Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
703
ISOLATION OF Cryptosporidium OOCYSTS FROM
SLAUGHTERED BROILER CHICKEN AND EXPERIMENTAL
INFECTION IN CHICKS
Atheer Kareem Kadhim , Mohammed Thabit Salih Al-Zubaidi
Department of Parasitology, College of Veterinary Medicine, University of Baghdad
Key Words: Cryptosporidium, Oocyst,, Chicks.
Corresponding Author: mohabood24@gmail.com
ABSTRACT
The aim of current study was investigate the isolation of Cryptosporidium oocysts
from 93 fecal samples of infected slaughtered broiler chicken in local markets of
some areas in Baghdad city, during the period from the beginning of November 2017
to end of April 2018. In this study three diagnostic techniques used, flotation by
Sheather's sugar solution, isolation and measuring of Cryptosporidium oocysts (by
ocular micrometer). For conform that the isolated species of the parasite from infected
cases belong to the Cryptosporidium baileyi, experimental infection done in, broiler
chicken chicks aged one week to study the histopathological lesion in infected organs
(intestinal, trachea, and bursa of Fabricius). The results of calibration of isolated
oocysts, showed that the mean of measurement size of Cryptosporidium oocysts was
6.2x 4.7 micrometers.
INTRODUCTION
Cryptosporidiosis is considered as a zoonotic parasitic disease caused acute
enteritis to the different types of animals and humans, the parasite detected by Jackson
Clark in 1895 in intestinal mucosa of rat (1). Tyzzer in 1910, called Cryptosporidium
on this parasite which it is a Greek term means hidden spores, because the difficulty
of diagnosing the four crescent sporozoite in the oocyst, which differ from other types
of coccidia, they do not contain Sporocyste (2). The infection occurs through
ingestion of mature oocysts with contaminated food and drinking water. The
importance of this parasite was increased in 1955 in poultry after the spread of it in
turkey fields, and the parasite caused high morbidity and mortality rates in birds in
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
704
Romania, with economic losses, then began give attention to the classification of
Cryptosporidium, and its species in the various vertebrates' hosts (3, 4, 5).
The parasite recorded for the first time in Iraq, in broiler chickens in Baghdad city
by Al-Attar and Abdul Aziz (6) which seen the parasite with infection rate 8.8%, and
isolate the Cryptosporidium from the bursa of fabricius, without any clinical signs.
The species of parasite which infect birds including poultry, are three species,
Cryptosporidium galli, which their oocysts measuring 8.3 × 6.3 micrometers, which
affects the real stomach Proventriculus of chickens and birds, C. meleagridis, which
their oocysts measuring 5.2 × 4.6 micrometers, that infects the small intestine of the
turkey and can infect humans, and C. baileyi which their oocysts measuring 6.2 x 4.6
micrometers, it affects the respiratory tract, small intestine, kidneys, bursa of fabricius
and cloaca, in poultry (2, 7, 8, 9).
The current study was designed to determine the causative species of
cryptosporidiosis in slaughtered broiler chickens from calibration of oocysts and study
the histopathological changes in site of infection (small intestine, trachea, and bursa of
fabricius) in experimentally infected broiler chicken chicks.
MATERIAL AND METHODS
Samples collection
Fecal samples collected from infected slaughtered broiler chicken in local markets
of some areas in Baghdad city, during the period from the November 2017 to end of
April 2018. The fecal samples were tacked directly from intestine content in a clean
plastic container (50ml size) and given sequential numbers. All information for the
animal included date of sampling, case history and clinical signs (if found) and the
name of region were recorded on containers of the samples, and these samples were
transported in refrigerated bag to the laboratory of parasitology ̸ College of Veterinary
Medicine-University of Baghdad.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
705
Laboratory examination
1. Floatation using Sheather's sugar solution
Fecal samples (2 g) were mixed will with 20 ml distilled water in a clean flask.
The mixture was then filtered through four layers of clean gauze to remove the fecal
debris. Afterwards, the suspension was collected in test tubes and was centrifuged
(2700 round/ minute) for 15 minutes. Then the supernatant was discarded, and few
amount of the suspension was kept with the sediment. Sheather's sugar solution (9 ml)
was added to test tubes and mixed well then centrifuged with the same rounds and,
then the surface layer was separated which contain the oocysts. One drop of the
surface layer was withdrawn by Pasture pipette and run on the a clean glass slide then
covered with the cover slip and scanned under light microscope ×40 and ×100 (10).
2-Ocular micrometer was used to determine the oocysts calibration or measurements
according to (11).
