Korean Journal of
Veterinary Service

Diarrhea is the most common disease in calves, and it causes severe productivity and economic loss to livestock farmers due to high morbidity and mortality (Bartels et al, 2010). Diarrhea is defined as the reduction in the bowel transit time of intestinal contents due to disorders of the digestive system that cause a reduction in water absorption and an increase in water content in feces and fecal amounts and times. Prolonged diarrhea results in dehydration, metabolic disorders, and dystrophy and can be fatal.

According to the National Animal Health Monitoring System for U.S. dairy, 57% of weaning calf mortality cases are due to diarrhea, and most cases occur in less than 1-month-old calves (US Department of Agriculture, 2008). In Korea, it has been reported that 97.6% of unweaned calves suffer from diarrhea at least once (Kim et al, 1990). In addition, Kang et al. (2001) reported that calf enteric diseases mostly occur in the 1-month-old group, and calves experienced less diarrhea with increasing age.

Cattle are important livestock in Korea and cattle farming accounts for 32% of the livestock industry (Korea Statistics, 2021). According to the 4th quarter data from the Korean National Statistical Office in 2021, the number of cattle in Korea was approximately 3.96 million including Hanwoo, beef, and dairy cattle; the number is increasing. Of these, 270,000 (7%) were raised in Chungcheongbuk-do (Chungbuk). Therefore, the prevention of diarrhea in calves is essential to prevent economic losses in the livestock industry.

Diarrhea is a difficult disease to treat because its causation is associated with multiple factors contribute. Diarrhea in calves is caused by a combination of infectious pathogens (viruses, bacteria, parasites, etc.) and non-infectious factors, including environmental factors, herd management systems, host nutrition status, and immunological conditions (Maes et al, 2003; Garaicoechea et al, 2006).

Among the various infectious pathogens, Escherichia coli K99, Salmonella spp., Clostridium perfringens type C, bovine coronavirus, bovine rotavirus, bovine viral diarrhea virus, Cryptosporidium spp., Giardia, and Eimeria are the main causes of bovine diarrhea (Cho et al, 2013; Choe et al, 2017). Because diverse pathogens contribute to bovine diarrhea, there are different methods to detect them, including microscopy, polymerase chain reaction, and enzyme-linked immunosorbent assay. Among these, rapid diagnostic kits are frequently used by field veterinarians, because they are easy to use and require a short time.

As calf diarrhea is attributable to various causes, different studies have investigated diarrheagenic pathogens in calves with diarrhea in Korea. However, these studies were carried out in different provinces, including Gyeongnam, Gangwon, and Gwangju, but not in Chungbuk, and previous studies tested a limited number of samples (Kim et al, 2016; Choe et al, 2017; Koh et al, 2019; Kim et al, 2021). Therefore, there are limited data on the prevalence of diarrheagenic pathogens in calves with diarrhea in Chungbuk, Korea.

Thus, the present study aimed to investigate the epidemiological characteristics of five diarrheagenic pathogens (coronavirus, rotavirus, E. coli K99, Cryptosporidium spp., Giardia spp.) in Hanwoo calves with diarrhea in Chungbuk, Korea, and contribute data for the establishment of a disease control policy against diarrhea in calves.

Sample collection

The present study collected fecal samples from Hanwoo calves (n=22,417) under the age of 1 year with diarrhea from 11 different regions of Chungbuk from January 2018 to December 2021 (Fig. 1). Samples were collected by licensed veterinarians for the diagnosis and treatment of diarrhea after obtaining oral consent from the owner.

Figure 1. Map of Chungcheongbuk-do, Korea and its administrative boundaries (www.chungbuk.go.kr).

For epidemiological analysis, data on the region, date, and age were collected. The ages of the calves were categorized into four according to the developmental stage (<1 month, 1∼3 months, 4∼6 months, and 7∼12 months). When the data were insufficient, they were indicated as “not available” and excluded from the statistical analysis.

