AL-Qadisiya Journal of Vet.Med.Sci. Vol./10 No.

/1 2011

Detection of Fascioliasis in sheep and cattle by using of

ELISA technique
A.M.A. Al-Khafajy
Coll. of Vet Med./Univ. of Al-Qadissiya
Abstract
The aim of present study detection of Fascioliasis in sheep and cattle immunologically
in dewania city by using ELISA test. The results showed high percentage of seropositive
sheep and cattle as 76.7% and 74.5% respectively .
The results of this study revealed an association between the percentage of infected animals
and age , so the cattle with age more than and less than one year give 82.3% and 17.6%
respectively while the sheep in the same above ages recorded 44.1% and 55.8% respectively .
Introduction
Bovine fasciolasis caused by the weeks . Adults appear in the bile ducts and
digenic trematoda Fasciola hepatica is a start to lay eggs (2).Liver damage and
world-wide parasitic disease common in acute disease especially in sheep are
ruminants, the two-host life cycle parasite caused by migrating immature parasites ,
is classically found in farms where all chronic disease occurs in cattle during the
conditions for the survival and the biliary phase ,the disease hampers
multiplication of the snail intermediate zootechnical characteristics , decrease milk
host (Lymnea truncata) are fulfilled. This yield weight loss , intermittent diarrhea.
snail is mainly found in damp meadows ( anemia and infertility problems , acute
watering-places , brooks , springs , ..) distomatosis of the sheep is characterized
(1).Fasciola egg shedding occurs with by anemia and sometimes sudden mortality
feces . Hatching follows in water and and chronic distomatosis by anemia ,
gives rise to infectious metacercariae fixed reduction of the dairy production .
on a plant holder, once the metacercaria reduction of the average daily profit and
are ingested by a ruminant, young flukes oedemas (3).This study aimed to detect of
migrate through the liver to reach bile the seropositive sheep and cattle of
ducts. The prepatent period is 8 to 10 facsiolasis in Diwaniya .
Material and Methods
Ninety four blood samples were the test serum , the conjugate remains
collected from sheep and cattle. bound to the microwell that contains the
Collection of samples:The cypress antigen and the enzyme catalyses the
Fasciola hepatica test uses 96 – well transformation of the colourless
microtitration plates sensitized by a chromogen into a pigmented compound.
monoclonal antibody specific to one The intensity of the resulting blue colour is
protein of Fasciola hepatica. This antibody proportional to the titer of specific
is used to trap the protein as well as to antibody in the sample the signal read off
purify it from lysate of the parasite.The test the negative control microwell is
blood sera are diluted in the buffer for subtracted from that of the positive
dilution . then incubate and washed. Then microwell sensitized by the antigen. The
the conjugate- a peroxidase labeled anti- interpretation of the results is done by
ruminant IgG1 monoclonal antibody-is comparing the signals of the samples
added to the wells (4).The plate is then serum with those of the positive controls.
incubated a second time at room Material :
temperature (18-24°c) , washed again and Microplates : Two 96 –well microtitration
the enzyme's substrate hydrogen peroxide plates . The columns are sensitized by the
and the chromogen tetramethylbenzidine Fasciola hepatica antigen that is captured
(TMB) were added.If specific Fasciola by a monoclonal antibody .
hepatica immunoglobulins are present in
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Washing solution : one 100 ml bottle of of its contents by flipping it over

20x concentrated washing solution . the sharply above a sink . Tap the
solution crystallizes spontaneously when microplate upside down against a
cold. If only part of the solution is to be piece of clean absorbent paper to
used, bring the bottle to room temperature remove all the liquid. Fill the used
until disappearance of all crystals. Mix the wells with the washing solution using
solution well and remove the necessary a squeeze bottle or by plunging the
volume. Dilute the buffer 1:20 with plate in a vessel of the right
distilled or dematerialized water . store the dimensions. Then empty the wells
diluted solution at 4°c . once more by turning the plate over
Dilution buffer: One 50 ml bottle of 5x above a sink .Repeat the entire
concentrated buffer for diluting the blood operation two more times, taking
sera, milks and conjugate . the bottle's care to avoid the formation of
contents are to be diluted with distilled or bubbles in the microwells. After the
demineralised water. this solution will plate has been washed three times go
keep at 4°c for at least 3 months on to the next step.
Conjugate : one bottle of anti-bovine 7. We diluted the conjugate 1:50 in the
immunoglobulin- peroxidase conjugate . buffer for dilution added 100 µl of
Positive reference : One bottle of Fasciola the dilute conjugate solution to each
hepatica positive serum . well. incubate at (18-24°c)
Chromogen solution:One 2 ml drop- temperature for 1hour.
dispenser bottle of the chromogen 8. Washed the plate as described in step 8
tetramethylbenzidine. Store at 4°c. above .
Substrate solution : One 30 ml bottle of 9. Prepared the indicator solution
the hydrogen peroxide substrate solution extemporaneously as follows :
Store at 4°c. Added 12 drops of chromogen to 9.5
Stopping solution : One 15 ml bottle of ml of the substrate solution . Mixed
the 1 M phosphoric acid stop solution . thoroughly , and then applied to the
Methods : plate immediately in volumes of 100
1. We brought all the reagents at room µl per microwell . At the time of
temperature ( 18-24°c) at least half distribution of the chromogen-
an hour before use. substrate mixture on the plates the
2. Removed the microplate from its solution must be completely
wrapper. colorless.
3. Placed 1ml aliquots of the dilution 10. Incubated for 10 minutes at room
solution, prepared as instructed in the temperature(18-24°c) this time is
"Compsition of the Kit " section, in 5 given as a guideline only , for in
ot 10 ml hemolysis tubes. Added 10 some circumstances it may be useful
ul of the serum sample to each of to lengthen of shorten the incubation
these tubes and shaked briefly on a time
mechanical agitator. Proceed in the 11. Added 50 µl of stop solution to each
same manner for the positive serum . microwell .
4. Added 100 ml aliquots of the 1:100 12. We readed the optical densities in the
diluted sample 2 in wells c1 and c2 microwell using a plate reader and a
etc .. 450 nm filter . results must be read
5. Incubated the plate at room trmperature fairly soon after the stopping solution
(18-24°c) for one hour. has been added since the chromogen
6. Rinsed the plate with the washing may crystallice in wells with strong
solution, prepared as instructed in signals and thereby distort the date
the" Composition of the Kit " section (4).
, as follows : Empty the microplate

