2.1 Sample collection and Processing From the selected farms all dead as well as live sick chickens were collected with detailed particular of the outbreaks of IBD including farm location, history, age, breed, total number of birds and affected birds in farm, intervals between the batches, vaccine schedule, daily mortality and total mortality and clinical signs of affected birds were also recorded. In each case sampling was done following standard sampling methods and send to the laboratory. Different organ like liver, bursa of fabricious, breast and thigh muscle, kidney were collected during necropsy for further study. 10% neutral buffered formalin, Xylene, Hematoxylin and Eosin stain. PBS, Distilled water etc. were used for necropsy and histopathology of collected samples. All the diagnostic works were carried under the Laboratory of Department of Pathology & Parasitology, Hajee Mohammad Danesh Science and Technology University (HSTU). Clinical diagnosis and in some cases necropsy examinations were carried out at the place of sampling whereas histopathology of all samples were done in the laboratory.
2.2 Preparation of Harris’ Hematoxylin Solution and other Chemicals Hematoxylin crystals-5g, Alcohol (100%)-50 ml, Ammonium or potassium alum-100 g, Distill water1L, Mercuric oxide (red)-2.5 g. Hemoatoxylin was dissolved in alcohol and alum in water by heat. The two solutions were thoroughly mixed and boiled as rapidly as possible. After removing from heat, mercuric oxide was added to the solution slowly. The solution was reheated to a simmer until it became dark purple, and then the vessel was removed from heat and immediately plunged into a basin of cold water until it became cool. 2- 4ml glacial acetic acid was added per 100 ml of solution to increase the precision of the nuclear stain. Before use, the prepared solution was filtered. For 1% stock alcoholic eosin taking Eosin Y, water-soluble- 1g, Distilled water- 20 ml, 95% alcohol80 ml and finally Eosin was dissolved in water and then 80 ml of 95% alcohol was added. For working eosin solution- Eosin stock solution- 1 part, Alcohol, 80%- 3 parts then 0.5ml of glacial acetic acid was added to 100 ml of working eosin solution just before use.
2.3 Necropsy Examination of Suspected Birds The necropsy was done on the selected birds taken from suspected flocks. At necropsy, gross changes were observed and recorded carefully by systemic dissection based on routine necropsy examination. The lesion-containing tissues and organs were also collected and preserved in 10% neutral buffered formalin for histopathology.
2.4 Histopathological Study During necropsy, Bursa of Fabricius was collected, preserved in 10% buffered neutral formalin for histopathological studies. Formalin fixed tissue samples were processed for paraffin embedding, sectioned and stained with hematoxylin and eosin according to standard method.
2.5 Processing of Tissue and Sectioning The tissues were properly trimmed into a thin section to obtain a good cross section of the tissue and washed under running tap water for overnight to remove the fixative. Dehydrated in ascending grades of alcohol to prevent shrinkage of cells using 50%, 70%, 80%, 90% alcohol, and three changes in absolute alcohol, for 1hr in each. Then cleaned in two changes in chloroform to remove alcohol, 1.5hr in each. After that tissues were embedded in molted paraffin wax at 56-600C for two changes, 1.5hr in each. Paraffin blocks containing tissue pieces were made using templates and molted paraffin. Then the tissues were sectioned with a microtome at 5- 6µm thickness. The sections were allowed to spread on lukewarm water bath (40-45°C) and taken on a glass slide. A small amount of gelatin was added to the water bath for better adhesion of the section to the slide. The slides containing sections were air dried and stored in cool place until staining.
2.6 Routine Hematoxylin and Eosin staining Deparaffinization of the sectioned tissues was done by 3 changes in xylene (3 minutes in each).Rehydration of the sectioned tissues was done through descending grades of alcohol (3 changes in absolute alcohol, 3 minutes in each; 95% alcohol for 2 minutes; 80% alcohol for 2 minutes; 70% alcohol for 2 minutes) and distilled water for 5 minutes. The tissues were stained with Harris’ hematoxylin for 10 minutes. Then sections were washed in running tap water for 10-15 minutes and staining was differentiated in acid alcohol (1part HCl and 99 parts 70% alcohol), 2-4 dips. The tissue sections were then washed in tap water for 5 minutes and dipped in ammonia water (2-4 times) until sections became bright blue and stained with eosin for 1 minute and then differentiated and dehydrated in alcohol (95% alcohol, 3 changes, 2-4 dips in each; absolute alcohol 3 changes, 2-3 minutes in each), The stained sections were then cleaned by 3 changes in xylene, 5 minutes in each and finally the sections were mounted with a coverslip using DPX. The slides were dried at room temperature and examined under a low (10X) and high (40X, 100X) power objectives.
2.7 Statistical Study In this study the mortality rate was calculated by the following statistical formula-
Mortality rate % = Deaths occurring during a given time period / Birds Population during the same time period x 100
Prevalence of disease was calculated by the following statistical formula-
Prevalence % = IBD infected birds during specified time period / Birds Population during the same time period x 100