Of the samples collected from 2018 to 2021, the specific age and co-infection status were verified for the partial ones collected in 2021 (n=2,234). Thus, analysis based on age and co-infection status was only performed for these.

Rapid diagnostic kit

The collected diarrheal fecal samples were immediately examined using a commercially available rapid diagnostic kit (Antigen Rapid BoviD-5 Ag Test Kit^Ⓡ; Bionote, Korea) to identify the causative agents of diarrhea. Using the kit, infections caused by rotavirus, coronavirus, E. coli K99, Cryptosporidium, and Giardia were simultaneously tested.

Based on the manual of the manufacturer, the sensitivity (SE) and specificity (SP) of the kit for the various pathogens were as follows: rotavirus: SE 96.4%, SP 95.9%; coronavirus: SE 96.0%, SP 95.3%; E. coli K99: SE 96.8%, SP 97.9%; Cryptosporidium: SE 96.8%, SP 98.2%; and Giardia: SE 96.5%, SP 97.3%.

Statistical analysis

The 95% confidence intervals were calculated for all the results. The chi-squared test, Fisher’s exact test, or Cochran-Armitage trend test were used to evaluate the association between prevalence and the variables (pathogens, season, age, and year) and implemented in R. P-values less than 0.05 denoted statistical significance. Following the analysis, standardized residuals were estimated to identify values responsible for a significant chi-square test. The values with standardized residual >±2 represent a significant deviation from the expected values.

Prevalence of diarrheagenic pathogens in calves

Of the 22,417 diarrhea fecal samples from Hanwoo calves raised in Chungbuk, Korea, 60.3% (n=13,518) were positive for five bovine diarrhea antigens using the rapid diagnostic kit (Table 1). Of the five tested pathogens, rotavirus was the most dominant pathogen in 7,739 (34.5%) samples. In addition, coronavirus, E. coli, Giardia, and Cryptosporidium were identified in 2,455 (11.0%), 1,828 (8.2%), 1,057 (4.7%), and 439 (2.0%) samples, respectively.

Table 1 . Prevalence of causative agents of calves’ diarrhea in Chungbuk, Korea according to season from 2018 to 2021.

Season	No. of tested	Causative agents (%, 95% C.I.)
		E. coli K99	Coronavirus	Rotavirus	Cryptosporidium	Giardia	Total
Spring (Mar∼May)	7,099	613 (8.6, 8.0∼9.3)	876* (12.3, 11.6∼13.1)	2,334* (32.9, 31.8∼34.0)	121 (1.7, 1.4∼2.0)	287* (4.0, 3.6∼4.5)	4,231 (59.6, 58.5∼60.7)
Summer (Jun∼Aug)	5,832	460 (7.9, 7.2∼8.6)	621 (10.7, 9.9∼11.4)	1,797* (30.8, 29.6∼32.0)	79* (1.4, 1.1∼1.7)	269 (4.6, 4.1∼5.2)	3,226* (55.3, 54.0∼56.6)
Autumn (Sep∼Nov)	4,953	481* (9.7, 8.9∼10.5)	537 (10.8, 10.1∼11.7)	1,745 (35.2, 33.9∼36.6)	112* (2.3, 1.9∼2.7)	314* (6.3, 5.7∼7.0)	3,189* (64.4, 63.1∼65.7)
Winter (Dec∼Feb)	3,317	184* (5.6, 4.8∼6.3)	270* (8.1, 7.2∼9.1)	1,358* (40.9, 39.3∼42.6)	82* (2.5, 1.9∼3.0)	145 (4.4, 3.7∼5.1)	2,039* (61.5, 59.8∼63.1)
Not available	1,216	90 (7.4, 5.9∼8.9)	151 (12.4, 10.6∼14.3)	505 (41.5, 38.8∼44.3)	45 (3.7, 2.6∼4.8)	42 (3.5, 2.4∼4.5)	833 (68.5, 65.9∼71.1)
Total	22,417	1,828 (8.2, 7.8∼8.5)	2,455 (11.0, 10.5∼11.4)	7,739 (34.5, 33.9∼35.2)	439 (2.0, 1.8∼2.1)	1,057 (4.7, 4.4∼5.0)	13,518 (60.3, 59.7∼60.9)

Not available, samples without information on the collected date..