3-Isolation and counting of oocysts for experimental infection:
Fecal samples (5 g) was mixed with 20 ml distilled water in a clean flask then
was filtered through four layers of clean gauze to remove the fecal debris. After that,
the suspension was collected in test tubes and were centrifuged (2700 round/ minute)
for 15 minutes.
a-The supernatant was discarded, and few amount of the suspension was kept with the
sediment and mixed well with Sheather's sugar solution (9 ml) then centrifuged with
the same procedure mentioned previously.
b-All the supernatant was collected then diluted with distilled water at a rate of 1:10
to prevent the effect of Sheather's sugar solution on the oocysts.
c-The diluted solution was sedimented by centrifugation (2700 round/ minute) for 15
minutes.
d-The sediment was collected in a clean test tube and added an equal volume of 1%
sodium Hypochlorate Solution, mixed well and left for 5 minutes and then added a
double volume of distilled water gradually on the wall of the test tube by a syringe of
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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1 ml size. A clear layer was separated above the solution, pulled by a Pasteur pipette
and moved to other test tubes.
e-The clear layer washed by centrifugation several times until the disappearance of
the chlorine smell from the solution.
f-The oocysts which isolated by this method from infected cases storage in potassium
dichromate solution v/v, and the number of oocysts calculated in 1 ml of suspended
oocysts solution by using haemocytometer slid which used for white blood cells
calculation in the eight squares of the two chamber of this slid, then the total number
of oocysts per 1ml calculate according to the following equation: Number of oocysts
in 1 ml = (1000 x calculated oocysts number) / 8. (12, 13, 14) (Fig: 1).
Fig: (1). Cryptosporidium oocysts calculated by haemocytometer x40
4-Experimental Study
The experimental infection was conducted to study the histopathological effects of
parasite in small intestine, trachea and bursa of fabricius of experimentally infected
broiler chicken chicks, and observe the developmental stages of the parasite in these
infected organs and record the pathogenic lesions to identification of the parasite type,
by presence of these stages of it in the specific site of infection.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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For the experimental infection (12) chicks of broiler chickens (Rose type) aged one
week and their weights 100-120 g were used. These chicks divided into two groups,
each group consisted of six birds. The first group G1 infected orally with one ml of
suspended oocysts solution, which containing 1000 oocysts (15, 16), while the second
group G2, act as control group, which inoculated orally with one ml of normal saline
solution. The two groups were placed inside cages within a typical poultry breeding
hall, which prepared the appropriate heat and ventilation. All chicks groups before
experiment examined their feces for detection of Cryptosporidium oocysts to ensure
that the parasite was not presence.
Examination of Experiment infected chicks
Fecal samples of all two groups chicks were examined after 3 days post infection
(PI), to confirm the incidence of the infection and the initiation of oocysts shedding,
the control groups were monitored for the duration of the experiment and the clinical
signs of the infected chicks were observed.
Histopathological Examination of infected chicks
Three chickens from each groups of experiment were killed on the 9th day PI.
The second group remained after 14 days PI. Tissue samples were taken from trachea,
small intestine, and bursa of fabricius, and placed in formalin solution 10% for 24
hours for fixation, and histopathological sections were made according (17) for
histopathological examination.
RESULTS
The result of study recorded shape and calibration of Cryptosporidium oocyst,
through using sheather's sugar solution the Cryptosporidium oocysts appear
transparent oval shapes, surrounded by a bright halo and contain undistinguishable
four sporozoites (Fig: 2). The results of calibration of isolated oocysts, showed that
the mean of measurement size of parasite oocysts was 6.2x 4.7 micrometers (Fig: 3)
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
708
Fig: 2. Cryptosporidim oocysts isolated by sheather's sugar solution x100
Fig: 3. Cryptosporidim oocysts calibrated with ocular micrometer x100
The Results of Experimental Study:
The two groups of chicks of experiment, starting examined their feces in the 3th day PI.
The shedding of oocysts in infected group G1, started in 4th days PI, while the first clinical
signs, diarrhea, appear in the 6th days PI, than followed with other clinical signs such as,
dullness, increased water consumption, anorexia, while there is not found any clinical signs
on the control group G2.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Gross Lesion
The experiment infected and not infected or control group chicks divided in two groups
for killing, to seen the post mortem changes and histopathological lesion in some internal
organs, (intestine, trachea and bursa of fabricius), first killed group include three chicks from
G1and G2 in the 9th day PI, while second group also include the remained 6 chicks in the two
groups killed in 14th days PI. There is sever changes seen in the G1, while no post mortem
changes in G2 chicks, these changes including redness and thickening of intestinal wall with
yellow feces also thickening in air sacs.
