Epidemiological Survey on Eimeria spp. Associated with Diarrhea in Pre-weaned Native Korean Calves.

Bovine coccidiosis is one of the most important parasitic diseases affecting calf productivity. Here, we investi- gated the prevalence of Eimeria spp. in pre-weaned native Korean calves and determined the correlation between diar- rhea and Eimeria spp. Fecal samples were collected from individual calves (288 normal and 191 diarrheic) in 6 different farms. Of the 479 samples, Eimeria oocysts were detected in 124 calves (25.9%). Five Eimeria spp. were identified; E. zuernii (18.8%) was the most prevalent, followed by E. auburnensis (12.5%), E. bovis (7.5%), E. subspherica (5.8%), and E. bukidnonensis (1.0%). A significant correlation was observed between diarrhea and mixed infection with more than 2 Eimeria spp. (odds ratio [OR]=2.21; 95% confidence interval [CI]: 1.09-4.49; P=0.03) compared to single infection (OR = 1.29; 95% CI: 0.77-2.15; P = 0.33). Of the 5 Eimeria spp. identified, E. subspherica (95% CI: 1.24-5.61; P = 0.01) and E. bukidnonensis (95% CI: 825.08-1,134.25; P = 0.00) strongly increased the risk of diarrhea by 2.64-fold and 967.39-fold, respectively, compared to other species. Moreover, mixed infection with E. auburnensis and E. bukidnonensis was significantly associated with diarrhea (OR = 2,388.48; 95% CI: 1,009.71-5,650.00; P < 0.00) in pre-weaned native Korean calves. This is the first report to demonstrate the importance of E. bukidnonensis associated with diarrhea in pre-weaned native Korean calves. Further epidemiological studies should investigate the prevalence of E. bukidnonensis and the association between E. bukidnonensis and diarrhea.

Bovine coccidiosis is caused by Eimeria spp. and is one of the most important parasitic diseases, leading to substantial economic losses in the livestock industry worldwide. Of more than 20 Eimeria species, the most prevalent species are E. bovis, E. zuernii, and E. auburnensis [1,2]. In particular, E. bovis and E. zuernii are highly pathogenic and can cause mortality and morbidity by disturbing absorption mechanisms in calves and young animals [3-5]. The clinical manifestation of coccidiosis in calves is characterized by watery to hemorrhagic diarrhea, abdominal pain, fever, dehydration, malnutrition, anorexia, and weight loss [4,6-9], while in adult cattle, coccidiosis occurs in subclinical forms [10]. Coccidiosis infections with more than one species of Eimeria are generally observed in the field; however, single species infections have been reported in some cases [8,11].

In cattle, Eimeria spp. are transmitted via the fecal-oral route and disease incidence is associated with several factors such as animal age; fecal contamination of feed, water, or soil; housing conditions (indoor or grazing); overcrowding; and poor hygiene [1,12,13]. Coccidiosis mainly causes problems in young animals, especially between 4 to 7 weeks after birth, and infected calves can spread the infection to other animals through feces containing infective oocysts in the environment and are also more susceptible to secondary bacterial and viral infections [13,14]. Despite the importance of coccidiosis, awareness of this disease is relatively very low in the Republic of Korea (ROK). Currently, there is little information available regarding Eimeria spp. circulating in cattle in the ROK. Therefore, the objective of this study was to examine the prevalence of Eimeria spp. in pre-weaned native Korean calves and to determine the association between diarrhea and the Eimeria spp. identified in this study.

All pre-weaned native Korean calves used in this study were sampled with the approval of the animal ethics and welfare committee of Chonbuk National University (approval number 2017-00026). Consent was obtained from the participating cattle owners.

A total of 479 fecal samples (191 diarrheic samples and 288 normal feces) were individually collected from pre-weaned native Korean calves (1–60 days) raised in 6 different locations (Gimje, Samrye, Youngju, Sangju, Mungyung, and Asan) in the central region of the ROK, between April and November 2017. Each animal’s identification number, age, gender, and the number of animals in its herd were recorded. The fecal samples were directly collected from the rectum by practicing veterinarians and then suspended in a solution of 2.5% potassium dichromate and transported to the laboratory for diagnosis. All fecal samples were recorded and classified as normal, pasty, watery, or hemorrhagic, according to their physical characteristics.

Fecal samples were analyzed for the presence of oocysts using the flotation technique with Sheather’s solution (saturated sugar solution; specific gravity 1.28) and examined microscopically (×400 magnification) for parasitological objects. Identification of Eimeria species was based on the morphological features of the oocysts (including size, shape, color, and texture of the oocyst wall and the presence or absence of a micropyle and polar cap) [15,16].

