This document discusses diagnostic studies for Fasciola infection among cattle and buffaloes. It provides information on the taxonomy, morphology, and lifecycle of Fasciola parasites. It also discusses the epidemiology, pathogenesis, clinical signs, postmortem lesions, and economic impact of Fascioliosis. Methods for diagnosis discussed include coprological examination, ELISA serological testing, and Agar Gel Immuno-diffusion. The aims of the study presented are to determine prevalence of Fasciola infection using different diagnostic methods and compare the methods' accuracy. Control methods mentioned include anthelmintic drug usage, snail control, vaccination, and management practices.

3. Some Diagnostic Studies
On Fasciola Infestation
Among Cattle And
Buffalo
‫في‬ ‫بالفاشيول‬ ‫للاصابة‬ ‫التشخيصية‬ ‫الدراسات‬ ‫بعض‬‫في‬ ‫بالفاشيول‬ ‫للاصابة‬ ‫التشخيصية‬ ‫الدراسات‬ ‫بعض‬
‫والجاموس‬ ‫البقار‬‫والجاموس‬ ‫البقار‬

4. Presented by:
Vet: Seham Fawzy Hassan
BVSCS (2009(

8. Taxonomy
Kingdom : Animalia
Phylum : Platyhelminthes
Class : Trematoda
Subclass : Digenea
Order : Echinostomifomis
Family : Fasioloida
Genus : Fasciola
Species : hepatic
gigantica

9. Morphology
They are flat
leaf-like,
grayish brown
in color
Fasciola hepatica
Fasciola gigantica
they may
reach up to
7.5 cm length.
Averaging 30 mm
in length and 13
mm in width

10. Life cycle

11. Fascioliosis
Fascioliosis is one of the world wide parasitic disease
common in ruminants (sheep, goat, cattle, buffaloes, camels(,
swine, horses, donkeys and rabbits caused by Trematoda
called Fasciola spp characterized by liver damage, animal poor
condition, decrease in productivity beside it has zoonotic
importance.

12. EPIDEMILOGY OF FASCIOLIOSIS
1-The occurrence
Management
The occurance differed according to:
Season
Species
Age
Sex

13. ZoonoTic imporTance
In a study made by Basem Refat (2013(:
he found that the residence distribution of human
fascioliosis in Assuit Governorate was 8.33 % and in
New Valley Governorate was 8.70%.

14. economic imporTance
The economic loss represented by:
•Condemnation or down-grading of affected livers at abattoirs.
•Suboptimal live weight gain and reduced production and/or quality of meat
and milk in cattle with chronic liver fluke infections.
•Cost of prophylactic or therapeutic anthelmintic treatment.
•Animal welfare issues implications.
•Risk of zoonotic infection.
Death of animals acutely infected with large numbers of flukes (unusual in
cattle(.
The annual loss in the world due to fascioliasis is 3.2 billion dollars.
In Egypt losses in meat and milk due to fascioliasis was 30% per year= one
milliard pound (according to issue of June 1998 of the General Organization
of Veterinary Services, Ministry of Agriculture(.

15. Pathogenesis
Infection occur through ingestion of the encysted
metacercaria on green fodders.
The prehepaTic sTage:
the metacercaria excysted in the lumen of intestine.
Then cercaria begin it’s journey in the abdominal
cavity, newelly exysted juvenile penetrate the
intestinal mucosa and found in the abdominal cavity,
then begin it’s journey to the liver.
Flukes can be carry on to penetrate other organs as
the lung ,diaphram ,fetus in pregnant animal

16. The hepaTic sTages
During migeration of the fluke through the liver parenchyma it
causes arteritis ,inflammatory reaction ,fiberosis of the
parenchyma.
The flukes concenterated at the venteral aspect of the liver.
The bile duct thickened due to hypertrophy and fiberosis of the
wall, Ca depositition starts at the wall of the bile duct.
Stenosis or compelete obstruction of biliary ducts.