Histopathological lesion in organs of experimentally infected chicks:
The result of study showed sever histopathological lesion in affected organs, include
small intestine, trachea and bursa of fabricius, in G1chicks, while without any
histopathological changes in G2 chicks, which represented the control group.
Small Intestine Lesions
The intestinal histopathological lesions of the experimentally infected chickse showed
severe epithelial distraction accompanied with widespread of necrosis leding to loss of
mucous membranes with the accumulation of debris cell necrosis with hyperplasia of goblet
cells and presence of developmental stages of the parasite on the upper surface, as well as
infiltration of sub mucosa layer with mononuclear cells (macrophages and plasma cells) in
chicks of G1, after 9days and 14days PI (fig 4, 5) respectively, while there is no changes in
intestine of G2 chicks (fig 6).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Fig; 4. Cross section of small intestine of G1 chicks after 9 days PI, widespread of
necrosis with epithelial distraction accompanied which led to loss of mucous
membranes(Blue arrow), with a number of developmental stages of the parasite
(Black arrow), H&E stain X20.
Fig: 5. Cross section of small intestine of G1 after 14 days PI, severe epithelial
distraction of mucous membranes with the accumulation of debris cell necrosis with
widespread necrosis areas (Blue arrow), with a several developmental stages of the
parasite (Black arrow), H&E stain X20.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Fig: 6. Cross section of the small intestine of control group G1 shows the normal
appearance of the epithelial layer, H&E stain, x 20
Tracheal Lesions
The lesion of tracheal cross section of G1 chicks after 9 days PI recorded several
developmental stages of the parasites appear oval or round structures on the upper
surface of the epithelium, with deciliation of the mucous epithelium (fig: 7), while
sever pathological changes observe in chicks after 14days PI include sever trachietis
and proliferated of epithelial layer, with mucinus materials on the surface of the
tracheal epithelium and severe necrosis accompanied by debris and infiltration of
inflammatory cells as well as the presence of mucosal hyperplasia with goblet cell
hypertrophy and presence of, with subcutaneous cell infiltration with plasma cells and
heterophils as well as the proliferation of mononuclear cells (fig: 8), while there is no
any pathological lesions in trachea of G2 chicks (fig: 9).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig: 7. Cross section of Trachea of G1 after 9 days PI, showes of round or oval structures
of developmental stages of the parasites (Black arrow),, with deciliation of the mucous
epithelium appear on the upper surface of the epithelium(Blue arrow), with infiltration of
inflammatory cell (Red arrow) H&E stain X20.
Fig: 8. Cross section of Trachea of G1 chicks after 14 days PI, showed the parasite stages
on the upper surface with severe necrosis accompanied by debris (Black arrow), and wide
deciliation of the mucous epithelium (Blue arrow), and infiltration of inflammatory cells
(Red arrow) as well as the presence of mucosal hyperplasia with goblet cell hypertrophy,
with presence of mucinus materials on the surface of the tracheal epithelium H&E stain
X20
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig: 9. Cross section of trachea of G2 control group shows the normal appearance of
the epithelial layer, H&E stain, x 20
Bursa of Fabricius Lesions
experimentally infected G1chicks recoded some pathological lesion in bursa of
fabricius after 9 and 14 days PI, include found some developmental stages of the
parasite on the upper surface of the bursa and hypertrophy and hyperplasia of bursal
epithelial cell, and, infiltration of mononuclear cells (fig: 10, 11) while there is no
histopathological lesions in bursa of fabricius of G2 chicks (fig: 12).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig: 10. Cross section of bursa of fabricius of G1 chicks after 9 days PI, seen some
developmental stages of the parasite on the upper surface (Blue arrow), and
hypertrophy and hyperplasia of bursal epithelial cell (Blue arrow), as well as
infiltration of inflammatory cells (Red arrow), H&E stain X20.
Fig: 11. Cross section of bursa of fabricius of G1 chicks after 14 days PI, seen some
developmental stage of the parasite on the upper surface (Blue arrow), and
hypertrophy and hyperplasia of bursal epithelial cell (Blue arrow), as well as
infiltration of inflammatory cells (Red arrow), H&E stain X40.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig: 12. Histopathological Cross section of the bursa of fabricius of G2 control group
shows the normal appearance, and without presence of any developmental stages of
the parasite H&E stain, x 10
DISCUSSION
The results of current study by using sheather's sugar solution the oocysts of
Cryptosporidium appear transparent oval shapes, surrounded by a bright halo and
contain undistinguishable four sporozoites, this result agreed with (2, 18, 19), who
they found same characters of the parasite by using different traditional diagnostic
methods.
