https://doi.org/10.52973/rcfcv-e33249
Received: 22/03/2023 Accepted: 15/05/2023 Published: 18/06/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33249, 1 – 6
ABSTRACT
This study reports potential causes of diarrhea in neonatal calves,
leading to calf mortality, from the selected population of the three
Provinces of Turkey. A total of 300 fecal samples were collected
purposively from diarrheic neonatal calves distributed to the three
age groups (1–14 days, 15–29 days, and 30–90 days), from Konya,
Karaman, and Aksaray Provinces of Turkey. The fecal specimens
were examined for the existence of Cryptosporidium spp., rotavirus,
coronavirus, and Escherichia coli by commercially available capture
direct enzyme linked immunosorbent assay (ELISA) kit. The oocysts
and coproantigens of Cryptosporidium were identied in 109 (36.3%)
and 156 (52%) of the 300 calves, respectively. While, rotavirus, E. coli
and coronavirus antigens were detected (P<0.05) in 57 (19%), 17 (5.6%)
and 6 (2%) calves, respectively. Mixed infection of the study pathogens
has also been found in this report. These results provide a baseline
information on the frequent causes of neonatal calf diarrhea in the
studied Provinces which can be used to develop a prophylaxis plan.
Key words: Cryptosporidium spp.; coronavirus; rotavirus;
Escherichia coli; diarrheic calves
RESUMEN
Este estudio informa sobre las posibles causas de diarrea en terneros
recién nacidos, que conducen a la mortalidad de los terneros, de
la población seleccionada de las tres provincias de Turquía. Se
recolectaron intencionalmente un total de 300 muestras fecales
de terneros recién nacidos con diarrea distribuidos en tres grupos
según la edad (1–14 días; 15–29 días y 30–90 días), de las provincias
de Konya, Karaman y Aksaray de Turquía. Las muestras fecales se
examinaron para detectar la existencia de Cryptosporidium spp.,
rotavirus, coronavirus y Escherichia coli mediante un kit de ensayo
inmunoabsorbente ligado a enzimas (ELISA) de captura directa
disponible en el mercado. Los ooquistes y coproantígenos de
Cryptosporidium se identicaron en 109 (36,3 %) y 156 (52 %) de los 300
terneros, respectivamente. Mientras que se detectaron antígenos de
rotavirus, E. coli y coronavirus (P<0,05) en 57 (19 %), 17 (5,6 %) y 6 (2 %)
terneros, respectivamente. En este informe también se ha encontrado
una infección mixta de los patógenos del estudio. Estos resultados
brindan información de referencia sobre las causas frecuentes de
diarrea neonatal en terneros en las Provincias de estudio que pueden
utilizarse para desarrollar un plan de prolaxis.
Palabras clave: Cryptosporidium spp.; coronavirus; rotavirus;
Escherichia coli; becerros diarreicos
ELISA–based Point Prevalence of enteropathogens in diarrheic calves in
Central Anatolia Region of Turkey
Prevalencia puntual de enteropatógenos basada en ELISA en terneros diarreicos en la región de
Anatolia Central de Turquía
Nermin Işik–Uslu
1
* , Ozlem Derinbay–Ekici
1
, Oğuzhan Avci
2
1
University of Selcuk, Faculty of Veterinary Medicine, Department of Parasitology. Konya, Turkey.
2
University of Selcuk, Faculty of Veterinary Medicine, Department of Virology. Konya, Turkey.
*Corresponding author: nerminisik@selcuk.edu.tr
Assessment of Enteropathogens in diarrheic calves through ELISA-based prevalence / Uslu et al. ________________________________
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INTRODUCTION
Enteric infections involving diarrhea, dehydration, and weight loss
are considered to major health problems throughout the World and
are signicant issues on dairy farms [1]. The diarrhea becomes worse
if the host susceptibility is compromised, e.g. in neonatal period,
which includes the rst 28 days following birth, and is refered as the
neonatal calf diarrhea (NCD) [2]. The predominant causative agents
of the NCD include: bacterial (Escherichia coli K99, Clostridium spp.,
Salmonella spp. and Campylobacter spp.), parasitic (Cryptosporidium
spp. and Coccidia spp.), and viral (rotavirus and coronavirus) agents
[2, 3]. Immunological conditions (vaccination and receiving quality
of colostrum), management factors (environmental and hygienic
conditions) and nutritional factors (feeding of adequate amounts of
milk) also play a role in predisposing the animals towards the NCD [1].
Cryptosporidiosis has been reported as the cause of serious
problems in many types of animals such as calves (Bos taurus), lambs
(Ovis aries), and kids in the neonatal period. In particular, 1–3–week–old
calves are highly susceptible to the disease, as it has been reported
that oocysts can be found in calves as young as 4 days old, with severe
diarrhea after three days of ingesting oocysts, and lasting for 2–14
days [4, 5].
