Histopathological examination and optimization of Multiplex PCR protocol for diagnosis of viral respiratory diseases in commercial poultry

Abstract

The present work was to optimize a multiplex PCR protocol for rapid and accurate diagnosis of avian respiratory viral diseases. Four sets of specific oligonucleotide primers were used in this study that amplified products of predicted sizes from each virus in the sRT-PCR as well as in the mPCR assays (1023, 320, 647, 149 bp for AIV, NDV, ILTV, and IBV respectively) with the use of 1% agarose gel electrophoresis for PCR amplified products. A total of 48 specimen samples included trachea, lungs, liver, spleen, proventriculus etc. were collected from different dead birds that were tentatively diagnosed as respiratory viral infection on the basis of anamnesis and postmortem lesions by the expert veterinary clinician. Samples were preserved in 10% buffered formalin for histopathological study and in 100% alcohol with -80oC refrizaration for molecular study like RNA/DNA extraction for PCR or RT-PCR. A questionnaire was used during the period from August 2013 to March 2014 to record additional information and history of the cases such as age and type of birds, litter type, vaccination history, farm category, sex, pathological lesions, histopathology etc. Out of 48 cases 35.42% was tentatively diagnosed as AI, 33.33% was ND and 12.25% was IB where visceral gout was most prominent lesion. No ILT was diagnosed tentatively through necropsy. Mixed infection is common in commercial chicken flock, among the cases 4.17% was diagnosed as AI+ND, 12.25% and 2.08% was diagnosed as AI/ ND and IB/ND respectively. Among 48 test samples both the PCR screening process a total of 33(68.75%) samples showed positive band in 1% agarose gel electrophoresis where 15(31.25%), 16(33.33%) and 2(4.16%) samples were found positive for AI, ND and IB respectively. No ILT was found in this screening. Among the diseases AI was diagnosed accurately in 70.58% cases through necropsy and 81.25% and 33.33% for ND and IB respectively. ILT was not diagnosed tentatively which was also proved by molecular diagnosis that indicates the 100% accuracy of the tentative diagnosis. Microscopically congestion and hemorrhage in trachea, lungs, liver and spleen were found commonly in most of the samples, in addition with exudates in trachea and hemorrhagic kidney with urate crystal in renal tubules in IB infected sample. Hemorrhage in proventriculus is also found in only AI and ND infected cases.