Prevalence of Cryptosporidium Infection in Cattle in Isfahan, Iran
Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
randomly selected from 30 regions in Isfahan, Iran, were examined to investigate the prevalence of Cryptosporidium infection. Cryp-
tosporidium oocysts were identified by using sheather’s concentration and the Ziehl–Neelsen modified staining technique in 30 of 480
cattle ranging from less than 6mo of age to older than 1yr of age. Infected animals were found in 86.6% (26/30) of regions investigated.
Overall prevalence of infection was 6.25%, but higher in cattle less than 6mo of age (10.8%) and this was statistically significant
(Po0.05). Both sexes of cattle were infected with Cryptosporidium parasites, but prevalences were higher in diarrheic (56.7%) than in
non-diarrheic (39%) cattle. Cryptosporidium appears to be prevalent in cattle in Isfahan.
Key Words. Cattle, Cryptosporidium, epidemiology, prevalence.
Cryptosporidium are commonly identified as intestinal pathogens in humans and animals. Fecal samples from 480 cattle
and prevalence increase in immunodeficient humans, such as
those with AIDS, as well as in neonates of some mammal spe-
cies, such as ruminants. At present, the control of cryptosporid-
iosis is difficult, bearing in mind the lack of a clearly effective
drug therapy and the resistance of oocysts to environmental fac-
tors and chemical agents (O’Donoghue 1995; Quilez et al. 1996).
In cattle, Cryptosporidium parvum is recognized worldwide as
one of the most common enteropathogens causing neonatal diar-
rhea (Angus 1990). Neonatal enteric infections, especially during
the first month of life, have a negative effect on the growth rate of
calves which may results in significant economic loss.
In the majority of cases, cryptosporidiosis is diagnosed by
observing the oocysts in the feces by specialized techniques
(Casemore 1991). Over 20 methods of staining have been described
to enable identification of oocysts (Weber et al. 1991). However,
the microscopic methods may sometimes fail in asymptomatic
infections: infections with less than 50,000oocysts/g feces consti-
tute the detectable limit (Pilarczyk and Balicka-Ramisz 2002).
Cryptosporidium infection plays an important role in animal
production as this parasite can negatively influence growth, impair
feed conversion, and reduce milk production (Esteban and Ander-
son 1995; Olson et al. 1995). Cryptosporidium infections of cattle
may also act as an infection source for humans through direct
contact with contaminated feces or water. Pasture run off has also
been implicated with outbreaks of cryptosporidiosis in humans
Cryptosporidium infections in cattle in Iran are limited, although
infection has been reported in some regions, the aim of this study
was to determine the prevalence of Cryptosporidium infection in
cattle in Isfahan Province, Iran, and to assess whether cattle of
different ages had different infection rates. These data will enable
assessment of the risk of this disease to cattle and humans in this
region of Iran.
NTERIC cryptosporidial infection is a well-recognized cause
of diarrhea in humans and animals. Both severity of infection
MATERIAL AND METHODS
Faecal sampling. Between October 2003 and April 2004,
stool specimens were collected from 480 cattle randomly select-
ed from 30 regions in Isfahan province, Iran. Animals were clas-
sified based on their age range: less than 6mo of age (83), between
7–12mo of age (87), and greater than 1-yr-old (310). On average,
10% of the total number of animals in each region were sampled.
For each animal, a single fecal sample was taken from the rectum
by using a disposable plastic bag (Farid, Co. Inc., Isfahan, Iran). In
calves with diarrhea, disposable plastic containers were used and
the feces classified according to consistency: watery or formed.
The fecal samples were transported to the laboratory on the day
and preserved in 2.5% (w/v) potassium dichromate solution and
refrigerated until processed.