Neonatal period has also been identified at risk for rotavirus
infections [6, 7, 8]. Cattle of all ages are sensitive to rotavirus
infections; however, higher prevalence has been reported in calves
under one year [9]. Clinical ndings of rotavirus infection are like
to those of cryptosporidiosis [10]. Rotavirus infection involves only
the small intestine; however, coronavirus infection involves both the
small and large intestines and can lead to more severe outcomes and
higher mortality rates [11].
E. coli is normal intestinal ora of humans and animals having
various serotypes with different pathogenic courses, depending on
the animal species [12]. The E. coli K99 strain is the most well–known
enteropathogen in calves [10]. NCD E. coli–caused can be fatal due to
rapid and excess loss of water and electrolytes from the body leading
to severe weakness and hypothermia [13].
In this study, the aim was to identify the prevalent etiological
agents causing NCD in Konya, Karaman, and Aksaray Regions of
Turkey through Enzyme– linked immunosorbent assay (ELISA) for
coproantigen detection.
MATERIALS AND METHODS
Fecal samples
Fecal samples were collected from 300 diarrheic calves aged 0–3
months (159 calves were aged between 0–14 days, 85 calves were aged
between 15–29 days, 56 calves were aged between 30–90 days) from
farms in three different Provinces of Turkey (Konya, Karaman, and
Aksaray) between January 2014 and April 2014. The farms included in the
study had similar climatic conditions. All calves were fed with colostrum
after birth. Almost 20–30 grams (g) fecal samples were taken from the
rectum in 5 mL eppendorf tube (Eppendorf Co., Germany) and kept
in refrigerators (ThermoFisher Scientic, U.S.A.) at 4°C until tested.
Modied Ziehl–Neilsen (MZN) for Cryptosporidium oocysts
Fecal smears were made as per the standard protocols [14]. Briey,
fecal samples were mixed with 0.09% NaCl solution, spread thinly on
the glass slide and dried at room temperature followed by modied
Ziehl–Neilsen (MZN) stain to detect Cryptosporidium oocysts in the
fecal smears under microscope (BX43 Olympus, Japan) with oil
immersion lense (X100) [14].
The evaluation considered the number of oocysts in 20 randomly
selected microscope elds in the preparations. The severity of the
infection according to the Cryptosporidium oocyst density were
dened as negative (no oocyst observed), mild (1–5 oocyst), moderate
(6–10 oocyst) and severe (>10 oocyst) [15].
ELISA for coproantigen detection
A commercial ELISA kit (Bio–X Diagnostics, Belgium) was used
to detect the presence of antigens against Cryptosporidium spp.,
coronavirus, rotavirus and E. coli in feces from all 300 diarrheic calves.
Feces diluted in dilution buffer and incubated on the microplate for 1h
at 4°C. After incubation step, the plate washed and incubated for 1 h
with the conjugate room tempurature, a peroxidase labelled antigen
(Cryptosporidium spp., coronavirus, rotavirus and E. coli) specic
monoclonal antibody. After this second incubation, the plate is washed
again and the tetramethylbenzidine added and incubated 10 min in
room temperature. The enzyme substrate reaction stopped with a
stop solution and the results evaluated in a 450 nm ELISA reader.
Statistical analysis
The results of the research were evaluated by chi–square test
(SPSS 2.0). P<0.05 value statistic was considered signicant.
RESULTS AND DISCUSSION
Calves screened during the study were classified into three
categories viz; 0–14 days (n=159), 15–29 days (n=85) and 30–90 days
(n=56). The most common mixed infection was caused by the two
agents viz; Cryptosporidium spp. and Rotavirus in 8% of the studied
calves followed by Coronavirus–E. coli (0.3%) and Cryptosporidium spp.–
Rotavirus–E. coli (0.6%) mixed infections. Mixed infection also followed
the similar pattern of age wide distribution as did the Cryptosporidium
being highest (12.9%) in 15–29 days aged calves followed in order
by those aged 0–14 days (7.5%) and 30–90 days (1.7%). The only
Coronavirus–E. coli mixed infection was observed in the 30–90 days
age group; and Cryptosporidium spp.–Rotavirus–E. coli mixed infection
were detected in 0–14 days (0.6%) and 15–29 days (1.1%) age groups.
