ARTICLE ORIGINAL
The prevalence of Cryptosporidium parvum
infection in the diarrhoiec and non- diarrhoeic
calves
° F. SEVINC, °° K. IRMAK and °° M. SEVINC
° Dr., Selçuk University, Department of Parasitology, Faculty of Veterinary Medicine, Konya, Turkey.
°° Dr., Selçuk University, Department of Internal Medicine, Faculty of Veterinary Medicine, Konya, Turkey.
Corresponding author : Dr. Ferda Sevinç, Department of Parasitology, Faculty of Veterinary Medicine, Selcuk University, P. O. Box 42031, Campus, Konya, Turkey.
Fax : 90-332-2410063, e-mail : fsevinc@selcuk.edu.tr
SUMMARY
RÉSUMÉ
Faeces samples from 300 diarrhoeic and non - diarrhoeic calves were
screened for the presence of Cryptosporidium infections. The prevalence of
Cryptosporidium parvum was determined by using acid-fast staining
method (Ziehl Neelsen) and ELISA kit. Calves were grouped according to
their age as follows: 1-10, 10-20, 20-30, 30-45 and >45 days. The prevalence of infection in diarrhoeic and non diarrhoeic calves was 63.92% and
9.85%, respectively. Cryptosporidium infection was detected in 50.75%,
35.71%, 25.45%, 14.71% and 13.24% of the calves in the age groups, respectively.
La prévalence de l’infection à Cryptosporidium parvum chez les veaux
d i a r rhéiques et non-diarrhéiques. Par F. SEVINC, K. IRMAK et M.
SEVINC.
Échantillons de fèces provenant de 300 veaux diarrhéiques et non - diarrhéiques ont été examinés pour la présence des infections de
Cryptosporidium. La prévalence de Cryptosporidium parvum a été déterminée en utilisant la méthode de coloration de Ziehl Neelsen modifiée et le kit
ELISA. Les veaux ont été groupés selon leur âge comme suit : 1-10, 10-20,
20-30, 30-45 et > 45 jours. La prévalence de l’infection chez les veaux diarrheiques et non diarrheiques était respectivement 63.92 % et 9.85 %.
L’infection à Cryptosporidium a été détectée respectivement chez 50,75 %,
35,71 %, 25,45 %, 14,71 % et 13,24 % des veaux dans les groupes d’âge.
KEY-WORDS : Cryptosporidium parvum - calves.
MOTS-CLÉS : Cryptosporidium parvum - veaux.
Introduction
threatening in young (1-4 weeks old) and immunocompromised animals. Infected immunocompetent animals are usually
asymptomatic, and are potential reservoirs for the infection
of other farmed animals and humans [14, 15, 30, 38].
Diarrhoea is the most important cause of morbidity and
mortality in neonatal period of calves. Neonatal calf diarrhea
has a complex aetiology and various infectious agents such
as enteropathogenic viruses, bacteria and parasites either singly or in combination have been implicated as causes.
C ryptosporidium parv u m is an intestinal protozoan parasite
and primary pathogen causing acute diarrhoea in calves [10,
11, 17, 23, 30, 33]. The most evident symptom of
Cryptosporidiosis is diarrhoea, and furthermore may be non
specific signs such as dehydration, fever, anorexia, weakness
and progressive loss of condition. Diarrhoea is usually selflimiting in immunocompetent animals, however it can be life
Revue Méd. Vét., 2003, 154, 5, 357-361
There are a variety of methods, including microscopy,
immunological and molecular techniques for the detection of
C ry p t o s p o r i d i u m oocysts. Microscopic methods include
concentration techniques and staining of faecal smears.
There are difficulties in distinguishing Cryptosporidium
oocysts from other small particles in faecal and environmental specimens such as yeasts, moulds, algae, and plant debris
by routine faecal examination techniques [16]. Modified
acid-fast staining technique is useful for the oocysts appeared
as pink to red spherical to ovoid bodies on a blue or purple
358
SEVINC (F.) AND COLLABORATORS
background, stained smears are permanent and are able to
store a long time before examination when the samples are
very numbers [34]. Staining techniques, however, is time
consuming and require an experienced microscopist, and the
sensitivity and specificity vary when the shedding of oocysts
is intermittently or in low numbers [4, 8, 16, 20, 21, 25, 31,
32, 39, 44].