The results of calibration of isolated oocysts, showed that the mean of
measurement size of it was 6.2x 4.7 micrometers, this result of oocyst calibration
ryptosporidium oocysts, showed that the measurement size of it was, which resemble
the global size of C.bailey, (6.1x 4.5) micrometers. This result agrees with (1, 2, 15,
16, 20) who recorded same measurement size of Cryptosporidium baileyi oocysts in
poultry .
Experimental Study
The results of experimentally infected broiler chicken chicks shows sever
pathological changes in organs (intestine, trachea, and bursa of fabricius) of G1chicks,
while without any pathological lesion in G2chicks or control group. The examination
of their feces starting in the 3th day PI, and the shedding of oocysts in infected group
G1, started in 4th days PI, while the first clinical signs, diarrhea, appear in the 6th days
PI, than followed with other clinical signs such as, dullness, increased water
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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consumption, anorexia, while there is not found any clinical signs on the control
group G2, The study recorded some gross lesion in the experiment infected groupG1
chicks, these post mortem changes included redness of intestinal wall with yellow
feces also thickening in air sacs, while there is not found any gross lesion in control
G2 group, this results agreed with (15, 16, 21, 22, 23), which recorded same clinical
signs (dullness, increased water consumption, anorexia) , gross lesion (like redness
and thickening of intestinal wall with yellow feces), and pathological lesion in
intestine, trachea and bursa.
Histopathological lesion in organs of experimentally infected chicks:
Several experimentally infection studies (15, 16, 22, 23, 24) have been made by
researchers to prove the isolated species of Cryptosporidium parasite through study
the site of infection and pathological changes.
There was sever histopathological lesion shows in the result of study in the affected
organs, include small intestine, trachea and bursa of fabricius, in G1chicks, while
without any changes in chicks control group.
The intestinal histopathological lesions of the experimentally infected chickse showed
severe epithelial distraction accompanied with widespread of necrosis leading to loss
of mucous membranes with the accumulation of debris cell necrosis with hyperplasia
of goblet cells and presence of developmental stages of the parasite on the upper
surface, as well as infiltration of sub mucosa layer with mononuclear cells
(macrophages and plasma cells) in chicks of G1, after 9days and 14days PI , while
there is no changes in intestine of G2 chicks.
These results agreed with several studies which recorded same pathological
lesions in small intestine of infected poultry and birds with cryptosporidiosis, and
confirmed the occurrence of similar lesions attributed to C.baileyi infection on the
peaks of intestinal villi, and that the infiltration of inflammatory cells in the layers of
the intestine is only a response caused by the extensive damage and destruction of the
epithelial cells.(9, 15, 16, 21, 22, 23, 24,).
The results of tracheal cross section lesion of G1 chicks after 14 days is sever
more than chicks killed after 9 days PI which include found of several developmental
stages of the parasites appear oval or round structures on the upper surface of the
epithelium, with deciliation of the mucous epithelium , also seen trachietis and
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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proliferated of epithelial layer, with mucinus materials on the surface of the tracheal
epithelium and severe necrosis accompanied by debris and infiltration of
inflammatory cells as well as the presence of mucosal hyperplasia with goblet cell
hypertrophy and presence of, with subcutaneous cell infiltration with plasma cells and
heterophils as well as the proliferation of mononuclear cells, while there is no any
pathological lesions in trachea of G2 chicks. This result agreed with.(9, 15, 16, 22, 23,
25, 26, 27) who they found same pathological lesion.
.
The result of experimentally infection on Bursa of Fabricius in G1chicks recoded
some pathological lesion after 9 and 14 days PI, include found some developmental
stages of the parasite on the upper surface of the bursa and hypertrophy and
hyperplasia of bursal epithelial cell, and, infiltration of mononuclear cells, while there
is no histopathological lesions in bursa of fabricius of G2 chicks. These pathological
lesion agrees with Goodwin and Blaghum et al., (28) , Rhee et al., (29) in chicken and
Alex and Marcelo, (9), in birds, Al-Zubaidi et al.,(16) and Al-Khayat, and Al-
Zubaidi, (23) in broiler chicken chicks which recorded same lesion in bursa of
fabricius of infected chicken and birds with cryptosporidiosis, include found of some
developmental stages of the parasite on the upper surface of the bursa and
hypertrophy and hyperplasia of bursa epithelial cell.
CONCLUSION
The current study proved that the isolated species of Cryptosporidium from
slaughtered broiler chicken according to the global and local calibration or
measurements of oocysts and the histopathological lesion in (intestine, trachea and
bursa of fabricius) of natural infected chickens belong to the Cryptosporidium baileyi.
ACKNOWLEDGMENT
The authors are very grateful to Assistant Professor. Dr. Zainab. I. Ibrahim in
department of pathology for the examination of histopathological sections.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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