This study found that Cryptosporidium, rotavirus, coronavirus, and
E. coli were the enteropathogens involved in the etiology of NCD,
and that Cryptosporidium and rotavirus were the most important
enteropathogens, among these. It is also suggested that for NCD, it
should be determined if the infection is of mixed nature as the clinical
indications only are not sucient to provide information about more
than one pathogen involved. Furthermore, the diagnosis should be
promoted by different diagnostic techniques [16]. For pathogen
identification, staining methods such as: the MZN, Trichrome,
Acridine Orange, Modiye Köster and Kinyoun acid–fast stains (for
Cryptosporidium) were used. Tests like the uorescent antibody
test and ELISA techniques (for rotavirus and and coronavirus),
and bacteriological cultures for E. coli, are used [4, 17, 18]. The
distribution of enteropathogens identied through commercial
ELISA according to the age groups in the neonatal diarrheal calves
are demonstrated in TABLE I. Among these, Cryptosporidium spp.
and coronavirus were detected at the highest and lowest rates,
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respectively. Rotavirus and E. coli were found to cause a moderate
number of infections compared to Cryptosporidium spp. in calves
with diarrhea in these regions. In this study, rotavirus was the second
most common agent detected in 19% of diarrheic calves, while the
overall prevalence of E. coli was 5.6% in diarrheic calves. The lowest
infection rate detected was for coronavirus (2%). Age wide distribution
of coronavirus was found to be 2.5 and 3.5% in 0–14– and 30–90–days
groups, respectively; whereas, no coronavirus antigens were found
in calves (n=85) of 15–29 days group (TABLE I).
Although management factors (e.g., environmental, and nutritional
factors) for calves with concurrent infections of Cryptosporidium spp.
and other agents may affect the outcome of the disease, the results of
this research demonstrate the signicance of Cryptosporidium spp. as
the major pathogen responsible for acute diarrhea in neonatal calves.
Regarding the most commonly detected pathogen of NCD, variable
reports are present with rotavirus [19] and Cryptosporidium spp. as the
most prevalent agent [20]. In Turkey, prevalence of Cryptosporidium
spp. has been found 3.9–70.3% [21, 22, 23, 24]. In other Countries,
prevalence reports range from 3.1 to 86.4% [25, 26, 27, 28, 29]. Risks
factors like age and immune status are signicant host–related
determinants affecting the prevalence of cryptosporidiosis. The
prevalence of Cryptosporidium spp. as determined through shedding
of Cryptosporidium oocysts in different age groups is variable [30].
In this study, the severity of the infection according to the
Cryptosporidium oocyst density was detected as 43%. Infection is
often restricted to younger animals of 8 weeks or less and is most
prevalent in calves that are less than one month of age [31]. The
highest infection rates in this study were in calves of 15–29 days of
age which is different from other reports where calves aged 1–10
[15], 7–14 [32], 8–14 [22], and 8–21 [33] days had been found most
susceptible. The main reason for this was thought to be due to sudden
antibody changes in their immune systems in case the calves received
insucient colostrum due to national and seasonal differences. In
addition, rotavirus infection was found to be considerably positive
in animals in this age group. It is thought that the surface of the
intestinal mucosa may be colonized by Cryptosporidium oocysts due
to the destruction of the intestinal mucosa after rotavirus infection.
Although there are many vaccine studies against Cryptosporidiosis,
the lack of an effective commercial vaccine is another reason for
Cryptosporidium the high rate in NCD [34].
In the present study, rotavirus was found in 20.3% of the diarrheic
calves which falls within the range (10.4–53%) of infections reported
elsewhere in Turkey [35, 36, 37, 38]. Although the prevalence of
rotavirus infection varies depending on factors such as the frequency
of farm hygiene and protection and control measures, the detection
of the disease in one of every ve calves in the study group in this
region indicates that the NCD disease is a disease that requires
precautions. The distribution of rotavirus found in this research
(20.3%) was comparable with those reported in diarrheic calves of
Belgium (20%), Switzerland (59%), Netherlands (17.7%), and the Brazil
(6,37%) [19, 31, 39, 40]. The prevalence of rotavirus was found highest
in 15–29 days age–group calves in this study which is in agreement
with the report of Mukhtar et al. from Pakistan [41].
TABLE I
Prevalence (%) of enteropathogens in the different
age groups of diarrheic calves
Age (days) 0 –14 15–29 30–90 Total
Number of animals (n) 159 85 56 300
Cryptosporidium spp.
