صالح موسي مختار عبدالوهاب

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1 Dept. of Poultry Diseases, Fac. Vet. Med. Assiut University Assiut Vet. Med. J. Vol. 55 No. 121 April 2009 PREVALENCE OF AVIAN LEUKOSIS VIRUS IN CHICKEN FLOCKS IN UPPER EGYPT (With 7 Tables) By S. MOUSA and M.H. ABDEL-WAHAB* * Animal Production Research Institute. (Seds, Beni-Suef), Egypt (Received at 3/3/2009) دراسة مدي انتشار فيروس مرض الليوكوزس في قطعان الدجاج في صعيد مصر صالح موسي مختار عبدالوهاب لدراسة مدي انتشار أنتيجن فيروس مرض الليوكوزس في مختلف قطعان الدجاج في صعيد مصر تم تجميع عينات مصل وبيض من مختلف مزارع الدواجن والتي تمثل مختلف األعمار ومختلف نظم التربية وأنواع الدجاج المختلفة والتي تشمل دجاج بيض المائدة ودجاج التسمين وأمهات الدجاج الالحم وبعض السالالت المحلية. وتم اختبار عينات المصل والبيض للكشف عن أنتيجن فيروس مرض الليوكوزس بواسطة اختبار اإلليزا. أظهرت النتائج أن كافة قطعان الدجاج في صعيد مصر التي خضعت لإلختبار احتوت علي فيروس مرض الليوكوزس بنسب متفاوتة. وكانت النسب األعلي لتواجد الفيروس في بداري التسمين والسالالت البلدية. وكانت نسبة الفيروس الداخلي لمرض الليوكوزس أعلي من نسبة الفيروس الخارجي. SUMMARY To study the prevalence of ALV antigen in different poultry flocks in Upper Egypt, serum and egg were collected from poultry farms representing different ages, different raising systems and different s including table egg layers, meat type broilers, breeders and random native breeds. Samples were tested for detection of P.27 Common antigen of ALV by ELISA test. All chickens flocks possessed ALV antigen at various degrees. The highest percentage was detected in meat type chicken flocks and balady chickens. Titre of endogenous virus was greater than exogenous virus. Key words: Leukosis, exogenous leukosis INTRODUCTION 1

2 Tumours continue to be a major cause of economic losses among poultry industry in many countries including Egypt. Economic impact includes mortalities, condemnations and immune suppression. Avian Leukosis Viruses (ALV) known as avian retroviruses induce a variety of tumours (neoplasms) of which lymphoid leukosis is common. Based on the discovery of the importance of infection at embryonic stage, extensive control programmes were initiated to control the spread of ALV (Arshad et al., 1997a). Vertically infected chicks (at embryonic stage) and chicks horizontally infected (at 1-day-old stage or during the rearing period) are at risk to become carriers of ALV. These are able to shed large amounts of the ALV into the environment and form a source of infection to uninfected hatchmates. Infected birds are likely to develop tumours and a high percentage of laying hens among these birds will in turn transmit virus vertically. Thus, culling of hens that are viraemic and shed virus in eggs will greatly reduce the vertical transmission of ALV (Arshad et al., 1997b). The presence of virus is determined by the detection of ALV P27 by indirect biologic assays, such as complement fixation (CF) for avian leukosis (COFAL) (Sarma et al., 1964). ELISA for ALV (Crittenden et al., 1987; Fadly and Witter, 1998) phenotypic mixing (Okazaki et al., 1975) resistance inducing factor (Rubin, 1960) and nonproducer cell activation (Rispens, 1970). Of all such assays, ELISA-AVL is the most commonly used test. This work was planned to investigate the prevalence of tumours among different s and ages through the determination of ALV antigen in chicken sera and albumen by ELISA test. MATERIALS and METHODS Sera: Blood were collected from layers, breeders, balady and broiler flocks from different areas in Upper Egypt. Sera were subjected to ELISA test for detection of LAV antigen. Egg albumen: Eggs were collected from different areas in Upper Egypt. Albumen was withdrawn by punching a small hole midway between the middle and the small end of the eggs, care was taken to avoid obtaining yolk materials. The albumen was assayed for ALV by ELISA test. ELISA test: Reagents: (a) ALV control (b) Negative virus control 2