Statistical analysis was performed to assess the association between diarrhea and the following variables: Eimeria infection, Eimeria spp. single- or mixed infection. Binary univariate logistic regression models were constructed using the generalized linear mixed model (GLMM) in SPSS 24.0 software package (SPSS, Chicago, Illinois, USA), with farm as a random effect. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess the likelihood of association. A value of P <0.05 was considered significant.

Of a total of 479 fecal samples, Eimeria oocysts were detected in 124 fecal samples (25.9%, 124/479). As shown in Table 1, Eimeria infection was detected at a relatively higher level in diarrheic feces (P =0.03) than in normal feces, indicating that Eimeria infection was significantly associated with diarrhea. Eimeria infection increased the occurrence of diarrhea by 1.76-fold compared with non-infection (OR=1.76; 95% CI: 1.06–2.91; P =0.03). When comparing the relationship between Eimeria spp. single- and mixed infection and diarrhea, no association was observed between Eimeria single infection and diarrhea (P =0.33); however, the occurrence of diarrhea was significantly increased by 2.21-fold in mixed infections compared to single infection (95% CI: 1.09–4.49; P =0.03; Table 1). These results suggest that the occurrence of diarrhea observed in pre-weaned native Korean calves is closely associated with mixed infections involving more than 1 species of Eimeria, but not with single infection. As Eimeria infection was not detected in fecal samples from calves with diarrhea, the possibility of infections with other pathogens, such as bacteria, viruses, and protozoa (Cryptosporidium and Giardia), cannot be ruled out.

Table 1

Association between Eimeria spp. infection and diarrhea determined using the logistic regression models with random farm effect

Variable	Category	No. examined (n=479)	No. of diarrheic feces (n=191)	No. of normal feces (n=288)	P-value	OR (95% CI)
Eimeria infection (%)	No (Reference)	355 (74.1)	132 (69.1)	223 (77.4)	-	-
	Yes	124 (25.9)	59 (30.9)	65 (22.6)	0.03	1.76 (1.06–2.92)
	Single infection	54 (11.3)	22 (11.5)	32 (11.2)	0.33	1.29 (0.77–2.15)
	Mixed-infection	70 (14.6)	37 (19.4)	33 (11.5)	0.03	2.21 (1.09–4.49)

In this study, 5 Eimeria spp. were identified based on morphological criteria (Fig. 1). E. bukidnonensis has a micropyle and is the largest of the Eimeria spp. (Fig. 1A). The oocyst is over 40 μm in size, has a very distinct dark brown color, and is very thick, thus making it easy to distinguish it from other species. E. bovis has microtubules and a smooth and homogeneous oocyst wall. It is commonly found in cattle in the ROK and has a typical ovoidal shape (Fig. 1B). The size of the oocyst is approximately ≥30 μm. E. auburnensis has a micropyle and is yellowish-brown in color. It has an elongated shape and is ≥35 μm in size (Fig. 1C). There is a lot of space on both sides of the oocyst because the zygote is nearly in the center. E. zuernii has no micropyle and is the most common Eimeria species in cattle. Its size ranges between 15–20 μm (Fig. 1D); slightly larger than E. subspherica. Moreover, as the zygote exists only in part of the oocyst, there is a lot of space. E. subspherica is the smallest and is approximately 10 μm in size (Fig. 1E). It does not possess a micropyle, the oocyst wall is thin, and the zygotes nearly fill the interior.

Fig. 1

Direct micrographs of Eimeria oocysts. (A) E. bukidnonensis, (B) E. bovis, (C) E. auburnensis, (D) E. zuernii, (E) E. subspherica.

According to our results, E. zuernii (18.8%) was the most frequently detected species, followed by E. auburnensis (12.5%), E. bovis (7.5%), E. subspherica (5.8%), and E. bukidnonensis (1.0%). E. wyomingensis was not identified in this study. Although the infection rates of E. subspherica and E. bukidnonensis were relatively low compared to other species, both showed a significant correlation with diarrhea in pre-weaned native Korean calves (P =0.01 for E. subspherica and P =0.00 for E. bukidnonensis; Table 2). In addition, the occurrence of diarrhea was strongly increased in all animals infected with E. subspherica (OR=2.64; 95% CI: 1.24–5.61; P =0.01) and E. bukidnonensis (OR=967.39; 95% CI: 825.08–1,134.25; P =0.00) compared to those not infected with E. subspherica and E. bukidnonensis, respectively. However, we could not determine for certain whether the original infection rate of E. subspherica and E. bukidnonensis was low or whether the positive rate was low because of the small number of samples collected. Further studies are thus necessary to investigate the prevalence of E. subspherica and E. bukidnonensis through a larger epidemiological survey.