84
(52.8%)
ab
50
(58.8%)
a
22
(39.2%)
b
156
(52%)
A
Rotavirus
20
(12.5%)
a
34
(40%)
b
7
(12.5%)
a
61
(19%)
B
Coronavirus
4
(2.5%)
a
0
(0.0%)
a
2
(3.5%)
a
6
(2%)
D
E. coli
8
(5%)
a
4
(4.7%)
a
5
(8.9%)
a
17
(5.6%)
C
Cryptosporidium spp.,
Rotavirus
12
(7.5%)
ab
11
(12.9%)
a
1
(1.7%)
b
24
(8%)
C
Coronavirus, E. coli
0
(0.0%)
a
0
(0.0%)
a
1
(1.7%)
a
1
(0.3%)
D
Cryptosporidium spp.,
Rotavirus, E. coli
1
(0.6%)
a
1
1.1%)
a
0
(0.0%)
a
2
0.6%)
D
Different letters in the same line (
A,B,C,D
) and columns (
a,b
) are statistically signicant
(Chi–square test,
P<0.05)
The prevalence of cryptosporidiosis diagnosed by MZN and
commercial ELISA in diarrheal stool specimens was 36.3 and 52%,
respectively. Microscopic fecal examination of MZN–stained smears
revealed Cryptosporidium spp. oocysts in 109 (36.3%) cases. The
highest distribution (47%) was in 15–29 days age group followed in
order by 0–14 days (33.9%) and 30–90 days (26.7%) age groups of
calves. While the highest prevalence in this study was found in the
calves aged 15–29 days, the differences between the age groups was
statistically signicant (P<0.05) (TABLE II). It was determined that 43%
of the sick animals were severely infected (>10 oocyst) (TABLE III). All
acid–fast positive samples showed a positive reaction on the ELISA.
TABLE II
Prevalence (%) of Cryptosporidium spp. in the different
age groups of diarrheic calves by MZN
Age
Number of animals
(n)
Cryptosporidium spp.
Infection rate
(%)
0 –14 day 159 54 33.9
b
15–29 day 85 40 47
a
30–90 day 56 15 26.7
b
Total 300 109 36.3
Different letters in the same columns (
a,b
) are statistically signicant (Chi–square test,
P<0.05)
TABLE III
Infection score according to age groups in calves
infected with Cryptosporidium spp.
Amount of
oocysts
0–14 days 15–29 days 30–90 days Total
Negative
(no oocyst)
105 45 41 191
(does not apply)
Mild
(1–5 oocysts)
12 11 3 26
(23.85%)
Moderate
(6–10 oocysts)
18 10 8 36
(33.02%)
Severe
(>10 oocysts)
24 19 4 47
(43.11%)
Assessment of Enteropathogens in diarrheic calves through ELISA-based prevalence / Uslu et al. ________________________________
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E. coli is another signicant NCD causing agent [17]. Previous
studies in Turkey found that E. coli was detected in 9.4% of 192
diarrheal calves in East and Southeast of Turkey [33], 26% of 138
diarrheal calves in Sivas [42], and 24.81% of 133 diarrheal calves in
part of eastern Turkey [43]. The prevalence of E. coli in NCD cases
in other Countries has been reported as 0.9–56.3% [20, 44, 45].
Differences in ndings may be due to disparities in the methods
used or in the age groups studied, as well as regional differences in
E. coli serotypes. Factors like shorter shedding period of bacteria
may inuence the distribution of E. coli [31]. In addition, the low
rate of infection compared to other regions and studies indicates
that the farms in these 4 regions apply effective protection control
methods such as vaccination during pregnancy and hyperimmune
sera administration to newborn calf against E. coli K99.
It has been stated that calves are quite sensitive to coronavirus
during their first 3–21 days [46]. A yellowish, watery diarrhea,
sometimes with the presence of mucous but rarely blood, develops and
lasts for 3–6 days. Virus can be detected in the stool during this period
[47]. In many former studies in Turkey, the prevalence of coronavirus
infection in calves was reported between one and 37.1% [37, 38, 48,
49]. Hasoksuz et al. reported infection rates in 37.1, 25.6 and 18.2%
of the cows in the age groups 0–30 days, 4–12 months, and 2–7 years,
respectively [49]. In other Countries, prevalence of coronavirus in
diarrheic calves has been found between 1.9 and 8% [28, 31, 39, 40].
Lower coronavirus prevalence may be associated with the previous
use of antimicrobial drugs and/or cleaning of the housing area [31],
because coronavirus is an opportunistic infectious agent.