17. Clinical signs
Acute type I fascioliasis Occurs when the animal
ingests more than 5000 metacercariae, which may lead to
its sudden death, especially sheep and goats without
showing any previous clinical signs.
Acute type II fascioliasis: Infection occurs by the ingestion
of 1000-5000 metacercariae. the animal dies and showing signs
of pallor, loss of condition and ascites.
Subacute fascioliasis: occurs due to the ingestion of 800-
1000 metacercariae. The animal becomes weak, anemic and
weight loss may occur resulting in death of the animal.

18. Chronic fascioliasis
occurs when 200-800 metacercariae are ingested. Chronic
Fasciolasis is prolonged and does not have clear key
symptoms except for gradual weight loss, pallor of mucous
membranes, ventral oedema and wool break.
Loss of condition
Lethargy
Anaemia
Bottle jaw
Sub-optimal growth rates
jaundice

19. •Diarrhoea
•Metabolic disease in dairy cows
•Reduced milk yield in dairy cows
•Reduced fertility
•Signs are exacerbated by poor nutrition or gastro-
intestinal parasitism

20. Effect of fascioliosis on blood
components
1-Anaemia: which accepted that is hemorrhagic anemia.
2-Plasma protein: decrease in albumin concentration partly
due to decrease the rate of synthesis and increase expansion of
plasma volume.
3-Immunoglobulin: increase in immunoglobulin synthesis
including IgG1, IgG2, IgM, IgE.
4-Dramatic increase in eosinophils.
5-Billirubinemia:due to presence of adult flukes in the bile duct
which affect production and flow of the bile.

21. Postmortem lesions
-Emaciated, anemic, edematous and/or icteric carcass due to liver
damage.
-Liver enlargement with bumpy, raised and/or depressed areas, dark
blue to black discolorations, hardness in consistence.
--Hemorrhagic tracts of migratory immature flukes in the liver in an
acute infection.

22. -Black parasitic material (excrement) in the liver.
-Cirrhotic effect on the liver (scarred surface(.
-Enlarged, thickened and calcified bile ducts

23. Diagnosis
1-coprological diagnosis.
2-serological diagnosis.

24. The prevalence
In a study made by Sahar M. Selim And Azza Hassan
Mohamed (2008) on 362 sheep in Menoufiya
Governorate he found that the prevalence of fasciola
infection was 65.7% with ELISA and 42.3% by fecal
examination

25. Coprological examination
Coprological examinations involve examining the
feces of animals to identify the parasite eggs.

26. Coprological examination
Detection of fasciola egg is simple and confirmatory
but it is not useful diagnostic tool at low levels of adult
fluke burden.
Also it cannot detect infection at the prepatent
period, because eggs are found in feces when the fluke
are already matured usually between (10-14 weeks of
infection) by this time major damages to the liver
parenchyma, cause hemorrhages and damages to the
liver may have already occurred due to fasciola entering
to liver.

27. Coprological diagnosis
False negative:
1(Egg deposition of the parasite is irregular.
2-the incubation phase of the infection is shorter than that of the
prepatent period.
3-clinical findings of the disease may appear long before egg can be
found in feces.
4-in case of obstruction of the billiary ducts.
False positive:
In case of death of the worm the egg laying persist for 21 days after
death of the worm.

28. Serological diagnosis
Immunological examinations, such as indirect
immunofloursence , ELISA, and complement fixation test are
methods of identifying different kinds of parasites by
detecting the presence of their antigens or within the
parasite itself.
These diagnostic methods are used in conjunction with
coprological examinations for more specific identification of
different parasite species in fecal samples.

29. Serological method have been developed as an alternative
approach to fecal egg detection ,which can test large
number of sera at the same time and also detect
infection earlier than fecal egg examination there are
evidences to show that serodiagnosis can detect the presence of
infection as early as 2 weeks after infection.
during the chronic phase, in cases with low-level or
sporadic production of eggs ; in cases of ectopic infection,
in which eggs are not found in stool; and after treatment, to
assess the response
Serological diagnosis

30. Serological diagnosis
False positive:
In case of cross reaction with other
trematods.
False negative:
1-In case of aged animals.
2-In case of animals with exhausted immune
system

31. Aim of the work
1-Studying the prevalence of animal fascioliasis among
cattle and buffaloes in Assiut Governorate through
coprological examination and two different serological
methods (Agar Gel Diffusion Test and ELISA(.
2- comparing between the specificity, sensitivity,
accuracy of each method.
3- Rerecording the prevalence of fascioliasis in cattle and
buffaloes in Assuit in relation to species (cattle or
buffaloes),season, age and sex.