*Standardized residuals >±2..

Prevalence of diarrheagenic pathogens according to regions

Among the 11 regions of Chungbuk, 9 (Chungju-si and Eumseong-gun excluded) showed rotavirus as the most prevalent pathogen (Fig. 2 and Table S1). The most common pathogen in Chungju-si and Eumseong-gun was E. coli and Giardia, respectively.

Figure 2. Prevalence of five bovine diarrheagenic pathogens according to the region in Chuncheongbuk-do, Korea.

The prevalence of diarrheagenic pathogens in the different regions varied from 24.6% to 73.2% (Table S1). The highest prevalence was observed in Cheongju-si and the lowest prevalence was observed in Jecheon-si.

Prevalence of diarrheagenic pathogens according to season

Regarding the seasons, 7,099, 5,832, 4,953, and 3,317 samples were collected in spring, summer, autumn, and winter, respectively. Diarrheagenic pathogens were identified in 59.6% (n=4,231), 55.3% (n=3,226), 64.4% (n=3,189), and 61.5% (n=2,039) of the samples in spring, summer, autumn, and winter, respectively (Table 1). Regardless of the seasons, rotavirus was the most prevalent pathogen, and the prevalence of the others was in the following order: coronavirus, E. coli, Giardia, and Cryptosporidium.

When statistically analyzed, E. coli K99 showed higher prevalence in autumn and lower prevalence in winter. In the case of coronavirus, it showed higher prevalence in spring and lower prevalence in winter. Rotavirus showed higher prevalence in winter, and lower prevalence in spring and summer. Cryptosporidium showed higher prevalence in autumn and winter, and lower prevalence in summer. In the case of Giardia, it showed higher prevalence in autumn and lower prevalence in spring. Overall, the pathogens were more prevalent in autumn and winter and less in spring. All the data described here are statistically significant (P<0.05).

Prevalence of diarrheagenic pathogens according to age

Regarding age, 1,251, 812, and 133 fecal samples were collected from aged <1 month, 1∼3 months, and 4∼6-month-old groups, respectively (Table 2). The results showed that younger calves suffered from diarrhea more than older calves. In addition, the highest prevalence of diarrheagenic pathogens (81.9%, n=1,024) was observed in the <1-month group, and the prevalence decreased with age.

Table 2 . Prevalence of causative agents of calves’ diarrhea in Chungbuk, Korea according to age in 2021.

Age (Month)	No. of tested	No. of positive (%, 95% C.I.)	Causative agents (%, 95% C.I.)
			E. coli K99*	Coronavirus*	Rotavirus*	Cryptosporidium*	Giardia*	Total†
<1	1,251	1,024 (81.9, 79.7∼84.0)	76 (6.1, 4.8∼7.4)	159 (12.7, 10.9∼14.6)	701 (56.0, 53.3∼58.8)	41 (3.3, 2.3∼4.3)	131 (10.5, 8.8∼12.2)	1,108 (88.6, 86.8∼90.3)
1∼3	812	505 (62.2, 58.9∼65.5)	100 (12.3, 10.1∼14.6)	118 (14.5, 12.1∼17.0)	274 (33.7, 30.5∼37.0)	11 (1.4, 0.6∼2.2)	133 (16.4, 13.8∼18.9)	636 (78.3, 75.5∼81.2)
4∼6	133	67 (50.4, 41.9∼58.9)	37 (27.8, 20.2∼35.4)	29 (21.8, 14.8∼28.8)	16 (12.0, 6.5∼17.6)	2 (1.5, −0.6∼3.6)	8 (6.0, 2.0∼10.1)	92 (69.2, 61.3∼77.0)
7∼12	38	28 (73.7, 59.7∼87.7)	7 (18.4, 6.1∼30.8)	25 (65.8, 50.7∼80.9)	20 (52.6, 36.8∼68.5)	1 (2.6, −2.5∼7.7)	11 (29.0, 14.5∼43.4)	64 (168.4)
Total	2,234	1,624 (72.7, 70.9∼74.5)	220 (9.8, 8.6∼11.1)	331 (14.8, 13.3∼16.3)	1,011 (45.3, 43.2∼47.3)	55 (2.5, 1.8∼3.1)	283 (12.7, 11.3∼14.1)	1,900 (85.1, 83.6∼86.5)

*P<0.05, by Cochran-Armitage trend test..