Since more than one pathogen can be found in calf diarrhea, it
is emphasized that the severity of the disease is higher in mixed
infections [50]. The most common mixed infection was caused by
the two agents viz: Cryptosporidium spp. and rotavirus (8%) in the
present study. Previous studies also found that Cryptosporidium spp.
and rotavirus were the most prevalent infectious agents in diarrheic
calves [19, 33]. It has also been reported that Cryptosporidium spp.,
which is the main cause of neonatal calf diarrhea, is a risk factor
for the emergence of rotavirus infection [40, 51]. In the etiology of
diarrhea, various researchers have reported that coronavirus can play
a role with or without rotavirus [35, 52]. Conrady et al. reported that
the highest estimated mean Cryptosporidium–rotavirus prevalence
was identied in Ireland (16.7%) [53], the highest estimated mean
Cryptosporidium–coronavirus prevalence was detected in the United
Kingdom (4.3%), and the highest estimated mean Cryptosporidium–E.
coli prevalence in Turkey (4.7%). Alkan reported that seven out of 83
diarrheal calves (13.4%) had mixed infections with both rotavirus and
coronavirus [35]. In a study of 82 diarrheic calves in Konya, three
calves (3.6%) were tested positive for rotavirus and coronavirus
[54]. In a different study involving acute diarrhea in 30 calves from
1–28 days of age, one calf was positive for rotavirus and E. coli at
three weeks of age, and one for rotavirus and coronavirus at two
weeks of age [52]. In research carried out by Icen et al., the rates of
various dual infections were 15.6% (Cryptosporidium spp. rotavirus),
1% (coronavirus– rotavirus), 5.2% (E. coli K99–Cryptosporidium spp.),
and 7.3% (rotavirus–E. coli) [33]. Two different triple infection rates
were reported as 3.1% (Cryptosporidium spp.–E. coli K99–rotavirus)
and 1.0% (Cryptosporidium spp.– coronavirus–rotavirus). While E.
coli and coronavirus infections were less common in this region,
the frequency of rotavirus and Cryptosporidium spp. infections was
found to be higher in calves less than 30 days old.
CONCLUSIONS
This study concludes that the etiologic agents alone or in combination
may play a role in the frequency distribution of the neonatal calf
diarrhea. Hence, treatment protocols should be designed with a
consideration for mixed infections. The target agents are of primary
zoonotic factors for human infections, with cattle assuming the role
of reservoir host. Hence, the results of this study may be used in
screening of infections in reservoir hosts and for the development
of effective control strategies through better understanding of the
transmission dynamics. Other tools like providing colostrum to the
newborns and an awareness campaign can be useful as a preventive
management of the neonatal calf diarrhea.
ACKNOWLEDGMENTS
This study was supported by SUBAP (No. 14401045).
Ethıcs approval
The study protocol was approved by Ethical Committee of Faculty
of Veterinary Medicine, Selcuk University, Turkey (Approval no:14/03).
Conict of interest
The authors declare that they have no conict of interest.
BIBLIOGRAPHIC REFERENCES
[1] Lorenz I. Diarrhoea of the young calf: an update. Proceedings
of the XXIV World Buiatrics Congress. Nice, Oct. 15, 2006,
France. 2016; p 1–14.
[2] Millemann Y. Diagnosis of neonatal calf diarrhoea. Rev. Md.
Vt. 2009; 160(8/9):404–9. doi: https://doi.org/bspzp7
[3] Izzo M, Kirkland P, Mohler V, Perkins N, Gunn A, House J.
Prevalence of major enteric pathogens in Australian dairy calves
with diarrhoea. Aust. Vet. J. 2011; 89(5):167–73.
[4] İnci A. Sığırlarda Cryptosporidiosis, Ozcel MA. (ed). Veteriner
Hekimliğinde Parazit Hastalıkları. 2013; 135–42.
[5] Thomson S, Hamilton CA, Hope JC, Katzer F, Mabbott NA,
Morrison LJ, Innes EA. Bovine cryptosporidiosis: impact,
host–parasite interaction and control strategies. Vet. Res. 2017;
48:1–16. doi: https://doi.org/gbtg74
[6] Mickelsen WD, Evermann JF. In utero infections responsible
for abortion, stillbirth, and birth of weak calves in beef
cows. Vet. Clin. North Am. Food Anim. 1994; 10(1):1–14. doi:
https://doi.org/kdrq
[7] Almeida PR, Lorenzetti, Cruz RS, Watanabe TT, Zlotowski P,
Aleri AA, Driemeier D. Diarrhea caused by rotavirus A, B, and C
in suckling piglets from southern Brazil: molecular detection and
histologic and immunohistochemical characterization. J. Vet.
Diagn. Investig. 2018; 30(3):370–6. doi: https://doi.org/gdkms5
[8] Bok M, Alassia M, Frank F, Vega CG, Wigdorovitz A, Parreno V.
Passive immunity to control Bovine coronavirus diarrhea in a
dairy herd in Argentina. Rev. Argent. Microbiol. 2018; 50(1):23–30.
doi: https://doi.org/jnmr
[9] Collery P. Causes of perinatal calf mortality in the Republic of
Ireland. Ir. Vet. J. 1996; 49:491–6.