Although acid-fast stains were widely-used standard
method, antigen detection assays such as direct fluorescent
antibody and ELISA testing are increasingly available to diagnose Cryptosporidiosis and have the advantage of both
being more sensitive and less user-dependent when compared with routine acid-fast testing [26]. Enzyme immunoassays are commonly used for the diagnosis of
Cryptosporidiosis [1, 9, 24, 40, 41, 43]. It has been reported
that the Cryptosporidium ELISA can detect Cryptosporidium
in more specimens than the microscopic examination, and is
s u fficiently sensitive and specific to detect clinical cases of
Cryptosporidiosis [2, 12, 18, 19, 25, 28, 40, 44].
Some
reports describing the prevalence of
Cryptosporidium infection by conventional microscopic examination techniques in diarrhoeic and non diarrhoeic calves
in Turkey have been reported [3, 5, 6, 13, 35, 36]. However,
no study detecting faecal antigens has been conducted. The
purpose of this study was to determine the prevalence of
Cryptosporidium parvum infection in different age groups of
diarrhoeic and non-diarrhoeic calves in Konya province by
comparing ELISA with the conventional microscopy method
using the modified acid-fast staining.
Materials and Methods
A. Specimens: Stool specimens (300) were collected in
sterile plastic bottles directly from the rectum of calves who
presented with diarrhoea (n: 97) and non-diarrhoea (n: 203)
from 3 February to 28 May 2002. Faecal smears were prepared from stool specimens on glass slides with 0.9 % isotonic
NaCl solution for Modified acid-fast staining at the sampling
d a y, and then the specimens were preserved in sodium acetate acetic acid formaldehyde solution (1.5 g of Sodium acetate, 2 ml of Glacial acetic acid, 4 ml of 37-40 %
Formaldehyde, and 92 ml of distilled water) in plastic bottles
and stored at 4°C until tested by ELISA.
B. Staining: Microscopic diagnosis of Cryptosporidium
was performed at Parasitology laboratories in Ve t e r i n a r y
faculty of Selcuk University with a modified acid-fast technique (34). Briefly, thin faecal smears were dried in room
temperature and fixed in methanole for two min and then
flooded with basic fuchsin solution (4 g of basic fuchsin, 20
ml of 95 % ethanole, 8 ml of dissolved crystal phenol, and
100 ml of distilled water) for 5 min and rinsed in 50 % ethanole. Then, the slides were decolorized in 1 % sulphuric acid
for two minutes. Following washing, the slides were pooled
in methylene blue solution (0.3 g of methylene blue, 30 ml of
ethanole, and 100 ml of 0.01 % KOH) for 1 minute, washed
and air dried, and examined under 100 X objectives.
The intensity of infection was estimated semiquantitatively
according to the average number of oocysts in 20 randomly
selected fields at 1000× magnification. The categories established were: negative (absence of oocysts); slight (1-5
oocysts); moderate (6-10 oocysts); severe (>10 oocysts).
C. Enzyme immunoassay: Detecting of Cryptosporidium
p a rv u m coproantigens was used a commercial ELISA kit
(BioX diagnostics). Stool in sodium acetate-acetic acid-formaldehyde were processed according to the manufacturer’s
recommendations and results were read by using a plate reader and a 450 nm filter (Anthos htll). The samples that yield
a difference in optical density that is greater than or equal to
0.150 were considered positive, and the optical density that is
less than 0.150 was considered negative.
Results
Total number of positive specimens was 82 by ELISA; 62
of these were identified by the modified acid-fast staining
method, giving 27.33 % and 20.67 % prevalence, respectively. All of the acid-fast positive specimens showed positive
reaction with specific anti- Cryptosporidium parvum monoclonal antibody. Two hundred and eighteen of 238 faecal
samples by ELISA were negative, althought all of them gave
negative results by Modified acid-fast staining method (Table
I).
C ryptosporidium parv u m oocysts appeared as pink to red
spherical to ovoid bodies on a greenish or purple background
with Modified acid-fast staining (Fig. 1).
Over a 5-month period, 97 diarrhoeic and 203 non diarrhoeic calves were examined to detect C. parv u m infection.