Cryptosporidium oocyst detection technique. The feces were
vortexed to give a homogenous suspension and approximately 50g
of the preserved feces were weighed out. Then, 20g were filtered
through gauze, washed with phosphate-buffered saline (PBS), and
compressed to yield approximately 7ml of filtrate. The filtrate was
layered over 7ml of 1M sucrose solution (specific gravity 1.13)
and centrifuged for 5min at 800g on a fixed rotator centrifuge in
order to concentrate the oocysts at the sucrose–water interface. The
filtrate layer (upper layer) and the interface were removed
and centrifuged for 5min at 800g to pellet the particulate materi-
al and oocysts. The supernatant was decanted and discarded and the
pellet was suspended in 1ml of PBS. A 15ml sample of the sus-
pension spread on a clean microscope slide and then air dried at
room temperature. The slides were stained in carbol fuchsin
(Merck, Darmstadt, Germany) for 10min, rinsed in tap water for
2–3min, and decolorized for 2–3min in acid alcohol (1% [v/v] HCl
in 70% ethanol). Slides were then rinsed in tap water for 2–3min,
counterstained with 0.3% (w/v) methylene blue for 30s, rinsed in
tap water for 5min, and air dried (Mtambo et al. 1997). Using a
light microscope, the entire smear was examined at 100? magni-
fication for Cryptosporidium oocysts. Ocysts appeared as spherical,
red- to pale-pink shapes, measuring ?5mm diam.
As we and others (Maldonado-Carargo et al. 1998) have found
that some fecal samples contain small numbers of non-Cryptos-
poridium particulates that pick up the acid-fast stain, there is a risk
of false positive identifications. In order to minimize false positive
tests, a sample was recorded as positive only if there were ?5
oocysts per smear; otherwise, it was recorded as negative. Al-
though this diagnostic approach helps to minimize false positives,
it may have increased slightly the risk of false negatives, espe-
cially if a calf was just starting or ending shedding.
Statistical analysis. Data were computed using Epi info Ver-
sion 6 statistics system (Center for Disease Control). A w2test was
used to compare the differences in prevalences of Cryptosporid-
ium oocysts between age-groups of cattle at 5% level of signifi-
cance. The 95% confidence intervals on overall prevalences were
The overall prevalence of cryptosporidial infection in cattle
was 6.25%. (30/480). Infected animals were found in 86.6% (26/
30) of the regions. Infection rates were significantly higher in
cattle less than 6mo of age (10.8%) than in the remaining age
Corresponding Author: Mehdi Azami, Department of Parasitology
and Mycology, School of Medicine, Isfahan University of Medical Sci-
ences, Isfahan, Iran—Telephone number: 198-311-556 3553; e-mail:
J. Eukaryot. Microbiol., 54(1), 2007 pp. 100–102
r 2006 The Author(s)
Journal compilation r 2006 by the International Society of Protistologists
groups (Po0.05; Table 1). The size (4.5–5.4 ? 4.2–5.0mm) and
shape of the oocysts identified in positive samples were consistent
with that of C. parvum (Fayer, Morgan, and Upton 2000).
Microscopic examination showed that the cattle of both sexes
were infected with Cryptosporidium: 6.28% (11/175) male ani-
mals and 6.22% (19/305) female animals were infected. There
was no statistically significant difference between these preva-
All 480 samples of known fecal consistency, either with or
without oocysts, were used to determine the relationship between
Cryptosporidium infection and diarrhea. Out of 480 samples, 30
(6.25%) were positive and 450 (93.75%) were negative for Cryp-
tosporidium oocysts. While 56.7% (17/30) of the oocyst-positive
samples were diarrheic (watery), only 39% (175/450) of the oo-
cyst-negative samples were diarrheic (watery; Po0.05).
Little is known of the prevalences of bovine or wildlife cryp-
tosporidiosis in Iran for comparison purposes. However, studies
on bovine cryptosporidiosis have shown prevalence rates from
1.61% to 14.59% in other regions in Iran (Azami, M., unpubl.
data). The prevalence of Cryptosporidium oocysts in cattle in the
present study falls within this range. Prevalences of Cryptospo-
ridium in Iran are typically lower than those reported in other
countries: 25% in calves in Poland (Pilarczyk and Balicka-Ramisz
2002); 35% of calves were reported as shedding in the United
States (Santin et al. 2004); 19–36% of cattle in Germany (Joachim
et al. 2003); 93% of cattle in the Japan (Uga et al. 2000); 23% of
109 adult cattle in Scotland were positive (Bukhari and Smith
1996); 13% of calves in Sweden (Kasprzak 1995); 20% of calves
in Canada (O’Donoghue 1995), 41% of calves in Czechoslovakia
(Quilez et al. 1996); and 19.7% of calves in Spain (Panciera,
Tomassen, and Gordner 1971). One notable exception was the
prevalence of 0.5% of 94 cattle sampled in Tanzania reported by
Kusiluka et al. (2005), a prevalence lower than even those report-
ed for Iran. Thus, in terms of risk of cryptosporidiosis, it appears
that it is lower in Iran than in other parts of the world.