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33249, 1 – 6
5 of 6
[10] Snodgrass D, Terzolo HR, Sherwood, Campbell I, Menzies J,
Synge B. Aetiology of diarrhoea in young calves. Vet. Rec. 1986;
119(2):31–4. doi: https://doi.org/brz5st
[11] Ikemori Y, Ohta M, Umeda K, Icatlo Jr. FC, Kuroki M, Yokoyama H,
Kodama Y. Passive protection of neonatal calves against bovine
coronavirus–induced diarrhea by administration of egg yolk or
colostrum antibody powder. Vet. Microbiol. 1997; 58(2–4):105–11.
doi: https://doi.org/cwvzq6
[12] Blanco JE, Blanco M, Mora, Blanco J. Production of toxins
(enterotoxins, verotoxins, and necrotoxins) and colicins by
Escherichia coli strains isolated from septicemic and healthy
chickens: relationship with in vivo pathogenicity. J. Clin.
Microbiol. 1997; 35(11):2953–7. doi: https://doi.org/kdrr
[13] Butler DG, Clarke RC. Diarrhoea and dysentery in calves.
In: Gyles CL (ed). Escherichia coli in domestic animals and
humans. Wallingford (United Kingdom): CAB International Oxon.
1994; p. 91–116.
[14] Ok Ü, Girginkardeşler N, Kilimcioğlu A, Limoncu E. Dışkı inceleme
yöntemleri. In: Özcel, MA, Altintas, N. Parazit Hastaliklarinda
Tani. Türkiye Parazitoloji Derg. 1997; 15:1–61.
[15] Ekici Ö, Sevinç F, Coşkun A, Işık, Sevinç N. İshalli buzağılarda
cryptosporidiosisin yaygınlığı. Eurasian J. Vet. Sci.
2011; 27(2):123–6.
[16] Nussbaum DJ, Salord JR, Rimmele DD. Evaluation of quantitative
latex agglutination for detection of Cryptosporidium parvum,
E. coli K99, and rotavirus in calf feces. J. Vet. Diagn. Invest.
1999; 11(4):314–8.
[17] Khan A, Khan MZ. Aetiopathology of neonatal calf mortality. J.
Isl. Acad. Sci. 1991; 4 (2): 159–65.
[18] Ragsdale J. Diagnostic samples, tests for calf diarrhea. Vet.
Quart. 2004; 7(1):6.
[19] Lanz UF, Kaufmann T, Sager H, Albini ., Zanoni R., Schelling E,
Meylan M. Prevalence of four enteropathogens in the faeces of
young diarrhoeic dairy calves in Switzerland. Vet. Rec. 2008;
163(12):362–6. doi: https://doi.org/cn739k
[20] Gillhuber J, Rügamer D, Pster K, Scheuerle MC. Giardiosis
and other enteropathogenic infections: a study on diarrhoeic
calves in Southern Germany. B.M.C. Res. Notes. 2014; 7:1–9. doi:
https://doi.org/f5xwbh
[21] Değerli S, Çeliksöz Kalkan K, Ozcelik S. Prevalence of
Cryptosporidium spp. and Giardia spp. in cows and calves in
Sivas. Turk. J. Vet. Anim. Sci. 2005; 29(4):995–9.
[22] Güven E Avcıoğlu H, Balkaya I, Hayirli A, Kar S, Karaer Z.
Prevalence of Cryptosporidiosis and molecular characterization
of Cryptosporidium spp. in calves in Erzurum. Kafkas Univ. Vet.
Fak. Derg. 2013; 19(6):969–74. doi: https://doi.org/kdrs
[23] Gündüz N, Arslan MÖ. Kars Yöresinde Buzağılarda Cryptosporidium
Enfeksiyonları Prevalansının Asit Fast Boyama (mAF) ve ELISA
Yöntemleriyle Belirlenmesi. Turkiye Parazitol. Derg. 2017; 41:5–8.
doi: https://doi.org/kdrt
[24] Yildirim A, Sevinc F, Onder Z, Duzlu O, Ekici OD, Isik N, Ciloglu
A, Simsek , Yetismis G, Inci A. Comparison of three diagnostic
methods in the diagnosis of cryptosporidiosis and gp60
subtyping of Cryptosporidium parvum in diarrheic calves in
Central Anatolia Region of Turkey. EuroBiotech J. 2021; 5 2):63–9.
doi: https://doi.org/kdrv
[25] Gow S, Waldner C. An examination of the prevalence of and risk
factors for shedding of Cryptosporidium spp. and Giardia spp.
in cows and calves from western Canadian cow–calf herds. Vet.