^†Co-infected cases were counted separately. Detailed information is described in Table S2..

As the incidence of diarrhea decreases with age, the number of fecal samples (n=38) collected from calves aged >7 months was lower than those from the other groups. However, the prevalence of diarrheagenic pathogens was higher (73.7%, n=28) than those of the 1∼3 and 4∼6 months age groups. In contrast with other age groups, several cases of diarrhea in this group were caused by co-infection (Table S2).

When the pathogens were analyzed according to age, the prevalence of rotavirus and Cryptosporidium decreased (P<0.05), whereas coronavirus, E. coli, and Giardia increased (P<0.05) based on the Cochran-Armitage trend test.

Prevalence of diarrheagenic pathogens according to year

From 2018 to 2021, rotavirus was the most common, and Cryptosporidium was the scarcest pathogen identified in the samples (Table 3). After rotavirus, coronavirus and E. coli were commonly detected. In cases of Cryptosporidium and Giardia, the prevalence was low compared to that of other pathogens; however, they showed an increasing trend during the tested years.

Table 3 . Prevalence of causative agents of calves’ diarrhea in Chungbuk, Korea according to year from 2018 to 2021.

Year	No. of tested	Causative agents (%, 95% C.I.)					No. of negative (%, 95% C.I.)
		E. coli K99	Coronavirus	Rotavirus	Cryptosporidium	Giardia
2018	4,737	816* (17.2, 16.2∼18.3)	811* (17.1, 16.1∼18.2)	1,276* (26.9, 25.7∼28.2)	85 (1.8, 1.4∼2.2)	112* (2.4, 1.9∼2.8)	1,637 (34.6, 33.2∼35.9)
2019	5,282	377* (7.1, 6.4∼7.8)	480* (9.1, 8.3∼9.9)	1,617* (30.6, 29.4∼31.9)	95 (1.8, 1.4∼2.2)	129* (2.4, 2.0∼2.9)	2,584 (48.9, 47.6∼50.3)
2020	7,197	286* (4.0, 3.5∼4.4)	575* (8.0, 7.4∼8.6)	2,914* (40.5, 39.4∼41.6)	146 (2.0, 1.7∼2.4)	413* (5.7, 5.2∼6.3)	2,863 (39.8, 38.7∼40.9)
2021	5,201	349* (6.7, 6.0∼7.4)	589 (11.3, 10.5∼12.2)	1,932* (37.2, 35.8∼38.5)	113 (2.2, 1.8∼2.6)	403* (7.8, 7.0∼8.5)	1,815 (34.9, 33.6∼36.2)
Total	22,417	1,828 (8.2, 7.8∼8.5)	2,455 (11.0, 10.5∼11.4)	7,739 (34.5, 33.9∼35.2)	439 (2.0, 1.8∼2.1)	1,057 (4.7, 4.4∼5.0)	8,899 (39.7, 39.1∼40.3)

*Standardized residuals >±2..

When statistically analyzed, the prevalence of E. coli K99 varied every year (P<0.05). In the case of coronavirus, it was more prevalent in 2018 and less prevalent in 2019 and 2020. Rotavirus showed higher prevalence in 2020 and lower prevalence in the other years. In the case of Giardia, it showed higher prevalence in 2020 and 2021, and lower prevalence in 2018 and 2019. Cryptosporidium did not show a statistically significant difference according to the years.

