Disease investigation This investigation was carried out in the Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University (BAU), Mymensingh. A pullet farm of Cox’s bazar district had 2200 chickens that showed progressive weight loss, higher rate of morbidity and mortality. There was paralysis of legs and wings and the disease was suspected as a case of MD. Extensive investigation was, therefore, carried out for confirmatory detection of the specific cause of illness. A total of 10 pullets were investigated and samples were collected at necropsy during May 2016. Systemic dissection and analysis were carried out on to the liver, sciatic nerve, feather follicles, intestine, skeletal muscles, kidneys, spleen, heart, lungs, and skin to unveil the cause of illness. Histology of tissue sections The collected tissue samples were preserved in 10% buffered neutral formalin and processed for histopathological investigation. Thin sections of liver, spleen, heart, kidney, skeletal muscle, feather follicles, sciatic nerve, intestine and lungs were embedded in paraffin, sectioned at 4-5µm using a microtome, deparaffinized in xylene and stained with H&E (Luna, 1968). The stained sections on to the slides were mounted using DPX, air dried and studied under low and high power microscopic fields. Giemsa staining of smears The liver of sick and dead pullets was cut into fine pieces, soaked with paper towel and impression smears were made onto the clean slides. The slides were air dried, fixed in ice cool absolute methanol for 45 mins. Smears were also taken from the venous blood onto clean slides, fixed for 45 mins in ice cool methanol and air dried. The slides were stained with Giemsa’s for 45 mins (Luna, 1968), washed in distilled water, air dried and examined under 100x microscopic field. Special emphasis was given to identify the type of mononuclear cells distributed in the smears. Detection of Meq gene of MDV by PCR Sections of liver, spleen, heart, kidney, skeletal muscle, feather follicles, sciatic nerve, intestine and lungs were preserved at -20ºC. The tissues were used to extract genomic DNA and PCR detection of Meq gene of MD viruses. Traditional method was used to extract DNA (N=10) from tissues of suspected pullets. Briefly, about 200 mg of individual tissue was macerated on sterile mortar and pestle while still in frozen. The crushed samples were transferred into the eppendorf tube containing 600μl cell lysis buffer and incubated at 56ºC for an hour. The tissue suspension was then centrifuged at 5000g for 10mins. The supernatant was collected in a fresh tube, 3μl of RNase solution was added to the nuclear lysate and mixed by inverting the tube 2-5 times. The mixture was incubated for 25 mins at 37°C. Equal volume of phenol chloroform isoamyl alcohol (25:24:1) was added and vortexed vigorously for 20 seconds. The mixture was centrifuged at 10000g for 2mins and supernatant was collected. Then 1/10th volume of 0.5N NaCl and 2.5 times ice cool absolute ethanol was added and incubated on ice for 30 mins. The solution was centrifuged at 13000g for 15 mins at 4ºC and the supernatant was carefully discarded. The pellet was desalted twice with 70% ethanol by centrifugation at 13000g for 15 mins at room temperature. The ethanol was carefully aspirated, the DNA pellet was air dried and 50μl nuclease free water was added. The purity and concentration of extracted DNA was measured by using agarose gel (1.5%) electrophoresis and spectophotometry (A260/A280). The concentration of genomic DNA (100ng/μl) was adjusted by adding nuclease free water. Ratio of A260 and A280 greater than 1.8 was considered as high purity and used in PCR. Appropriate primer sequences for the PCR detection of Meq gene of MD viruses (Table 1) were used as described previously. PCR reactions was performed on each DNA sample in a 25μl volume consisting of reaction mixture (12.5μl), primers (forwards and reverse, 25 pmol/1μl each), template DNA (5μl) and nuclease free H2O (5.5μl). The thermal profile consisted of an initial denaturation for 2 mins at 94°C followed by 35 cycles of DNA amplification reaction in a Master Cycler (Master Cycler Gradient, Eppendorf, Germany). The condition of PCR amplifications were denaturation for 60 secs at 94°C, annealing for 60 secs at 55°C and extension for 3 mins at 72°C followed by a final extension for 5 mins at 72°C. The PCR reactions were held at 4°C and the reaction was terminated by adding 3µl 50 mM EDTA. The PCR amplicons were analyzed by electrophoresis in 1.5% agarose gel, stained with ethidium bromide and examined under UV light using an image documentation system (Cell Biosciences, Alphalmager HP, USA).