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Results
The results of the present study seropositve animal and their age group so
showed that seroposative cattle and sheep the rate of infection was increased in cattle
were (74.5%) and (76.7%)respectively with ages more than and less than one year
while the negative results of cattle and which recorded (82.3%) and (17.6%)
sheep were (25.4%) and (23.2%) respectively table (2).also in sheep and in
respectively table (1).The results showed the same way revealed (44.1%) and
also there were relationship between (55.8% ) respectively table ( 3 ) .

Table ( 1 ) the percentage of seropositive animal of F . hepatic in sheep and cattle

sample Serum N. + % - %
Cattle 51 38 %74.5 13 %25.5
sheep 43 33 %76.7 10 %23.3

Table ( 2 ) the relationship between seropositive cattle and age group .

Age of Sever Moderate Positive Suspected Total
0
cattle infection +++ infection ++ + +\- N. %
cattle ≤ 1
0 1 3 3 2 9 17.6%
year
cattle > 1
8 7 11 5 11 42 82.3%
year
51

Table ( 3 ) the relationship between seropositive sheep and age group .

Age of Sever Moderate Positive Suspected Total
0
sheep infection +++ infection ++ + +\- N. %
Sheep ≤ 1
3 6 5 3 7 24 55.8%
year
Sheep > 1
7 2 6 1 3 19 44.1%
year
43

12

10

8 +++
++
6
+
4 +\-
0
2

0
cattle ? 1 cattle ? 1
year year

Fig ( 1 ) the relationship between seropositive cattle and age group.

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7
6
+++
5
++
4
+
3
-
2 0
1
0
Sheep ? 1 Sheep ? 1
year year

Fig ( 2 ) the relationship between seropositive sheep and age group.

Discussion
F . hepatica is a wordwide parasitic after 7 – 11 weeks since the beginning of
disease common in sheep and cattle infection, it is possible to find parasite egg
.Diagnosis of F.hepatica in cattle can only in feces , it have been showen that patients
performed after 8 -10 weeks by ingestion animals liver containing parasite
coprological examination of fecal material egg and produce false positive result in
however sometimes even repeated fecal stool microscopic examination ( pseudo
examination cannot identify any F . fascioliasis ) ( 5 ) .So one of definitive
hepatica infection due to lack of diagnosis is ELISA test the employment of
sensitivity of this method .The result of ELISA test for diagnosis of fasciolasis
this study showed high perevalance of make possible after 2 - 4 weeks post
seropositive sheep and cattle 76.7% & infection and the sensitivity of ELISA is
74.5% respectively which were agreement about 98% ( 6 ) .(7) they reported that the
slitly with (4) who founded 90% positive result in ELISA and fecal
seroposative result . this results higher than examination were 86% and 51%
our results may be due to variation in respectively and that agreement with our
geographical area and climatic condition study .Also the ability to diagnose and treat
and presence of low lying area which is of infection is high advantage of the
important to complete the life cycle.(4) ELISA because it minimize tissue damage
mention that the sensitivity and spescifity in the infected animals caused by immature
of ELISA in detection of fascioliasis in flukes as they migrate through the liver ,
sheep and cattle were 90% and 80% early treatment prevent shedding of egg in
respectively , It has been conducted that feces . thus contributing to effective
ELISA is an authentic test for diagnosis of management by reducing the rate of
infected animals with F . hepatica .The infection ( 8 ) .The result of this study
only confirmatory test for diagnosis of related to age group and seropositivity
fascioliasis is finding the parasite egg in relationship showed that cattle more than
feces or duodenal lavage of infection one year and less than one year age group
patient , however other test like 82.3% and 17.6% respectively while
complement fixation test and skin test are sheep more than one year and less than one
also used for diagnosis of F . hepatica but year 44.1 % & 55.8% respectively . ( 9 )
they character by lower sensitivity than recorded that detection of F . hepatica in
above confirmatory test , for example only human by ELISA were all positive cases
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occurred in children between the age of 9 – they are responsible for taking herds of
12 years old because children may become sheep to grazing field and that in
incontact with F . hepatica metacercaria agreement with our study as infection
by drinking contaminated water or eating occur when there is suitable condition and
vegetables growing near ditches because suitable chance to complete the life cycle .
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ELISA
74.5% 76.7%
17.6% 82.3%
55.8 % 44.1 %

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