Overview on the diagnostic methods of Cryptosporidiosis in bovine calves

Overview on the diagnostic methods of Cryptosporidiosis in bovine calves
El-Kelesh E.A., Saba S.E.R., Asmaa M. El Nady, Balegh A.A., Hegab A.A.
Parasitology Department, Animal Health Research Institute, Agricultural Research 
Centre

ABSTRACT
C
ryptosporidiosis is one of the most important causes of neonatal enteritis and diarrhea in calves leading to significant morbidity and 
mortality rates globally. This study was conducted to highlight the 
diagnostic methods of cryptosporidiosis in pre-weaned cattle calves. For 
this purpose, a total of 88 diarrheic faecal samples of pre-waned calves at 
Giza and El-Fayoum governorates, Egypt, were microscopically examined 
using Modified Ziehl-Neelsen staining (MZN), Immunochromatographic 
test (ICT) and Enzyme linked Immunosorbent Assay (ELISA). The prevalence of infection was 71.59% (63/88), 73.86% (65/88), and 75.00%
(66/88) for MZN, ICT, and ELISA respectively. The Sensitivity was 
96.9%, 100%, and 100% whereas; Specificity was 100%, 100%, and 
95.7% for MZN, ICT, and ELISA, respectively.

INTRODUCTION
The zoonotic protozoan parasites of the 
genus Cryptosporidium are obligate, intracellular parasites that infect the epithelial cells 
lining the luminal surfaces of the digestive and 
respiratory tracts of a wide variety of vertebrates, including humans, livestock, wild animals, and birds (Fayer et al. 2000). Cryptosporidium parvum was first described in 1907
by Tyzzer in the small intestine of mice 
(Chalmers and Katzer, 2013). Cryptosporidiosis was first reported in cattle in early 1970
(Panciera et al. 1971), but the observed clinical disease could not be solely attributed to 
Cryptosporidium as there was evidence of confection with other viral bacterial pathogens. 
Neonatal diarrhoea in experimentally infected 
calves with Cryptosporidium species was reported as the single infective agent (CastroHermida et al. 2002). Four species of Cryptosporidium are commonly found in cattle: C. 
parvum, C. bovis, C. ryanae, and C. andersoni, 
but only C. parvum is associated with clinical 
disease in neonatal calves (Thomson et al. 
2017). C. parvum predominates in pre-weaned 
calves, C. bovis and C. ryanae in post-weaned 
calves, and C. andersoni in older calves and 
adult cattle (Robinson et al. 2006). Cryptosporidium infection in dairy calves can lead to villous atrophy in the small intestine mucosa and 
Received in 5/3/2023
Received in revised from 
12/4/2023
Accepted in 26/4/2023
Keywords: 
Cryptosporidium 
Cattle calves
Sandwich ELISA
MZN
ICT
Egyptian Journal of Animal Health
P-ISSN: 2735-4938 On Line-ISSN: 2735-4946
Journal homepage: https://ejah.journals.ekb.eg/
*Corresponding author: Aliaa B. Baleg , Parasitology Department, Animal Health Research Institute - Agriculture Research Center (ARC) 
E-mail address: aliaabaleg@yahoo.com
DOI: 10.21608/EJAH.2023.300350
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El-Kelesh et al., Egyptian Journal of Animal Health 3, 3 (2023), 19-27
increase intestinal permeability (Thomson et 
al., 2017). Consequently, these pathologies can 
lead to diarrhoea and an increased risk of mortality from dehydration (Delafosse et al. 2015). 
Cryptosporidium parvum infections in 
calves present as profuse watery diarrhoea 
with acute onset, and can be accompanied by 
depression, weakness, and anorexia (Santín, 
2013). Some infected cattle exhibit reduced 
weight gain compared with uninfected controls, and another study had found that infection may interfere with milk production in 
dairy cows (Robinson et al. 2006).
Cryptosporidium is a monoxenous parasite, 
undergoing all life cycle stages, including sexual and asexual reproduction, within a single
host and producing thick-walled, environmentally hardy oocysts (Brown, 2014). Naturally 
infected calves can shed in excess of 
3.89 × 1010 infective oocysts over a 6-day period which leads to widespread contamination 
of grazing lands, water sources, and the general environment (Nydam et al. 2001). Cryptosporidium oocysts are transmitted between 
hosts via the faecal-oral route, either directly 
via contact with faeces from infected hosts, or 
indirectly through environmental contamination or ingesting of contaminated food or water (Niine et al. 2018; Thomson et al. 2017).
Autoinfection with Cryptosporidium was reported due to thin-walled oocyst production 
(Leitch and He, 2011). Importantly, cryptosporidiosis is not only a hazard for animal 
health and production but also its zoonotic 
characteristics represents a life-threatening 
disease (Elmahallawy et al. 2020).
Cryptosporidium can be diagnosed by a 
number of techniques including microscopic 
examination, either by the wet mount preparation or staining the smears with modified acidfast stain, or by fluorescent stains (Khurana 
and Chaudhary, 2018). Immunological diagnosis using either antibody or antigen detection in faecal samples is available. Examples 
of these tests are Enzyme-Linked Immunosorbent Assays (ELISA), Immunochromatographic tests (ICT), and Immunofluorescence 
assays (IFA). Various molecular methods for 
the detection of DNA are also available 
(Aboelsoued and Abdel Megeed, 2022). The 
modified acid-fast staining is broadly applied 
for clinical diagnosis owing to its simplicity 
