Genetic variation and pathological changes of Koi Herpesvirus (KHV) Infection of Cyprinus carpio

Abstract

KHV has made mass mortality in koi and common carp, and spread out to many countries. In Indonesia, KHV has caused outbreaks of mass mortality in many provinces, and cause economic losses and significant social matter. The aim of this research are to recognize and to analyze genetic variation of KHV that infects Cyprinus-carpio and to recognize its biogeographic distribution in Indonesia as well as to recognize and to analyze tissues pathological changes which infected by KHV. In this research, the fish that have been used are koi and common carp which suspected infected by KHV according to the clinical symptoms derived from 20 provinces in Indonesia. In every fish sample was taken its gill to PCR examination and then was done sequencing DNA (for KHV positive samples), for histopathology and immunohistochemistry examination, the organs that were taken: gill, kidney, spleen, intestine or digestive tract, liver, heart, and brain. Based on the results of sequencing DNA KHV and phylogenetic tree construction that has been made, there are 17 variants from 18 samples KHV positive was found. Those variants can be grouped into two clusters the main branch which consisted of group 1 includes variants KHV from South Kalimantan, Lampung, West Papua, West Kalimantan, West Java, Bali, East Nusa Tenggara. Then group II consists of KHV variants from North Sumatra, West Kalimantan, West Nusa Tenggara, Riau, East Kalimantan, and DKI Jakarta. Related to the infections of KHV variants on koi and common carp, pathological changes were found in such organs (gill, spleen, kidney, intestine and digestive tract, liver, heart, and brain). Pathological changes in gill organ was found proliferation of epithelial cells and fusion of secondary lamella, hypertrophy epithelial cells of gill lamella, telangiectasis, the inclusion body, edema, proliferation of hyaline layer and fibrosis at the base of the gills. Spleen pathological change was found infiltration of inflammatory cells or lymphocytes, appeared MMC, hemorrhage, congestion, and edema. Kidney pathological change was found hemorrhage, proliferation of cells in the interstitial, MMC, thickening of the tubule, inflammation of the glomerulus, the inclusion body, congestion, edema, fibrosis, necrosis of the glomerular. Intestine and digestive tract was found hemorrhage, enteritis, proliferation, goblet cells, fusion of the villi, deposit enterolit on gastric, congestion, edema, and necrosis in krypta. Liver pathological change was found hydropic degeneration, perivascular cuffing, congestion, and fibrosis. Heart pathological change was found infiltration of inflammatory cells or lymphocytes in the endocardium, hemorrhage, epicarditis, pericarditis, myocarditis, vacuolization, congestion, edema, fibrosis, and necrosis.

KHV diketahui telah menyebabkan kematian masal pada golongan ikan mas dan koi. Virus ini telah tersebar di berbagai negara di dunia. Di Indonesia, KHV dilaporkan telah menimbulkan wabah kematian masal di banyak provinsi, dan menyebabkan kerugian ekonomi dan sosial yang cukup besar. Berkaitan dengan hal tersebut, penelitian ini bertujuan untuk mengetahui dan menganalisis variasi genetik KHV yang menginfeksi Cyprinus carpio dan mengetahui sebaran biogeografisnya di Indonesia, mengetahui dan menganalisis perubahan patologis jaringan yang terinfeksi KHV, serta mengetahui dan menganalisis penyebaran KHV pada jaringan organ-organ target yang terinfeksi KHV. Dalam penelitian ini, ikan uji yang digunakan adalah ikan mas dan koi yang diduga positif terinfeksi KHV berdasarkan pada gejala klinis yang berasal dari 20 provinsi di Indonesia. Pada setiap ikan sampel diambil organ insang untuk pemeriksaan PCR, dan selanjutnya dilakukan sekuensing DNA (untuk sampelsampel positif KHV). Untuk pemeriksaan histopatologi dan uji imunohistokimia, organ yang diambil terdiri dari insang, ginjal, limpa, usus/saluran pencernaan, hati, jantung, dan otak. Pemeriksaan sampel dengan metoda PCR dimulai dengan melakukan ekstraksi DNA KHV dari insang, selanjutnya dilakukan amplifikasi produk DNA, elektroforesis, serta pengamatan dan dokumentasi. Hasil menunjukkan positif KHV jika terlihat ada pita DNA pada ukuran fragmen 290 bp. Uji PCR dilakukan untuk meneguhkan diagnosa KHVD terhadap ikan mas dan koi sampel yang menunjukkan gejala klinis melalui pengamatan secara makroskopik. Selain menggunakan metoda PCR dan DNA Sequencing, pemeriksaan KHV juga dilakukan dengan pemeriksaan histopatologik dan uji imunohistokimia. Pemeriksaan histopatologik dilakukan untuk mengetahui perubahan patologik pada organ-organ yang terinfeksi varian-varian KHV, dan dilakukan dengan menggunakan pewarna rutin hematoksilin eosin (HE). Selanjutnya, pada uji imunohistokimia dilakukan aplikasi dengan kromogen DAB. Perubahan warna menjadi cokelat/kecokelatan menunjukkan jaringan positif terinfeksi KHV.