ELISA in Comparison With Conventional Methods for Detection Of

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Animal Health Research institute, New Valley, Egypt

ELISA in comparison with conventional methods for detection of

Trypanosomiasis in camels

.

(With one table and one figure)

By

Osman, F. A; Abd-el Rhman, M. A (a); Raghib, M. F(b); Sadiek, A. H (b)

a-Department of parasitology, faculty of Medicine, Assiut University, Egypt

b-Department of Animal Medicine, Assiut University, Egypt

430)(

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SUMMARY

The aim of the present study was to evaluate the efficiency of Enzyme Linked

Immuno-Sorbent Assay (ELISA) in comparison with the parasitological

method for the diagnosis of the trypanosome evansi in camels. A total of 430

camels selected from private farms at different localities in (Al-kharga & Al-

Dakla) of New Valley Governorate were investigated in this study.The camels

were divided into three groups. Group 1 included 370 animals with clinical

manifestations of recurrent fever, progressive anemia, losses of condition and

edema and was used as suspected group. Group 2 included 30 camels that

were proved free from trypanosomiasis by both clinical, parasitological and

serological tests and was used as negative control group. Group 3 included

another 30 camels proved to be infected by Trypanosoma evansi by

parasitological examination and was used as positive control group. The resultsrevealed that; whereas parasitological examination was positive in 100 out of

430 (23.2%). Ag-ELISA test has detected 98 (20.6%) but with Ab-ELISA test

detected 140 (32.5%). The results indicated the feasibility of the ELISA

technique as a potential immunodiagnostic assay for rapid, accurate diagnosis

where antibody assays would enable an overall assessment of the population

exposed to infection; antigen assays enable identification of individuals with

active infections.

INTRODUCTION

The camel is a creature of the desert; a habitat generally not conducive

to the development and transmission of parasites. In spite of this the camel

harbors a surprisingly diverse of parasites. Camel trypanosomiasis (surra) is a

disease of camels caused by Trypanosoma evansi. The disease is the most

important single cause of economic losses in camel rearing areas, causing

morbidity of up to 30.0% and mortality of around 3.0% (Njiru et. al., 2000).

The causative Trypanosoma evansi, was discovered by Griffith Evans in 1880

in infected camels in the Dar Ismail- Khan district of Punjab. Since then

studies have shown that the parasite can infect all species of domesticated

livestock, although the principle host varies geographically (AL-

RAWASHDEH et. al., 1999). In Africa, tse tse fly is the most important host

(Luckin A.G.1988). Because of the range of agro-ecological zones and the


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diverse farming systems in which the disease occurs as well as its debilitating

effects on affected camels. Surra has attracted international symposiums on

strategies for research and control of Surra.

MATERIAL AND METHODS

1- Animals

A Total number of 430 camels were investigated during the current

study (Julie 2006 to August 2007). The camels were selected from private

farms of different localities, in New Valley governorate-Egypt. The camels

were divided into three groups. Group 1 included 370 camels with apparent

clinical manifestations of Surra (Pyrexia with progressive anemia, loss of

condition and lassitude, recurrent episodes of fever and edema particularly of

the lower parts of the body) and was used as suspected group. The second

group included 30 camels that were proved free from trypanosomes by

clinical, parasitological and immunological (ELISA) studies and was used as

negative control group. The third group included 30 camels proved infested by

trypanosomiasis by parasitological examination and was used as positive

control group.

2- Samples1- Heparinized whole blood was sampled from both peripheral and deep blood

vessels in vacuumed tubes for parasitological tests and animal inoculation.

2-Serum samples (blood without anticoagulant were obtained from studied

camels), used for immunological test (ELISA).

3-Adopted methods

A- Trypanosoma evansi antigen preparation.

The blood of heavily parasitaemic mice is collected and Trypanosoma

evansi separated on a Diethyl amine ethyl (DEAE) cellulose column and

washed three times by centrifugation in cold Phosphate buffer solution with


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1% glucose (PSG). The final pellet was suspended in cold PSG to

concentration of 4% and briefly ultrasonicted on ice for two minutes until

disintegration of the organisms is completed. This preparation was centrifuged

at 4c and 40,000g for 60 minute and the supernatant was diluted in water so

as to obtain a protein concentration of 1mg/ml. The obtained reagent was

stored in small aliquots at - 70c until used.

B- Parasitological diagnosis.

1-Examination of fresh Jiemsa stained blood films from suspected field cases

as described by Paris et al., 1982).

2-Mouse inoculation was used to reveal sub clinical (non patent) infection in

camels where the heparinized blood of positively infected camels was

inoculated intraperitoneally (0.5ml ) into 3 mice . Inoculated animals were

bled from the tail three times a week to detect parasitism by stained blood

films.The blood of heavily infected mice were used to prepare Trypanosoma

Antigen.

C-Enzyme-linked immunosorbent assay (ELISA)

The principle of this technique depends on the enzyme conjugate that

binds to the antigen/ antibody complex and then react with a suitable substrate

to yield characteristic color. Elisa was carried out to detect the presence of

antibodies (Ab-ELISA ) or to detect Ag (Ag-ELISA ) ofTrypanosoma.