C. parvum was detected in 62 (63.92 %) of diarrhoeic calves
by ELISA (51 of 62 ELISA-positive faecal samples were
also positive by acid-fast staining) and in 20 (9.85 %) of non
diarrhoeic calves by ELISA (11 of 20 ELISA-positive faecal
samples were also positive by acid-fast staining) (Table II).
The age distribution of the infection was 50.75 %, 35.71 %,
25.45 %, 14,71 % and 13.24 % in the age groups, respectively (Table II).
T ABLE I — Prevalence of C ryptosporidium parv u m infection in calves by the acid-fast staining method and
ELISA.
Revue Méd. Vét., 2003, 154, 5, 357-361
THE PREVALENCE OF CRYPTOSPORIDIUM PARVUM INFECTION IN THE CALVES
Discussion
Diarrhoea is the most pronounced clinical feature of
Cryptosporidiosis. Several studies observed a higher prevalence of C. parv u m infections among diarrhoeic calves as
compared with non diarrhoeic calves [6, 7, 11, 27, 33, 42]. In
the present study, C. parv u m was detected in 62 (63.92 %)
and 20 (9.85 %) of 97 diarrhoeic and 203 non diarrhoeic
calves, respectively (Table 2). The intensity of oocysts was
significantly higher in diarrhoeic animals than in non-diarrhoeic ones. Thus, there was a significant association between C. parvum infection and diarrhoea (χ2,P < 0.001).
Cryptosporidiosis is associated with age and immunity, and
is commonly seen in calves less than 1 month of age [27, 29,
30]. In this study, the majority of the diarrhoeic calves
(79.38%) were less than one month of age and 53 (68.83.%)
of these were positive for Cryptosporidium parvum infection.
The infection was detected in 34 (50.75 %), 15 (35.71 %), 14
(25.45 %), 10 (14.71 %) and 9 (13.24 %) of calves in the age
groups; 1-10, 10-20, 20-30, 30-45 and 45 < days old, respect i v e l y. Indeed, C ryptosporidium parv u m infection was most
commonly detected in 1-10 days of age calves than that of the
other age groups (Table II). The high prevalence of
Cryptosporidium parvum in 4-10 days of age calves is similar to previous reports [33, 42]. The occurrence of high infection rates in this category may be attributed to the immatured
immunity.
Cryptosporidiosis can be life threatening in immunocompromised calves [30]. During this study, severe diarrhoeic
and dehydrated 5 calves died. These animals were average 7
days old, early born and not sufficiently feed with colostrum.
C. parvum were detected in 3 death calves. The other calves
may be died due to the other infectious agents.
During this study, 62 stool specimens were positive, and
203 stool specimens were negative by the modified acid-fast
staining. These results were compared with the results obtained by the Cryptosporidium parvum ELISA. All the positive
acid-fast specimens were also positive by the
Cryptosporidium parvum ELISA (Table I). 218 of 238 faecal
samples by ELISA were negative, althought all of them gave
negative results by Modified acid-fast staining method (Table
I). Twenty (8.40 %) negative acid-fast specimens were moderately positive by the C ryptosporidium parv u m ELISA.
According to these findings; sensitivity, specificity of the C.
parvum ELISA and compatibility with the modified acid-fast
staining method were determined as 100%, 91.60%, and
93.33 %, respectively. Thus, the C ryptosporidium parv u m
ELISA appears to be more sensitive and specific than the
acid-fast test. These findings support the reports of others
with this technique [19, 22, 37].
Acid-fast staining detected C. parvum in 51 of 62 ELISApositive faecal samples in diarrhoeic calves. In non diarrhoeic calves, 11 of 20 faecal samples gave positive results
by acid-fast staining, but all of them were positive by ELISA.
The specificity of ELISA was higher than diarrhoeic calves
in non-diarrhoeic calves.
The present survey showed that C ryptosporidium parv u m
infection was commonly seen in 4 to 30 days old and diarrhoeic calves, and although the compatibility of both of the
techniques was quite high, ELISA was more sensitive and
specific than the modified acid-fast stained technique.
Further studies are needed determining the concomitant
infections and the other factors in aetiology of neonatal calf
diarrhoea.
Acknowledgements
This work was supported by The Coordination of Scientific Research
Projects, University of Selcuk (No. of Project: 2001/141).
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