In this study, five diarrheagenic pathogens were investigated in Hanwoo calves aged <1 year with diarrhea in Chungbuk, Korea. Of the five pathogens, rotavirus had the highest prevalence at 34.5% in diarrheic calves. Because diarrhea is one of the most common diseases in calves, there have been several different studies in Korea. However, since diarrhea itself is not a report-required disease in Korea, an investigation has been performed by limited number of researchers or government institutes in specific regions or pathogens. For example, Koh et al, (2019) and Kim et al, (2021) reported similar results to this study which showed the most common causative pathogen for calf diarrhea was rotavirus in Gwangju-si and Jeollabuk-do. On the contrary, Kim et al, (2016) and Choe et al, (2017) showed that Giardia was the most common pathogen in calves with diarrhea. It is difficult to determine the reason for inconsistent results, and we think factors including experimental methods, age, and studied regions could affect the results.

On analyzing the prevalence according to season, we found that the highest prevalence of diarrheagenic pathogens was in autumn (64.4%), and the results are consistent with a previous study that reported the highest incidence of diarrhea in Hanwoo calves in Gwangju-si (Koh et al, 2019). The results could be due to the low immune status caused by rapid temperature changes in autumn. In our study, rotavirus was more prevalent in winter than in the other seasons, which is consistent with previous studies. Nourmohammadzadeh et al, (2012) reported that the rate of rotavirus incidence was higher in winter and postulated that low temperature and relative humidity may increase the survival of rotavirus (Brandt et al, 1982).

Rotaviruses are known to have an age-dependent incidence. A study conducted by Monney et al, (2018) showed that the prevalence of rotavirus infection was the highest in 10∼25-day-old calves. Other studies also reported a higher occurrence of diarrhea caused by rotavirus in neonatal calves from 1 week to 2 months of age (Alfieri et al, 2006; Papp et al, 2013; Masuda et al, 2014). This study also found that rotavirus was the most dominant diarrheagenic pathogen in unweaned calves. In addition, the prevalence of rotavirus infection in calves younger than 1 month was the highest among the tested groups, and it decreased with age (P<0.05).

Diarrhea in calves frequently occurs in newborns within the age of 1∼3 months, and several studies on calf diarrhea target less than 3 months of age (Björkman et al, 2003; Alfieri et al, 2006; Monney et al, 2018; Singh et al, 2020; Wei et al, 2021). Likewise, the results of this study showed that the proportion of diarrheic feces collected from calves less than 3 months old was 92.3% (n=2,063) of 2,234 samples with confirmed age. The numbers significantly decreased with the calves’ age. Interestingly, the proportion of diarrhea cases in the age group older than 7 months was low; they were usually co-infection cases.

The prevalence of E. coli and coronavirus increased with age; on the contrary, rotavirus infections had the highest incidence in calves less than 1 month, which decreased with age. These findings suggest that rotavirus is the main cause of pre-weaned calf diarrhea, whereas E. coli and coronavirus are the main causes of after-weaned calf diarrhea. Our study demonstrated that the dominant target enteric pathogen varies with calf age; eventually, it is necessary to treat calf diarrhea differently according to age.

Cryptosporidium and Giardia are zoonotic intestinal protozoan parasites (Lee et al, 2016a; Lee et al, 2016b). Generally, diagnosis of Cryptosporidium and Giardia is performed by microscopic examination with staining or by molecular method, however, not so many studies have been done in Korea. In the case of Cryptosporidium, previous studies reported a 4.4∼9.9% prevalence in calves with diarrhea by the molecular method (Lee et al, 2016b; Lee et al, 2019). In addition, 5.6∼13.1% of calves with diarrhea were infected by Giardia tested by the molecular method (Lee et al, 2016a; Lee et al, 2018; Oh et al, 2021). Both cases showed relatively higher prevalence than this study, and it might be affected by the experimental method because those studies examined the pathogens by the molecular method which shows high sensitivity.