- Antibody-enzyme linked immunoassay (Ab-ELISA )

Anti-Trypanosoma antibody levels in test sera from camels were assayed in

micro plate ELISA using Trypanosomal lysate antigen, rabbit anticamel IgG-

peroxidase conjugate and orthophenylen diamine as achromagen (Olaho-

Mukani,1989).

- Antigen -enzyme linked immunoassay (Ag-ELISA )

Monoclonal antibody (TEA/23.4.6) and its peroxides conjugate (1/23.4.6


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Peroxides) were used for the test. The test carried were used out in 96- well

polystyrene micro plate ELISA to assay circulating Trypanosomal antigens in

tested camel sera (LUCKINS, 1991).

RESULTS

Parasitological examination.

This test was carried out by the help of the Dept. of Parasitologly,

Faculty of Medicine, Assuit University. The test revealed that: A total of 100

camels were parasite-positive (70 camels from group 1 and 30 camels from

group 3). All the infecting trypanosomes belonged to T. evansi (fig. 1). The

results were summarized in table (1).

Serological tests (Ag- and Ab-ELISA ).

Of the 430 tested samples, 98 samples (20.6%) were found to be antigen

positive. Most of the tested sera were found positive forTrypanosoma

antigen where Ag-ELISA detected 98 parasite-positive cases out of 100 (98%)

from the three groups (table 1). There was a statistical difference (p


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finally by appropriate modification it should be possible to adapt the assay for

use under field conditions.

Antibody assays would enable an overall assessment of the population

exposed to infection; antigen assays would enable identification of individuals

with active infections (Kosum, et al.,2002). In the past many serological tests

depend upon demonstration of antibodies (Nantulya 1990), deposit the fact that

they persist in the circulation after effective cure (Luckins 1988). However, the

Ag-ELISA described here usually gave positive results only in camels with

active infection as described by Voller and Savigny (1981). These findings are

in accordance with that reported by Omer et al. (1998). It could be concluded

that ELISA technique is considered as a potential immunodiagnostic assay for

rapid, accurate diagnosis where antibody assays would enable an overall

assessment of the population exposed to infection; antigen assays enable

identification of individuals with active infections.

REFERENCES

AL-Rawashdeh O. F.,Sharif L.A., AL-Qudah k., AL -Ani F.

K.(1999):Trypanosome evansi infection in camels in Jordan.Revu

Elev.MDd.Vet.pays trop.1999,52 (3-4);233-237

Kosum Chansiri, Sintawee Khuchareontaworn and Nopporn (2002):

PCR-ELISA for diagnosis of Trypanosoma evansi in animals and

vector: Molecular and Cellular Probes, Vol. 16, Issue 3: pp: 173-177

Luckins, A. G. (1991): Antigen detection ELISA for Trypanosoma evansi

using group -specific monoclonal antibodies in improving the diagnosis

and control of trypanosomiasis and other vector -born disease of

African livestock using immunoassay methods, third Research

coordination meeting, Abidgan,20 -25 may.1991

Luckins,A.G. (1988):Trypanosomaevansi in Asia, Parasitol.Today, 4; 137 -


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142

Nantulya,V. M. (1990), Trypanosomiasis in domestic animals;the problems

of diagnosis Rev.Sci.Tech.Int.Epiz,9; 357 -367

Njiru Z.K., Ole Mapeny I.M., Ouma J.O.,Ndung J.M., OLaho-MUKani

W. (2000): Prevalence of trypanosomiasis in camel,calves;a pilot study

in laikipia district of kenia. Revu Elev. MDd. Vet. Pays trop.2000, 53

(2); 183 -186

Olaho-Mukani,W.(1989): Development and characterization of monoclonal

antibodies to T. evansi and their application as immunodiagnostic

reagents. Ph.D. thesis, university of Nairobi.

Omer O H,Magzoubs M, Haroun EM, Mahmoud OM, Abdel Hamid YM.

(1998): Diagnosis of trypanosome evansi in Saudi Arabian camels by

the passive hamagglutination test and Ag-ELISA. Zentrabl

veterinarmed 1998; 45; 627-633

Paris.g.Murray, M,and mcOdimba,F. (1982): A comparative evaluation of

the parasitological techniques currentely available for the diagnosis of

African trypanosomiasis in cattle. Acta Trop.39, 307-317.

Voller,A.and De Savigny,D. (1981 ): Diagnostic serology of tropical disease.

Immunol methods 46,1-29.

Table (1); Comparison of parasitological and ELISA methods for detection of

trypanosomiasis in camels in New Valley governorate.

ELISAMethods

Groups Parasitological Ag Ab

Group (1) (370) 70 18.9% 70 (18.9%) 110 29.7%

Group (2) (30) - ve 0% -ve ( 0%) -ve 0%


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Group (3) (30) 30 100% 28 (93.3%) 30 100%

Total (430) 100 23.2% 98 ( 20.6%) 140 32.5 %

Group (1) act as suspected group.

Group (2) act as negative control animals.

Group (3) act as positive control animals.

Fig. 1: Trypanosoma evansi in a a Giemsa stained blood film


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ELISA in Comparison With Conventional Methods for Detection Of