Elisa In Comparison With Conventional Methods For Detection Of

‫‪Animal Health Research institute, New Valley, Egypt‬‬

‫‪ELISA in comparison with conventional methods for detection of‬‬ ‫‪Trypanosomiasis in camels‬‬ ‫ﻣﻘﺎرﻧﺔ اﺧﺘﺒﺎر اﻻﻟﻴﺰا ﺑﺎﻟﻄﺮق اﻻﻋﺘﻴﺎدﻳﺔ ﻟﻠﻜﺸﻒ ﻋﻦ ﻃﻔﻴﻞ اﻟﺘﺮﻳﺒﺎﻧﻮﺳﻮﻣﺎ ﻓﻲ اﻻﺑﻞ‪.‬‬ ‫)‪(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 ‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬ ‫اﺳ ﺘﻬﺪﻓﺖ اﻟﺪراﺳ ﺔ ﻣﻘﺎرﻧ ﺔ وﺗﻘﻴ ﻴﻢ آﻔ ﺎءة اﺧﺘﺒ ﺎرات اﻻﻟﻴ ﺰا ﺑ ﺎﻟﻄﺮق اﻻﻋﺘﻴﺎدﻳ ﺔ ﻟﺘ ﺸﺨﻴﺺ ﻣ ﺮض‬ ‫اﻟﺘﺮﻳﺒﺎﻧﻮﺳﻮﻣﺎ )اﻟﻬﻴﺎم( ﻓﻲ اﻻﺑﻞ واﺳﺘﺨﺪم ﻟﻬﺬا اﻟﻐﺮض ﻋﺪد ‪ 430‬ﻣﻦ اﻻﺑﻞ اﻟﺒﺎﻟﻐﺔ ﻣﻦ آ ﻼ اﻟﺠﻨ ﺴﻴﻦ وﺗ ﻢ‬ ‫اﺧﺘﻴﺎرهﺎ ﻣﻦ اﻟﻤﺰارع واﻟﺘﺠﻤﻌﺎت اﻟﻤﻮﺟﻮدة ﻓﻲ ﻣﺤﻴﻂ ﻣﺪﻳﻨﺘﻲ اﻟﺨﺎرﺟﺔ واﻟﺪاﺧﻠﺔ ﺑﻤﺤﺎﻓﻈﺔ اﻟﻮادي اﻟﺠﺪﻳﺪ‬ ‫ﺣﻴﺚ ﺗﻢ ﺗﻘﺴﻢ هﺬﻩ اﻟﺤﻴﻮاﻧﺎت اﻟﻰ ‪ 3‬ﻣﺠﻤﻮﻋﺎت‪ .‬ﻣﺠﻤﻮﻋﺔ ‪) 1‬ﻣﺠﻤﻮﻋﺔ اﺷﺘﺒﺎﻩ ﻣﺮض اﻟﻬﻴﺎم( وﺷﻤﻠﺖ ﻋﺪد‬ ‫‪ 370‬رأﺳﺎ وآﺎﻧﺖ ﺗﻌﺎﻧﻲ ﻣﻦ أﻋﺮاض ﻧﻮﺑﺎت اﻟﺤﻤ ﻰ‪ ،‬اﻻﻧﻴﻤﻴ ﺎ اﻟﻤﺘﺰاﻳ ﺪة‪ ،‬اﻟﻨﺤﺎﻓ ﺔ واﻻودﻳﻤ ﺎ‪ .‬ﻣﺠﻤﻮﻋ ﺔ ‪2‬‬ ‫وﺷ ﻤﻠﺖ ‪ 30‬رأﺳ ﺎ وﺛﺒ ﺖ ﺧﻠ ﻮهﻢ ﻣ ﻦ ﻃﻔﻴ ﻞ اﻟﺘﺮﻳﺒﺎﻧﻮﺳ ﻮﻣﺎ ﺑ ﺎﻟﻔﺤﺺ اﻟﻄﻔﻴﻠ ﻲ اﻟﻤﻌﺘ ﺎد واﻟﻔﺤ ﺺ اﻟﻤﻨ ﺎﻋﻲ‬ ‫أﻳﻀﺎ واﺳﺘﺨﺪﻣﺖ آﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻂ اﻟﺴﻠﺒﻲ واﻟﻤﺠﻤﻮﻋﺔ ‪ 3‬واﻟﺘﻲ ﺷﻤﻠﺖ ﻋﺪد ‪ 30‬رأﺳ ﺎ ﻣ ﻦ اﻻﺑ ﻞ واﻟﺘ ﻲ‬ ‫ﺛﺒﺖ اﺻﺎﺑﺘﻬﺎ ﺑﻄﻔﻴﻞ اﻟﺘﺮﻳﺒﺎﻧﻮﺳﻮﻣﺎ ﺑﺎﻟﻔﺤﺺ اﻟﻄﻔﻴﻠﻲ اﻟﺘﻘﻠﻴ ﺪي واﺳ ﺘﺨﺪﻣﺖ آﻤﺠﻤﻮﻋ ﺔ اﻟ ﻀﺎﺑﻂ اﻻﻳﺠ ﺎﺑﻲ‪.‬‬ ‫أﺛﺒﺖ اﻟﺪراﺳﺔ ان اﻟﻔﺤ ﺺ اﻟﻄﻔﻴﻠ ﻲ آ ﺎن اﻳﺠﺎﺑﻴ ﺎ ﻓ ﻲ ‪ 100‬ﺣﺎﻟ ﺔ ﻣ ﻦ اﻟﻌ ﺪد اﻟﻜﻠ ﻲ )‪ (% 23.2‬ﺑﻴﻨﻤ ﺎ أﻇﻬ ﺮ‬ ‫اﻟﻔﺤﺺ اﻟﻤﻨﺎﻋﻲ ﻟﻠﺒﺤ ﺚ ﻋ ﻦ اﻻﻧﺘﻴﺠ ﻴﻦ )اﻟﻤﺴﺘ ﻀﺪ( اﺻ ﺎﺑﺔ ‪ 98‬ﺣﺎﻟ ﺔ ﻓﻘ ﻂ )‪ (% 20.6‬واﻇﻬ ﺮ اﻟﻔﺤ ﺺ‬ ‫اﻟﻤﻨﺎﻋﻲ ﺑﺎﻻﻟﻴﺰا ﻟﻠﺒﺤﺚ ﻋ ﻦ اﻻﺟ ﺴﺎم اﻟﻤ ﻀﺎدة اﺻ ﺎﺑﺔ ‪ 140‬ﺣﺎﻟ ﺔ )‪ .(%32.5‬وﺗﺆآ ﺪ ه ﺬﻩ اﻟﻨﺘ ﺎﺋﺞ اهﻤﻴ ﺔ‬ ‫اﺳ ﺘﺨﺪام اﻻﻟﻴ ﺰا آﺎﺧﺘﺒ ﺎر ﻣﻨ ﺎﻋﻲ ﻓﺎﻋ ﻞ ﻻﺟ ﺮاء اﻟﻤ ﺴﺢ اﻟﻤﻨ ﺎﻋﻲ واﻟﺘ ﺸﺨﻴﺺ اﻟ ﺴﺮﻳﻊ واﻟ ﺪﻗﻴﻖ ﻟﻠﻤ ﺮض‬ ‫وأوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ أﻳﻀﺎ ان اﺧﺘﺒﺎر اﻻﻟﻴﺰا اﻟﻤﻌﺘﻤﺪ ﻋﻠﻰ اﻟﺒﺤﺚ ﻋ ﻦ اﻻﺟ ﺴﺎم اﻟﻤ ﻀﺎدة أآﺜ ﺮ اﻓ ﺎدة ﻻﺟ ﺮاء‬ ‫ﺗﻘﻴ ﻴﻢ ﻋ ﺎم ﻟﺤﺠ ﻢ اﻟﻤ ﺮض واﻟﺤﻴﻮاﻧ ﺎت اﻟﺘ ﻲ ﺗﻌﺮﺿ ﺖ ﻟﻼﺻ ﺎﺑﺔ ﻳﻨﻤ ﺎ ﻳﻜ ﻮن اﺧﺘﺒ ﺎر اﻟﻠﻴ ﺰا ﻟﻠﺘﻌ ﺮف ﻋﻠ ﻰ‬ ‫اﻻﻧﺘﻴﺠﻴﻦ )اﻟﻤﺴﺘﻀﺪ( أآﺜﺮ اﻓﺎدة ﻟﻠﺘﻌﺮف ﻋﻠﻰ اﻻﺻﺎﺑﺎت اﻟﻨﺸﻄﺔ ﻟﻠﻤﺮض‪.‬‬