Parasitol. 2006; 137(1–2):50–61. doi: https://doi.org/crfp9w
[26] Singh BB, Sharma R, Kumar H, Banga H, Aulakh RS, Gill JPS,
Sharma JK, Prevalence of Cryptosporidium parvum infection
in Punjab (India) and its association with diarrhea in neonatal
dairy calves. Vet. Parasitol. 2006; 140(1–2):162–5. doi:
https://doi.org/bt93g7
[27] Cai M, Guo Y, Pan B, Li N, Wang X, Tang C, Yaoyu F, Xiao L.
Longitudinal monitoring of Cryptosporidium species in pre–
weaned dairy calves on five farms in Shanghai, China. Vet.
Parasitol. 2017; 241:14–9. doi: https://doi.org/gbkz2v
[28] Caffarena RD, Casaux ML, Schild CO, Fraga M, Castells M, Colina
R, Maya L, Corbellini LG, Riet–Correa F, Giannitti F. Causes of
neonatal calf diarrhea and mortality in pasture–based dairy
herds in Uruguay: a farm–matched case–control study. Braz.
J. Microbiol. 2021; 52(2):977–88. doi: https://doi.org/kdrw
[29] Hoque S, Mavrides DE, Pinto P, Costas S, Begum N, Azevedo–
Ribeiro C, Liapi M, Kvac M, Malas S, Gentekaki E, Tsaousis AD.
High occurrence of zoonotic subtypes of Cryptosporidium
parvum in Cypriot dairy farms. Microorganisms. 2022; 10(3):531.
doi: https://doi.org/kdrx
[30] Markovics A, Pipano E. Shedding of cryptosporidial oocysts by
naturally infected calves. Isr. J. Vet. Med. 1987; 43:46–9.
[31] Bartels CJM, Holzhauer M, Jorritsma R, Swart WA, Lam TJ.
Prevalence, prediction and risk factors of enteropathogens in
normal and non–normal faeces of young Dutch dairy calves. Prev.
Vet. Med. 2010; 93(2–3):162–9. doi: https://doi.org/ch7mpc
[32] Díaz–Lee A, Mercado R, Onuoha E., Ozaki L, Muñoz P,
Muñoz V, Martinez FJ, Fredes F. Cryptosporidium parvum in
diarrheic calves detected by microscopy and identified by
immunochromatographic and molecular methods. Vet. Parasitol.
2011; 176(2–3):139–44. doi: https://doi.org/cvmxkh
[33] Içen H, Arserim NB, Işik N, Özkan C, Kaya A. Prevalence of Four
Enteropathogens with Immunochromatographic Rapid Test in
the Feces of Diarrheic Calves in East and Southeast of Turkey.
Pak. Vet. J. 2013; 33(4): 496–99
[34] Thomson S, Hamilton CA, Hope JC, Katzer F, Mabbott NA,
Morrison LJ, Innes EA. Bovine cryptosporidiosis: impact,
host–parasite interaction and control strategies. Vet. Res. 2017;
48(42):1–6. doi: https://doi.org/f6zz
[35] Alkan F. Buzağı ishallerinde rotavirus ve coronavirusların
rolü. Ankara. Üniv. Vet. Fak. Derg. 1998; 45(1):29–37. doi:
https://doi.org/kdrz
[36] Sakli GU, Bulut O, Hasöksüz M, Hadimli HH. Investigation of
bovine coronavirus and bovine rotavirus by rapid diagnosis kit
and RT–PCR in diarrheic calf feces. J. Istanbul. Vet. Sci. 2019;
3(3):57–63. doi: https://doi.org/kdr2
Assessment of Enteropathogens in diarrheic calves through ELISA-based prevalence / Uslu et al. ________________________________
6 of 6
[37] Atasoy MO, Isidan H, Turan T. Genetic diversity, frequency
and concurrent infections of picobirnaviruses in diarrhoeic
calves in Turkey. Trop. Anim. Health Prod. 2022; 54(2):127. doi:
https://doi.org/kdr3
[38] Keleş İ, Ekinci G, Tüfekçi E, Citil M, Güneş V, Aslan Ö, Onmaz AC,
Bekdik IK, Varol K, Deniz O. Etiological and predisposing factors
in calves with neonatal diarrhea: A clinical study in 270 case
series. Kafkas Univ. Vet. Fak. Derg. 2022; 28(3):315–326. doi:
https://doi.org/kdr4
[39] De Graaf DC, Vanopdenbosch E, Ortega–Mora LM, Abbassi H,
Peeters JE. A review of the importance of cryptosporidiosis
in farm animals. Int. J. Parasitol. 1999; 29(8):1269–87. doi:
https://doi.org/frgq9t
[40] Cruvinel LB, Ayres H, Zapa DMB, Nicaretta JE, Couto LFM, Heller
LM, Bastos TSA, Cruz B, Soares VE, Teixeira WF, Oliveria JS,
Fritzen JT, Aleri AA, Freire R.L, Lopes WDZL. Prevalence and
risk factors for agents causing diarrhea (Coronavirus, Rotavirus,
Cryptosporidium spp., Eimeria spp., and nematodes helminthes)
according to age in dairy calves from Brazil. Trop. Anim. Health
Prod. 2020; 52:777–91. doi: https://doi.org/kdr5
[41] Mukhtar N, Yaqub T, Munir M, Nazir J, Aslam A., Masood A, Tahir
Z, Javed M, Nadeem A. Prevalence of group a bovine rota virus
in neonatal calves in Punjab, Pakistan. The J. Anim. Plant. Sci.
2017; 27(2):379–83.
[42] Küliğ C, Coşkun A. Sivas ve ilçelerindeki neonatal ishalli
buzağılarda E. coli, Cryptosporidium, Clostridium perfringens,
Rotavirüs ve Coronavirüs prevalansı. Turk. Vet. J. 2019; 1(2):69–73.
[43] Cengiz S, Adiguzel MC. Determination of virulence factors and
antimicrobial resistance of E. coli isolated from calf diarrhea,
part of eastern Turkey. Ankara Univ. Vet. Fak. Derg. 2020; 67
(4): 365–71. doi: https://doi.org/kdr6
[44] Garcıa A, Ruiz–Santa–Quiteria J, Orden J, Cid D, Sanz R, Gómez–
Bautista M, Fuente R. Rotavirus and concurrent infections with
other enteropathogens in neonatal diarrheic dairy calves in
Spain. Comp. Immunol. Microbiol. Infect. Dis. 2000; 23(3):175–83.
doi: https://doi.org/dp5mtg
[45] Ryu J, Kim S, Park J, Choi KS. Characterization of virulence genes
in Escherichia coli strains isolated from pre–weaned calves in
the Republic of Korea. Acta Vet. Scand. 2020; 62(1):1–7. doi:
https://doi.org/h3mf
[46] Tsunemitsu H, Yonemichi H, Hirai T, Kudo T, Onoe S, Mori K,
Shimizu M. Isolation of bovine coronavirus from feces and nasal
swabs of calves with diarrhea. J. Vet. Med. Sci. 1991; 53(3):433–7.
doi: https://doi.org/dm9nkk
[47] Gulliksen SM, Jor E, Lie K, Hamnes I, Løken T, Åkerstedt J,
Østerås O. Enteropathogens and risk factors for diarrhea in
Norwegian dairy calves. J. Dairy Sci. 2009; 92(10):5057–66. doi:
https://doi.org/cffm8s
[48] Erdoğan HM, Ünver A, Güneş V, Çitil M. Frequency of rotavirus
and coronavirus in neonatal calves in Kars district. Kafkas Univ.
Vet. Fak. Derg. 2003; 9(1):65–8.
[49] Hasoksuz M, Kayar A, Dodurka T, IlgazA. Detection of respiratory
and enteric shedding of bovine coronaviruses in cattle in
Northwestern Turkey. Acta Vet. Hung. 2005; 53(1):137–46. doi:
https://doi.org/dcp755
[50] Pospischil A. Pathologie und Pathogenese infektiöser
Durchfallerkrankungen beim Kalb. Vet. 1989; 5:27–32.
[51] Garro CJ, Morici GE, Tomazic ML, Vilte D, Encinas M, Vega C, Bok
M, Parreno V, Schnittger L. Occurrence of Cryptosporidium and
other enteropathogens and their association with diarrhea in
dairy calves of Buenos Aires Province, Argentina. Vet. Parasitol.
Reg. Stud. Reports. 2021; 24:100567. doi: https://doi.org/kdr7
[52] Al M, Balıkçı E. Neonatal ishalli buzağılarda rotavirus, coronavirus,
E. coli K99 ve Cryptosporidium parvum'un hızlı test kitleri ile
teşhisi ve enteropatojen ile maternal immünite ilişkisi. F. Ü.
Sağ. Bil. Vet. Derg. 2012; 26(2):73–8.
[53] Conrady B, Brunauer M, Roch FF. Cryptosporidium spp. Infections
in Combination with Other Enteric Pathogens in the Global Calf
Population. Anim. 2021; 11(6):1786. doi: https://doi.org/kdr8
[54] Ok M, Güler L, Turgut K, Ok Ü, Şen I, Gündüz I, Birdane MF,
Guzelbektas H. The studies on the aetiology of diarrhoea in
neonatal calves and determination of virulence gene markers
of Escherichia coli strains by multiplex PCR. Zoon. Publ. Health.
2009; 56(2):94–101. doi: https://doi.org/fvg7v3