3 (c) Rabbit anti-p27 peroxidase conjugate (d) Dilution buffer (e) ABTs-hydrogen peroxidase substrate (f) Stop solution (g) Wash solution ELISA reader: 96 well plate reading ELISA reader with 405 nm filter. Reader was a Biotek SL 311. Preparation of controls: A p27 and negative controls were provided with the kit. in a reading-to-use form. The p27 and negative control were allowed to equilibrate to room temperature. Preparation of conjugate solution: A horseradish peroxidase conjugated rabbit anti-p27 was supplied in 50 glycerol. 200 l of the conjugate solution were diluted in 10 ml dilution buffer. ELISA test procedure: According to manufacture plates were processed as follows: (a) An anti-p27 antibody coated test plate was removed from the protective bag and labelled. (b) 100 l negative control were directly added to wells As, H10 & H12 without dilution. Pipette tip was discarded each time. (c) 100 l control was directly added to wells A1, A3 and H11 without dilution. (d) 100 l of unknown sample (either albumen or serum were added to each well. (e) Plate was incubated for 30 minutes at room temperature. (f) Plates were washed. (g) Addition of anti-p27 peroxidase conjugate, substrate and solution. Processing of DATA: (a) Plates were read using an ELISA plate reader set at 405 nm and concentration of P27 antigen per sample was calculated by the following equation: SP (Sample absorbance) (average ve control absorbance) corrected ve control (CPC) absorbance (b) By means of a software proflock produced by KPL USA, the titres standard deviations and coefficient of variation were calculated. RESULTS 3

4 Results of ELISA test for detection of P-27 common ALV antigen are shown in tables (1-7). Tables (1-3) summarize the findings in correlation to age. It is clear that birds aged 3-5 months constituted the highest rate of cases (66.4). On the other hand, tables (4-6) show the rate in correlation with. Layers showed the least rate (15.7) while meat-type chickens, breeders and balady chickens showed higher rate (66.7 and 67.8) respectively. For detection of exogenous virus disseminated in eggs, a total of 1311 were collected from eggs of different sources. Results illustrated in table 10 show that 32.3 of cases were for presence of exogenous virus. Table 1: Results of detection of ALV antigen in sera of chickens up to 2 months of age Source & Menia layers Kena type Assiut breeders meat Assiut balady Total Table 2: Results of detection of ALV antigen in sera of chickens of 3-5 months of age. Assiut balady Assiut breeders Total

5 Table 3: Results of detection of ALV antigen in sera of chickens more than 6 months of age Kena meat type Assiut breeders Menia layers Assiut layers Total Table 4: Detection of ALV antigen in sera of layer flocks. Assiut Minia Menia

6 Table 5: Detection of ALV antigen in sera of meat-type and breeder flocks Kena (meat type) Assiut (1) Assiut (2) Assiut (3) Total Table 6: Detection of ALV antigen in sera of balady chickens. chicken type Assiut Assiut Total

7 Table 7: Results of detection of exogenous ALV antigen in eggs (albumen): chicken type Assiut breeders Assiut layers Assiut layers Assiut layers Menia layers Menia layers Menia layers Assiut Balady Assiut Balady Assiut Balady Assiut Balady Assiut Balady Assiut Balady Assiut Balady Assiut Balady Assiut Balady Assiut Balady Total DISCUSSION An enzyme-linked immunosorbant assay (ELISA) was developed for the serological diagnosis of big liver and spleen disease by Todd et al. (1993). The test utilizes specific antigen recovered from the livers of infected hens. This specific antigen is fractionated by gel-filtration chromatography and immobilized on micro-titre plates. Coated plates are produced commercially by Proflock U.S.A. (KPL). In this work antibodies against a common antigen (P27) was used to detect presence of ALV in tested. Results showed great variation among tested flock. The percentage of cases was higher in native breed hens and in parent meat type chicks than in egg type chicks. This variation may be attributed to either differences in susceptibility among different breeds or variation on hygienic precautions and biosecurity. 7