‫‪1‬‬

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 & AlDakla) 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 results revealed 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 2

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- Samples 1- 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 3

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 4◦c 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 - 70◦c 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 ) of Trypanosoma. - 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 IgGperoxidase conjugate and orthophenylen diamine as achromagen (OlahoMukani,1989). - Antigen -enzyme linked immunoassay (Ag-ELISA ) Monoclonal antibody (TEA/23.4.6) and its peroxides conjugate (1/23.4.6 4

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 for Trypanosoma 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 <0.0001 ) in Ag-ELISA optical density (OD) values between parasite-positive and parasite negative camels but no statistical difference was observed in the AbELISA (OD) values between parasite- positive and parasite-negative camels.

DISCUSSION For control of camel trypanosomiasis, it is essential that accurate epidemiological information should be obtained. Such information depends on the use of sensitive diagnostic tests. The limitations of parasitological diagnosis necessitates the application of immunodiagnostic assays to ensure that, and for a number of reasons ELISA tests are preferred to used. First, it is possible to produce defined antigens and monoclonal antibodies. Second, the ELISA is easy to automate in order to screen large number of samples and 5

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): Trypanosoma evansi in Asia, Parasitol.Today, 4; 137 6

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. Methods Groups

ELISA Parasitological

Ag

Group (1)

(370)

70

18.9%

70 (18.9%)

Group (2)

(30)

- ve

0%

-ve ( 0%)

7

Ab 110 29.7% -ve

0%

Group (3)

(30)

30

100%

28 (93.3%)

30

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

8

100%

>b

9