The high prevalence of Cryptosporidium in young calves is
similar to previous reports that Cryptosporidium infection is more
common in newborn or unweaned calves (Castro-Hermida et al.
2002; Lefay et al. 2000). The occurrence of high infection rates in
this age category is attributed to poor immunity in newborn calves
and ease (Castro-Hermida et al. 2005; McCluskey, Greiner, and
Donovan 1995) of oocyst contamination through bucket-feeding.
In the current study, infection rates were statistically signifi-
cantly higher in diarrheic cattle. Thus, it can be concluded that
Cryptosporidium infection is associated with the occurrence of
diarrhea in these cattle. Other workers have also reported a statis-
tically significant association between cryptosporidial infection
and diarrhea (Pilarczyk and Balicka-Ramisz 2002; Sobieh et al.
1987; Uga et al. 2000). However, Snodgrass et al. (1986) and
Kaminjolo et al. (1993) found no statistically significant associ-
ation between infection and diarrhea, although cryptosporidia
were detected more frequently in diarrheic than in healthy calves.
The present study has clearly demonstrated that Cryptosporid-
ium is prevalent in Iranian livestock, although at lower levels than
in most other parts of the world. Nevertheless, measures to control
and prevent Cryptosporidium infection should be considered in
animal production facilities, both to increase productivity of the
animals and also to reduce the risk of human infection.
I wish to thank Dr. Fabio Tosini and Dr. Giovanni Widmer. This
study received financial support from the Department of Parasit-
ology,School ofMedicine, Isfahan University ofMedical Sciences.
Angus, K. W. 1990. Cryptosporidiosis in ruminants. In: Dubey, J. P.,
Speer, C. A. & Fayer, R. (ed.), Cryptosporidiosis of Man and Animals.
CRC Press Inc, Boca Raton. p. 83–104.
Bukhari, Z. & Smith, H. V. 1996. Detection of Cryptosporidium muris
oocysts in the faeces of adult dairy cattle in Scotland. Vet. Rec., 138:
Casemore, D. P. (ed.), 1991. Laboratory Methods for Diagnosing Crypto-
sporidium and Cryptosporidiosis. CRC Press Inc, Boca Raton, FL.
Castro-Hermida, J. A., Gonzalez-Losada, Y. A., Mezo-Menendez, M. &
Ares-Mazas, E. 2002. A study of cryptosporidiosis in a cohort of neo-
natal calves. Vet. Parasitol., 106:11–17.
Castro-Hermida, J. A., Pores, I., Poupin, B., Ares-Mazas, E. & Chartier, C.
2005. Prevalence of Giardia duodenalis and Cryptosporidium parvum
in goat kids in western France. Small Ruminant Res., 56:259–264.
Esteban, E. & Anderson, B. C. 1995. Cryptosporidium muris: prevalence,
persistency and detrimental effects on milk production in a dry lot dairy.
J. Dairy. Sci., 78:1068–1072.
Fayer, R., Morgan, U. & Upton, S. J. 2000. Epidemiology of Cryptospo-
ridium: transmission, detection and identification. Int. J. Parasitol.,
Joachim, A., Krull, T., Schwarzkopf, J. & Daugschies, A. 2003. Preva-
lence and control of bovine cryptosporidiosis in German dairy herds.
Vet. Parasitol., 112:277–288.
Kaminjolo, J. S., Adesiyun, A. A., Loregnard, R. & Kitson-Piggott, W.
1993. Prevalence of Cryptosporidium oocysts in livestock in Trinidad
and Tobago. Vet. Parasitol., 45:209–213.
Kasprzak, W. 1995. Wykrywanie koproantygenow w chorobach paso-
zytniczych [diagnosis of coproscopical]. Wiad. Parazytol., 41:19–24.
Kusiluka, L. J. M., Karimuribo, E. D., Mdegela, R. H., Luogu, E. J., Mu-
nishi, P. K. T., Mlozi, M. R. S. & Kambarage, D. M. 2005. Prevalence
and impact of water-borne zoonotic pathogens in water, cattle, and hu-
mans in selected village in Dodoma, rural and bagamoyo districts, Tan-
zania. Physics. Chemistry. Earth. Parts A/B/C., 30:818–825.