32. Protocol of workProtocol of work

33. 1-source of the sample
• Live animals from different farms
and households (feces and blood
samples) , slaughtered animals from
different slaughter houses.

34. Sample collection and
handling
• From each live animal (from different
farms and different household animals)
blood and fecal sample will be taken
after clinical examination and infected
slaughtered aniamls recording it and
taking the fasciola worms from
infected livers for antigen preparation.

35. 1-Faecal samples
About 10-20 gm feces from each cattle and
buffaloes will be collected for parasitological
examination directly from the rectum of each
animal before slaughtering and freshly
defecated feces will be put in plastic bags
with gloved hand.

36. 2-Blood samples:
10 ml of whole blood samples will be taken from
jugular vein of each cattle and buffaloes to serum
tubes and be allowed to clot. Sera separating by
centrifugation at 3000 rpm for 15 minutes after
being keeping it in the refrigerator overnight. Sera
will be kept at (-20) c until used.

37. 3-Adult worms
Routine postmortem examination of
each slaughtered animal will be
carried out to check the presence of
fasciola, then the adult worms
collected for antigen preparation.

38. Fasciola antigen
1-somatic antigen:
It is an antigen located in the body of the worm.
2-secretory and execretory antigen:
a) Proteases: which used by the worm in migration through host
tissue, acquisitation of nutrition , evasion of the host immune
response.
b) Fluke hemoglobin: which is involved in aerobic respiration.
c) Proline : amino acid used in proliferation of the epithelium of
the bile ducts.
d) Tegumental execretion :which produced due to osmoregulation.

39. 1-Macroscopic examination:
The physical characters of the feces
studied (color, consistency, presence of
blood /or mucus(.
2-Microscopic examination
Using sedimentation technique
Fecal examination

40. Egg of fasciola
The eggs are yellow-brown
and operculated with a
thickening of the shell at
the poles; their dimensions
are 130 to 145 µm by 70
µm to 90 µm.
operculum

41. Serological diagnoSiS
1-Antigen preparation.
2-ELISA.
3-Agar Gel Immuno-diffusion.

42. 1-Antigen preparation.
Preparation of somatic antigen:
Antigen preparation from adult fasciola worms according to
(J.B. W. J. Cornelissen et al., 1992(
1-Adult flukes (10 g), collected from the bile ducts of infected
slaughtered bovine.
2-washing 3-4 times at room temperature for 1 h in 0.01 M
phosphate-buffered saline (PBS) (pH 7.0(.
3-The flukes will be homogenized and the ground flukes will be
extracted overnight in 100 ml of a mixture of phosphate
buffered saline with 100 U/ml Na penicillin, 0.25 mg/ml
streptomycin, 100 U/ml nystatin at 4 c.

43. 1-Antigen preparation.
5-The suspension will be centrifuged at 10,000 x g for 20 min at
4°C. The supernatant filtered and stored at -70 °C until used.
6-The protein concenteration of antigens will be determined.

44. 2-ELISA

45. ELISA
1-Coating ELISA Plates :(Voller et al., 1977(
Coating is achieved through passive adsorption of the
antigen to the assay microplate . This process occurs
though hydrophobic interactions between the micro
titer plate and fasciola antigen.
1-The most common method for coating plates involves adding
a 0.1-10 μg/ml solution of protein dissolved in an alkaline
buffer such as phosphate-buffered saline (pH 7.4) or
carbonate-bicarbonate buffer (pH 9.4) but the technique
applied for fasciola antigen the concenteration of the antigen
is 0.5-2 microgram/ml.