Table 2

Association between the different Eimeria infections and diarrhea determined by the logistic regression models with random farm effect

Eimeria species	Total No. (n=479)	No. of diarrheic feces (n=191)	No. of normal feces (n=288)	P-value	OR (95% CI)
E. zuernii (total)	90 (18.8)	42 (22.0)	48 (16.7)	0.10	1.76 (0.89–3.46)
E. auburnensis (total)	60 (12.5)	26 (13.6)	34 (11.8)	0.35	1.22 (0.80–1.87)
E. bovis (total)	36 (7.5)	19 (9.9)	17 (5.9)	0.14	2.12 (0.78–5.76)
E. subspherica (total)	28 (5.8)	16 (8.4)	12 (4.2)	0.01	2.64 (1.24–5.61)
E. bukidnonensis (total)	5 (1.0)	5 (2.6)	0 (0)	0.00	967.39 (825.08–1,134.25)

The mixed infection patterns associated with diarrhea were also analyzed. Although E. bovis was not associated with diarrhea as a single infection, it demonstrated a significant relationship with diarrhea in mixed infections with E. subspherica, E. zuernii, and E. auburnensis (P <0.00). Similar to E. bovis, E. zuernii did not cause diarrhea as a single infection; however, a mixed infection of E. zuernii with E. auburnensis; E. subspherica; or E. bovis, E. auburnensis, and E. subspherica significantly increased the occurrence of diarrhea (2.5-fold and 5.1-fold, respectively) compared with E. zuernii single infection (Table 3). Moreover, mixed infection with E. auburnensis and E. bukidnonensis was strongly associated with diarrhea (OR=2,388.48; 95% CI: 1,009.71–5,650.00; P <0.00; Table 3). Importantly, to date, there have been no reports that mixed infection with E. auburnensis and E. bukidnonensis is associated with diarrhea. Taken together, we conclude that although Eimeria is not related to the occurrence of diarrhea in pre-weaned calves as a single infection, mixed infection with more than 2 Eimeria spp. appears to have an important effect on the occurrence of diarrhea. Consequently, of the 5 Eimeria spp. identified in the present study, our results suggest that mixed infections with E. aubernensis and E. bukidnonensis are significantly associated with the occurrence of diarrhea in pre-weaned native Korean calves and that the impact of Eimeria infection on calf diarrhea cannot be ignored.

Table 3

Concurrent Eimeria spp. significantly associated with diarrheic feces determined by the logistic regression model with random farm effect

Reference	Concurrent Eimeria spp.	P-value	OR (95% CI)
E. bovis	E. subspherica	<0.00	Not tested
	E. zuernii + E. auburnensis + E. subspherica	<0.00	Not tested
E. zuernii	E. auburnensis + E. subspherica	0.01	2.46 (1.28–4.74)
	E. bovis + E. auburnensis + E. subspherica	<0.00	5.10 (2.40–10.82)
E. auburnensis	E. bukidnonensis	<0.00	2,388.48 (1,009.71–5,650.00)

In the present study, the overall prevalence of Eimeria was 25.9% in pre-weaned native Korean calves regardless of diarrhea. The prevalence of Eimeria spp. in this study was lower than previously reported [1,2,4,6,17]. These differences may be attributed to calf age, breed, the sampling time, and geographical location. The risk of Eimeria infection increases until 3 months of age; however, problems are most common in young animals (3 weeks to 6 months) [9]. Therefore, further studies are necessary to investigate the prevalence of Eimeria in calves of various ages.

Several studies have shown that E. zuernii and E. bovis are the most prevalent species known to cause clinical coccidiosis in calves [1,6,10,18,19]. However, our results demonstrate that E. bovis was detected less frequently than E. auburnensis and that both E. zuernii and E. bovis were not associated with diarrhea (P =0.1 and P =0.14, respectively), while E. bukidnonensis was significantly associated with diarrhea in pre-weaned native Korean calves. To date, there have been no reports of E. bukidnonensis causing diarrhea in calves. Based on our results, E. bukidnonensis is strongly associated with diarrhea in mixed infection with E. auburnensis (P <0.00), as well as a single infection (P = 0.00). To the best of our knowledge, this is the first study to demonstrate that E. bukidnonensis is associated with diarrhea. Although mixed infection with E. bukidnonensis and E. subspherica was not observed in the present study, it may play an important role in causing diarrhea in pre-weaned calves, as E. bukidnonensis and E. subspherica were strongly associated with diarrhea as a single infection.

In conclusion, the present study identified the 5 Eimeria spp. in pre-weaned native Korean calves and showed that Eimeria appears to strongly cause diarrhea in mixed infections with 2 or more species (≥ 2), but not in single infection. Contrary to previous studies, E. zuernii and E. bovis were not related to diarrhea. Further studies are necessary to identify the importance of Eimeria infection in calf diarrhea in the ROK via epidemiological surveys and to investigate other factors affecting the development of coccidiosis.