Lefay, D., Naciri, M., Poirier, P. & Chermett, R. 2000. Prevalence of
Cryptosporidium infection in calves in France. Vet. Parasitol., 89:1–9.
LeChevallier, M. W., Norton, W. D. & Lee, R. G. 1991. Occurrence of
Giardia and Cryptosporidium sp. in surface water supplies. Appl. Env-
iron. Microbiol., 57:2610–2616.
Maldonado-Carargo, S., Atwill, E. R., Saltijeral-Oaxaca, J. A. & Herrera-
Alonso, L. C. 1998. Prevalence of and risk factors for shedding of
Cryptosporidium parvum in Holstein Friesian dairy calves in central
Mexico. Prevent. Vet. Med., 36:95–107.
McCluskey, B. J., Greiner, E. C. & Donovan, G. A. 1995. Patterns of
Cryptosporidium oocysts shedding in calves and a comparison of two
diagnostic methods. Vet. Parasitol., 60:185–190.
Mtambo, M. M. A., Sebatwale, J. B., Kambarage, D. M., Muhairwa, A. P.,
Maeda, G. E., Kusiluka, L. J. M. & Kazwale, R. R. 1997. Prevalence of
Cryptosporidium sp. oocysts in cattle and wildlife in Morogoro region,
Tanzania. Perven. Vet. Med., 31:185–190.
O’Donoghue, P. J. 1995. Cryptosporidium and cryptosporidiosis in man
and animals. Int. J. Parasitol., 25:139–195.
Table 1. Prevalence of Cryptosporidium sp. infection according to the
Age rangeNumber of
AZAMI—CRYPTOSPORIDIUM INFECTION IN CATTLE IN ISFAHAN, IRAN
Olson, M. E., McAllister, T. A., Deselliers, L., Morck, D. W., Buret, A. G.,
Cheng, K. J. & Ceri, H. 1995. The effect of giardiasis on production in a
domestic ruminant (sheep) model. Am. J. Vet. Res., 56:1470–1474.
Panciera, R. J., Thomassen, R. W. & Gordner, F. M. 1971. Cryprosporidial
infection in a calf. Vet. Pathol., 8:479–484.
Pilarczyk, B. & Balicka-Ramisz, A. 2002. Prevalence of Cryptosporidium
sp. in western Pomerania. Elect. J. Polis Agricult. Univ. Anim. Hus-
Quilez, J., Sanchez-Acedo, C., Cacho, E-del., Clavel, A., Causape, A. C. &
Del-Cacho, E. 1996. Prevalence of Cryptosporidium and Giardia in-
fections in cattle in Aragon (Northeastern Spain). Vet. Parasitol.,
Santin, M., Trout, J. M., Xiao, L., Zhou, L., Greiner, E. & Fayer, R. 2004.
Prevalence and age-related variation of Cryptosporidium species and
genotypes in dairy calves. Vet. Parasitol., 122:103–117.
Snodgrass, D. R., Terzolo, H. R., Sherwood, D., Campbell, I., Menzies, J.
D. & Synge, B. A. 1986. A etiology of diarrhoea in young calves. Vet.
Sobieh, M., Tacal, J. V., Wilcke, B. W., Lawrence, W. & El-Ahraf, A.
1987. Investigation of cryptosporidial infection in calves in San Ber-
nardino Country, California. J. Am. Vet. Med. Assoc., 191:816–818.
Uga, S., Matsuo, J., Kono, E., Kimura, K., Inoue, M., Rai, S. K. & Ono, K.
2000. Prevalence of Cryptosporidium parvum infection and pattern of
oocyst shedding in calves in Japan. Vet. Parasitol., 94:27–32.
Weber, R., Bryan, R. T., Bishop, H. S., Wahlquist, S. P., Sullivan, J. J. &
Juranek, D. D. 1991. Threshold of detection of Cryptosporidium oocysts
in human stool specimens: evidence for low sensitivity of current di-
agnostic methods. J. Clin. Microbiol., 29:1323–1327.
Received: 05/07/06, 06/15/06, 09/16/06; accepted: 10/26/06
J. EUKARYOT. MICROBIOL., VOL. 54, NO. 1, JANUARY–FEBRUARY 2007