8 Variation in suspectibility among different breeds or chickens lines were proven by many workers (Fadly and Payne, 2003). Virus of avian leucosis / sarcoma group seemed to be prevalent in commercial chickens but high rate of these chicks seemed to be carrying endogenous virus. Practically it was of greater importance to detect congenitally transmitting hens than those carrying ALV antigen. The egg albumen was used for detection of exogenous ALV antigen. This method proved to be more efficient in detection of transmission of ALV from dams to their embryos and for shedding ALV into eggs. Results of ELISA test showed higher titers among meat type breeders with a mean of and lower titer in table egg layers with mean titer of 104. Nearly all examined flocks were carrying ALV antigen in their sera but examination of egg albumin to detect exogenous virus lower incidence and nearly all examined layer flocks were negative while higher incidence was detected in native breeds than in meat type breeders. Detection of ALV antigen in sera seemed to be correlated with age and small proportion was found in young ages as compared with adult birds. This may be due to exposure after lateral transmission or multiplication of virus in vertically infected chicks. REFERENCES Fadly, A.M. and Payne, L.N. (2003): In U.M. Saif, H.J. Barnes; A.M. Fadly; J.R. Glisson; L.R. McDouglad and D.E. Swayne. Diseases of Poultry, 11 th Ed. Iowa State Press, U.S.A. Arshad, S.S.; Bland, A.P.; Hacker, S.M. and Payne, L.N. (1997a): A low incidence of histiocytic sacromatosis associated with infection of chickens with the HPRS-103 strain of subgroup. J. Avian Leukosis virus. Avian Dis., 41: Arshad, S.S.; Howes, K.; Barron, G.S.; Smith, L.M.; Russell, P.H. and Payne, L.N. (1997b): Tissue tropism of the HPRS-103 strain of J subgroup avian leucosis virus and of a derivative acutely transforming virus. Vet. Pathol. 34: Crittenden, L.B.S.; McMahon, M.S. Halpern and Fadly, A.M. (1987): Embryonic infection with the endogenous avian leucosis virus Rous-associated virus-0 alters responses to exogenous avian leucosis virus infection. J. Virol. 612:

9 Fadly, A.M. and Witter, R.L. (1998): Oncornaviruses: Leukosis / Sarcoma and reticuloendotheliosis. In Glisson, J.R.; Jackwood, D.J.; Pearson, J.E.; Reed, W.M. and Swayne, D.E. (eds). A laboratory manual for the isolation and identification of Avian Pathogens. 4 th ed. Am. Assoc. Avian Pathologists: Kennet Square, PA, Okazaki, W.H.G. Purchase and Brumester, B.R. (1975): Phenotypic mixing test to detect and assay avian leucosis viruses. Avian Dis. 19: Rispens, B.H.; Long, P.A.; Okazaki, W. and Brumester, B.R. (1970): The NP activation test for assay of avian leucosis / sarcoma viruses. Avian Dis. 14: Rubin, H. (1960): A virus in chick embryos which induces resistance in vitro to infection with Rous sarcoma virus. Proc. Natl. Acad. Sci. United States, 46: Sarma, P.S.; Turner, H.C. and Huebner, R.J. (1964): An avian leucosis group-specific complement fixation reaction. Application for the detection. Tsukamoto, K.; Hasebe, M.; Kakita, S.; Tanigichi, Y.; Hihara, H. and Kono, Y. (1992): Sporadic congenital transmission of avian leucosis virus in hens dischanging the virus into the oviducts. J. Vet. Med. Sci., 54 (9): Todd, D.; Mawhinney, K.A.; McAliden, V.A. and Douglas, A.J. (1993): Development of an enzyme-linked immunosorbent assay for the serological diagnosis of big liver and splkeen disease. Avian Dis. 37: