Azhar Nazir

Comparative Study Of Different Techniques For Diagnosis Of Lymphoid Leucosis In Commercial Layer Chickens - 1997

This project was designed to study and compare the results of ELISA, impression smears of visceral organs, haematological (TLC and DLC) and histopathological examination for diagnosis of lymphoid leucosis in commercial layers. For this purpose a total of 90 birds were selected from different commercial layer farms, having the history of lymphoid leucosis, age ranged from 5 to 9 months and having enlarged liver and bursa of Fabricious on palpation.

After the selection of birds, their blood was obtained and serum was seprated avoiding the lysis of RBCs. The serum thus obtained was used to detect the lymphoid leucosis antigen with the help of proflock kit of ELISA by KPL USA. Ninety serum samples were put into the ELISA plate well coated with an anti-P27 antibody. The anti-P27 antibody formed a antibody antigen complex. After washing the plate, an affinity purified rabbit anti-P27 peroxidase conjugate was added to each well. The antibody antigen complex remaining from the previous step reacted with the conjugate, brief incubation period was provided and then unreacted conjugate was washed through second wash step. Substrate which contained a chromogen ABTS), was added to each well. Chrornogen colour was changed (from clear to green-blue), which occurred in the presence of peroxidase enzyme. The relative intensity of colour developed in 15 minutes (Compared to control) which is directly proportional to the quantity of P27 antigen in the sample. Now the substrate was incubated, stop solution was added to each well to terminate the reaction. The plate was read using an automatic ELISA reader, which also calculated the SP (sample to positive) values for each sample tested.

The birds having titres of 1000 or above were found positive.

Twenty birds out of total 90 were found positive by ELISA.

These ELISA positive birds were purchased from each farm and the same number of healthy birds from the same flock were subjected to organ body weight ratio and haematological examination (TLC, DLC) and histopathology of positive cases.

The total leukocytic count of the ELISA positive birds showed increase in the leukocytic count when compared with the healthy birds of the same flock. The mean values for ELISA +ve birds of flock number 1, 2, 3 and 4 were 44.93±0.40, 45.90±0.41, 45.90±0.41 and 45.04±0.63, respectively with mean TLC values of 27.32±0.23, 27.38±0.35, 27.38±0.35,27.24±0.29 of healthy birds of flock number 1, 2, 3 and 4, respectively, where the differential leukocytic count showed mild increase in lymphocytes and basophilis and a marked increase in monocyte. The mean value for lymphocytes of the ELISA +ve flock numbers 1, 2, 3 and 4 were 57.35±0.14, 57.30±0.15,57.30±0.15 and 57.76±0.44 respectively compared with the healthy birds of flock numbers 1, 2, 3 and 4 which were 56.0±0.20, 56.0±0.29, 56.20±0.29 and 57.44±0.3. The mean basophilic value for ELISA +ve flock number 1, 2, 3 and 4 were 1.61±0.60, 1.56±0.24, 1.56±0.24 and 1.48±0.37 respectively, compared with the mean value of healthy birds of flock numbers 1, 2, 3 and 4 which were 1.57±0.68, 1.48±0.68, 1.48±0.59 and 1.60±0.37, respectively.
The mean monocyte value for ELISA +ve flock were 14.50±0.13,14.50±0.10, 14.50±0.10 And 14.56±0.44 for flock number 1, 2, 3 and 4, respectively, mean value of the healthy birds were 9.19±0.57, 9.10±0.44, 9.10±0.44, 9.10±0.44 For flock numbers 1, 2, 3 and 4 respectively.

The organ body weight ratio showed a highly significant
increase in the organ body weight indices of affected liver, kidney, spleen, ovary and bursa of Fabricious.

The gross lesions mainly were great enlargement of the liver, spleen, kidney, ovary and bursa of Fabricious, with grayish white, or creamy white foci. In some organ the nodular tumor growth has also seen.
Histopathological studies of the affected birds revealed the infiltration of the lymphoblasts and other blood cells in the affected organ. Areas of necrosis at some places in affected organ were observed. The tumors cells showed the aggrigates of lymphoblast. The impression smears made from the fresh specimen of liver, spleen, kidney, ovary and bursa of Fabricious, stained with Giemsa stain, confirmed the lymphoid leucosis due to the presence of lymphoblast.

Out of 20 positive cases, liver was affected in all the cases, spleen in 18 cases, ovary in 15 cases, kidney in 11 and bursa of Fabricious in 14 cases.

It was concluded that ELISA can be used as a most reliable and effective method of diagnosis in live birds, whereas in dead birds, disease can be confirmed by the presence of lymphoblasts in impression smears of liver and spleen. Blood changes can be use as aid in early diagnosis, whereas histopathology is a time consuming procedure.




Department of Pathology

0514,T


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