and cost-effectiveness (Jafari et al. 2015; 
Mahmoudi et al. 2021). 
The incidence rate of Cryptosporidium in 
calves, all over the world ranged from 3.4 to 
96.6% (Thomson et al. 2017). In Egypt, the 
highest prevalence rate of Cryptosporidium in 
calves was 56.32% (Essa et al. 2014), while, 
the lowest one was 9.2% (Mahfouz et al. 
2014). 
The objectives of this study were 
to investigate the rate of Cryptosporidium infection in pre-weaned cattle calves in the Giza 
and El Fayoum governorates, Egypt. Additionally, the diagnostic methods by acid-fast staining, immunochromatographic, and ELISA 
were compared in terms of statistical factors, 
duration of the laboratory experiment, and the 
cost-effectiveness of testing, to determine the 
superior method for the detection of Cryptosporidium in the infected calves.
MATERIALS AND METHODS
For this study, 88 Fresh faecal samples were 
collected directly from the rectum of diarrheic 
pre-weaned cattle calves in Giza (n=54) and El 
Fayoum governorates (n-34), Egypt between 
2021 and 2022. The age of sampled calves 
ranged from two days to two months old. Each 
sample was kept in a labeled clean container 
and transferred in ice boxes to the Parasitology 
Laboratory at Animal Health Research Institute Dokki, Giza on the same collection day.
Laboratory Diagnosis 
The collected samples were concentrated 
using faecal flotation with a Sheather’s sugar 
solution (Singh et al. 2006).
Detection of oocysts using Modified ZiehlNeelsen staining method (MZN)
A Floated material of concentrated faecal 
smear from each sample was transferred to a 
glass slide and allowed to dry at room temperature. Following fixing by methanol (2 min), 
the slides were flooded with basic carbolfuchsin for 5 min. After a brief rinse with tap 
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El-Kelesh et al., Egyptian Journal of Animal Health 3, 3 (2023), 19-27
water, the slide was decolorized with acid alcohol until the disappearance of red color (45–
60 s) and rinsed again. Malachite green 0.5%
or methylene blue 1.4% solution (1 min) was 
used as the counterstain (El Kelesh et al. 
2009; Smith et al. 2008). The smear slides 
were washed with tap water and air-dried. Finally, all slides were examined using the oil 
immersion lens (100×), and Cryptosporidium 
oocytes were identified (4 - 6 microns, pink to 
red cells in the green or blue background). 
The duration of the MZN stain was 20 min 
(including procedure and examination).
Immunochromatographic test (ICT): 
 An immunochromatography test strips kit 
was used following the manufacturer's instructions to discover Cryptosporidium parvum
antigens in faeces (BIO K 387, Belgium). A 
spoonful of liquid faecal samples was diluted 
with the liquid contained in the bottle, and homogenized well, taking care to prevent foam 
formation. A device was plunged into the diluted samples of the bottle for 10 minutes. 
Positive sample for Cryptosporidium appears 
as 2 lines while negative one appesrs as one 
line. The duration of ICT method was 20 min 
for detection of Cryptosporidium antigens. 
ELISA Method 
A commercial sandwich, double wells ELISA kit (BIO-X Diagnostics, Belgium) was 
used to detect Cryptosporidium antigens following the manufacturer's instructions. The 
test was performed on 88 maintained faecal 
samples at 4 °C without preservative. The 
plate is coated with monoclonal antibodies. 
Faecal samples were diluted and added to 
each coated well. After 1 hour incubation at 
37 °C the conjugated monoclonal antibody 
was added. Following the incubation, the reaction was visualized by tetramethylbenzidine 
(TMB) and the results were read at 450 nm 
using a Microplate ELISA reader. The duration of ELISA method was 2 hours for detection of Cryptosporidium antigens. 
Statistical Methods:
Diagnostic accuracy was assessed via two 
methods. The first is the Composite Reference 
Standard (CRS), where the sensitivity and 
specificity were calculated for each of the 
three tests, considering the combined results 
from at least TWO individual tests as the diagnostic ‘GOLD’ standard. The second is Latent 
class analysis (LCA), which is used to identify 
a set of discrete, mutually exclusive latent 
classes (diseased and not diseased) based on 
the observed results of the samples to a set of 
categorical variables (positive and negative). 
The model with the best fit was chosen to estimate the conditional probability Pr (positive) 
to represent the sensitivity of the test and the 
conditional probability Pr (negative) to represent the specificity of the test. Agreement between each pair of tests was assessed using the 
k statistic. CRS and agreement were done using IBM© SPSS© Statistics version 22
(IBM© Corp., Armonk, NY, USA). LCA was 
done using RStudio 2022.12.0+353 for Windows Mozilla/5.0, with the po LCA package.
RESULTS
Microscopical examination
Microscopic examination of fecal stained 
smears using MZN was confirmed to be infected with Cryptosporidium oocysts which appeared as pink spherical bodies 4-6µ against a 
green or blue background, Figure (1). 63 out of 
88 calves were found to be infected with Cryptosporidium spp., with an overall prevalence of 
71.59% in Egypt; at Giza governorate was 
70.37% (38/54) and at El Fayoum was 73.52
(25/34).