In the present study, 39.7% of the tested samples showed negative results for the rapid diagnostic kit. This result should be carefully interpreted because of false-negative results due to the sensitivity of the kit and an insufficient number of pathogens at the early stage of the disease. To overcome this limitation, a final laboratory test should be performed to target various calf diarrheagenic pathogens. Because 34.6∼48.9% of the diarrheal fecal samples were negative every year, we believe that there may be infections caused by other viruses, bacteria, or protozoa undetectable by the kit. Moreover, management/environmental factors, such as overfeeding, poor hygiene, colostrum deprivation, and individual animal susceptibility, maybe another reason for diarrhea (Alfieri et al, 2006).

When the data were analyzed according to the regions in Chungbuk, rotavirus was the most prevalent pathogen in nine regions. In contrast, the prevalence of E. coli was highest in Chungju-si and Giardia in Eumseong-gun. A commercially available rotavirus-coronavirus vaccine is commonly used in Chungbuk (Table S3). Although there is commercially available rotavirus-coronavirus-E. coli vaccine, due to cost issues, it is not commonly used in Chungbuk. The result of this study showed the necessity of providing rotavirus-coronavirus-E. coli vaccine, for which E. coli infection is common. In addition, Giardia was more prevalent than any other pathogen that poses a risk to public health in Eumseong-gun, and more attention is needed to manage farm hygiene. Thus, the accurate diagnosis of bovine diarrheagenic pathogens prevents the misuse and abuse of animal drugs, prevents the spread of diseases through proper treatment, and contributes to the production of safe livestock products.

The Ministry of Agriculture, Food and Rural Affairs (MAFRA) introduced a 50% state-funded livestock disease treatment insurance system as a pilot project for regular treatment by veterinarians to reduce the burden of medical expenses on cattle farms due to livestock diseases from 2018 to 2024. According to a press release by MAFRA, calf diarrhea and enteritis treatment accounted for the highest proportion (50%) of diseases responsible for indemnity, which suggests that calf diarrhea is prevalent in Korea (Ministry of Agriculture, Food and Rural Affairs, 2019). Nevertheless, only 410,000 (21.1%) cattle were supported for vaccination against bovine diarrhea, which is significantly low, given the 1,947,000 fertile cows in Korea (Korea Statistics, 2021). Currently, the government of Chungbuk supplies a bovine diarrhea vaccine for 27,000 cattle every year; however, it covers only 19% of fertile cows older than 1 year (n=145,000) raised in Chungbuk (Korea Statistics, 2021). Therefore, to prevent economic losses due to calf diarrhea, a sufficient amount of bovine diarrhea vaccines should be supplied.

In this study, the highest prevalence was observed in rotavirus and followed by coronavirus, E. coli K99, Giardia, and Cryptosporidium. Intriguingly, the trend was consistent from 2018 to 2021. In addition, when the data were analyzed according to the regions, most regions in Chungbuk showed similar trends. It is difficult to analyze the reason for this phenomenon, considering previous studies also showed similar results, this might be a common situation throughout Korea. Additional epidemiological analysis and pathogenesis study on diarrheagenic pathogens should be followed.

In conclusion, the present study is the first large epidemiological study to examine the prevalence of five bovine diarrheagenic pathogens in Hanwoo calves with diarrhea over 4 years (2018∼2021) throughout Chungbuk. The findings of this study could be helpful for early diagnosis and treatment based on the calves’ region, season, and age to efficiently prevent diarrhea.

Ethical review and approval were waived for this study as the study focused on fecal samples. This study was performed for the diagnosis and treatment of the animals, and animal contact during the examination was minimal, consistent with routine physical examination procedures, and conducted under the supervision of a licensed veterinarian with appropriate handling. All the treatments were neither harmful nor against animal welfare.

The authors appreciate the cooperation of the veterinarians. This research was supported by Chungbuk National University, Korea National University Development Project (2021).

The online version contains supplementary material available at https://doi.org/10.7853/kjvs.2022.45.3.201.

The authors declare no conflicts of interest. The opinions expressed by the authors do not necessarily reflect the opinions of the Department of Animal Health, Agricultural Policy Bureau, Chungcheongbuk-do, Cheongju, Korea, with which the authors are affiliated.