caused diarrhea in Korean native calves (Park et al. 2011). Another possibility is that vaccination for BoRVA has been well implemented in the ROK, which has caused a reduction in BoRVA infection rate among calves. This reflects the importance of vaccination. Several reports have indicated that the most prevalent genotypes of BoRVA are G6, G8, and G10 in combination with P, P, P, and P, with G6P being the most prevalent in cattle among several countries (Alfieri et al. 2004; Badaracco et al. 2013; Collins et al. 2014; Fukai et al. 2004; Park et al. 2011). All our isolates detected in this study were assigned to G6P. Moreover, to our knowledge, the P-III and P- VIIIlineages have been identified for the first time in the present study, and the P-VIII lineage is dominant in pre-weaned Korean native calves. Molecular characterization of RVA strains prevalent among animals is of great importance in terms of both animal and public health. Although the prevalence of BoRVA was low in pre-weaned calves, BoRVA should be continuously surveyed to determine BoRVA genotypes circulating in Korean native calves because of interspecies transmission and reassortment events.
BToV was originally isolated from diarrheic calves (Lojkic et al. 2015) and is now distributed worldwide (Gulacti et al. 2014; Hoet et al. 2002; Koopmans et al. 1991; Park et al. 2008). Our results revealed that the prevalence of BToV was 2.2%, which was slightly lower than that previously reported in the ROK (Park et al. 2008). This could be attributed to the different sample collection regions and the different target genes used for detecting BToV as well as calf age. BToV infection was high in Mungyeong farm compared with the other farms. The pathogens detected varied according to the farms from which samples were collected (Table 2), likely owing to the difference in hygiene levels and management systems in farms. BToV alone acts as a primary enteric pathogen in calves aged < 30 days (Aita et al. 2012). However, our result showed that BToV infection was more prevalent in calves aged ≤ 30 days, but this prevalence was not statistically significant (P = 0.137) and was also not associated with diarrhea (P = 0.662). Our findings were inconsistent with those of a previous study (Aita et al. 2012). Here, BToV was found only in co-infections with BCoV or BNoV (Table 4), and these co-infections did not cause diarrhea. To date, there is little available information regarding BToV infection causing diarrhea in the ROK. Phylogenetic analysis revealed that our isolates were more closely related to Japanese strains than to the Breda 1 strain (Fig. 4). Although BToV infection has been reported in the ROK, owing to differences in the target gene, we could not reveal genetic diversity between the isolates identified in this study and the previously reported Korean isolates. Further studies involving large-scale epidemiologic investigation of several genes are warranted to identify the prevalence of BToV and its association with diarrhea.
The results of this study revealed that BNoV is most frequently detected in pre-weaned Korean native calves. Of the viral pathogens examined, BCoV and BoRVA appear to be the major viral pathogens contributing to CD in the ROK, and the prevalence of BCoV is higher than that of BoRVA. The prevalence of BCoV, BNoV, BoRVA, and BToV was not significantly associated with calf age. The association of BNoV and BToV with the pathogenesis of diarrhea in pre-weaned calves remains inconclusive. Therefore, monitoring and surveillance of BCoV and BoRVA in cattle populations are recommended for preventing and controlling diarrhea in calves.
The authors would like to thank Mr. Jeong-Byoung Chae and Du-Gyeong Han for helping to collect feces.
This research was supported by the National Research Foundation of Korea (NRF), funded by the Korea government (MSIP) (No. 2018R1D1A1B07048271).
Compliance with ethical standards
This study was approved and performed in accordance with the ethics guidelines and procedures of the policy of the review to Kyungpook National University Animal Care and Use Committee. Each farmer also provided their written informed consent prior to inclusion in our study.
Conflict of interest
The authors declare that they have no conflict of interest.
- Athanassious, R., Marsolais, G., Assaf, R., Dea, S., Descoteaux, J.P., Dulude, S., Montpetit, C., 1994. Detection of bovine coronavirus and type A rotavirus in neonatal calf diarrhea and winter dysentery of cattle in Quebec: evaluation of three diagnostic methods. Canadian Veterinay Journal 35, 163–169.Google Scholar
- Badaracco, A., Garaicoechea, L., Matthijnssens, J., Louge Uriarte, E., Odeon, A., Bilbao, G., Fernandez, F., Parra, G.I., Parreno, V., 2013. Phylogenetic analyses of typical bovine rotavirus genotypes G6, G10, P and P circulating in Argentinean beef and dairy herds. Infection, Genetics and Evolution 18, 18–30.PubMedCrossRefGoogle Scholar
- Bidokhti, M.R., Traven, M., Ohlson, A., Baule, C., Hakhverdyan, M., Belak, S., Liu, L., Alenius, S., 2012. Tracing the transmission of bovine coronavirus infections in cattle herds based on S gene diversity. Veterinay Journal 193, 386–390.Google Scholar
- Ferragut, F., Vega, C.G., Mauroy, A., Conceicao-Neto, N., Zeller, M., Heylen, E., Uriarte, E.L., Bilbao, G., Bok, M., Matthijnssens, J., Thiry, E., Badaracco, A., Parreno, V., 2016. Molecular detection of bovine Noroviruses in Argentinean dairy calves: circulation of a tentative new genotype. Infection, Genetics and Evolution 40, 144–150.PubMedCrossRefGoogle Scholar
- Ito, M., Tsuchiaka, S., Naoi, Y., Otomaru, K., Sato, M., Masuda, T., Haga, K., Oka, T., Yamasato, H., Omatsu, T., Sugimura, S., Aoki, H., Furuya, T., Katayama, Y., Oba, M., Shirai, J., Katayama, K., Mizutani, T., Nagai, M., 2016. Whole genome analysis of Japanese bovine toroviruses reveals natural recombination between porcine and bovine toroviruses. Infection, Genetics and Evolution 38, 90–95.PubMedCrossRefGoogle Scholar
- Matthijnssens, J., Ciarlet, M., McDonald, S.M., Attoui, H., Banyai, K., Brister, J.R., Buesa, J., Esona, M.D., Estes, M.K., Gentsch, J.R., Iturriza-Gomara, M., Johne, R., Kirkwood, C.D., Martella, V., Mertens, P.P., Nakagomi, O., Parreno, V., Rahman, M., Ruggeri, F.M., Saif, L.J., Santos, N., Steyer, A., Taniguchi, K., Patton, J.T., Desselberger, U., Van Ranst, M., 2011. Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG). Archives of Virology 156, 1397–1413.PubMedPubMedCentralCrossRefGoogle Scholar
- Midgley, S.E., Banyai, K., Buesa, J., Halaihel, N., Hjulsager, C.K., Jakab, F., Kaplon, J., Larsen, L.E., Monini, M., Poljsak-Prijatelj, M., Pothier, P., Ruggeri, F.M., Steyer, A., Koopmans, M., Bottiger, B., 2012. Diversity and zoonotic potential of rotaviruses in swine and cattle across Europe. Veterinary Microbiology 156, 238–245.PubMedCrossRefGoogle Scholar
- Smiley, J.R., Hoet, A.E., Tråvén, M., Tsunemitsu, H., Saif, L.J., 2003. Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses Journal of Clinical Microbiology 41, 3089–3099.PubMedPubMedCentralCrossRefGoogle Scholar
- Thomas, C., Jung, K., Han, M.G., Hoet, A., Scheuer, K., Wang, Q., Saif, L.J., 2014. Retrospective serosurveillance of bovine norovirus (GIII.2) and nebovirus in cattle from selected feedlots and a veal calf farm in 1999 to 2001 in the United States. Archives of Virology 159, 83–90.PubMedCrossRefGoogle Scholar