46. N.B. The buffer used for dilution contains no other
proteins that might compete with the fasciola antigen
for attachment to the micro titer plate.
2-Then washing the plate 3 times with washing buffer.
3-The plate wells will be blocked with 5% bovine serum
albumin (200 microliter in each well) for blocking all the
spaces to prevent adsorbtion of any protein other than
fasciola antigens
4-incubation of the coated plate 1 hour at 37 c. then the
plate will be washed 3 times with washing buffer.

47. ELISA
2-Addition of the serum:
100microliter of diluted serum samples (1:50) with phosphate
buffer serum (PBS) pH 7.2 will be added in each well and incubated
30 minutes at room temperature .Then washing the plate 5 times
with washing buffer.
3-Addition of the conjugate:
Dilution of enzyme conjugate to (1: 500) with dilution sample
(P.B.S).Then 100 microliter of diluted enzyme conjugate will be
transferred to each well and incubation for 1 hour at 37 c . After
incubation, plate will be washed and 5 times with washing buffer.

48. ELISA
4-Addition of the substrate
Washing the plate and adding the 3,3,5,5 Tetramethyl benzidine
dihydrochloride (TMB) in each well, then incubate the plate in the
dark for 30 min at room temperature.
5-Stopping the reaction
The color reaction stopped with 50 microliter 0.1 M sulphoric
acid per well.
6-Reading the result
Color changes will be measured in ELISA reader at 450 nm filter.

49. Adult worms of fasciola sp. Will be collected from the bile
ducts of infected cattle and buffaloes. The worms will be
washed with physiological saline and storing it at – 20 c
until examination.
The antigen will be prepared by homogenizing 0.1 g of each
adult fluke for 30 min in 5 ml of physiological saline. the
emulsion will be frezed and thawed twice and centrifuged
at 5000 rpm for 30 min. the supernatant of each emulsion
used as the antigen.The protein concenteration of antigens will be
determined.
1-Antigen preparation.
Preparation of somatic antigen: acc to
linh et al., (2003(

50. 2-Agar gel Immunodiffusion
The passive diffusion of soluble antigen and
it’s specific antibody towords each other to
make precepitation line between each other

51. Agar gel Immunodiffusion

52. Agar gel Immunodiffusion

53. E) Stastical analysis:
The data obtained will be tabulated in relation to
difference in species, sex, age, locality, season
and the sensitivity, specificty, accuracy of the
methods of diagnosis.

54. Control
1-Antifasciolia drugs usage
(Rafoxanide ,tricalbendazole,
albendazole(.
2-Snail control (Niclosamide(.
3-Vaccination
4-Managemental control

55. Refrences
Ash, L. R. and Orihel, T. C. (1987): Parasites: A Guide to Laboratory Procedures and Identification.
American Society of Clinical Pathologists Press, Chicago. pp. 4-30.
Hansen, J. and Perry, B., (1994): The Epidemiology, Diagnosis and Control of Helminth Parasites
of Ruminants, in Africa, A Hand Book, International Laboratory for Research on Animal Diseases
(ILRAD) Nairobi, Kenya, pp. 25-45.
J.B. W. J. Cornelissen , W. A. de Leeuw& P. J. van der Heijden (1992):comparison of an
indirect haemagglutination assay and an Elisa for diagnosing Fasciola hepatica in experimentally and
naturally infected sheep,Veterinary Quarterly, 14:4, 152-156.
Linh, B. K.; Thuy ,D. T.; My, L. N.; Sasaki, O. and Yoshihara, S. (2003):"Application of Agar
Gel Diffusion Test to the Diagnosis of Fascioliosis in the Cattle and Buffaloes in the Red River Delta of
Vietnam." J. A. R. Q.37 (3): 201-205.
Soulsby, E.J.L. (1982):Helminths, Arthropods and Protozoa of Domesticated Animals. 7th
ed. London,
England. Bailliere Tindall, pp: 42-50, 800- 809.
Voller, A., Bidwell, D. E., and Bartlett, A. (1977a): "The Enzyme Linked Immunosorbent Assay
(ELISA). "pp. 24-26. Flow-line Puplications,Guernsey.