Detection of Renibacterium salmoninarum and Yersinia ruckeri by the peroxidase-antiperoxidase immunohistochemical technique in melanin-containing cells of fish tissues

Immunohistochemical enzyme techniques are important tools for detection of microbial antigens in tissues. Such methods may substantially aid diagnostic work, especially for organisms which are slow-growing or difficult to culture. Since detection of infectious agents in fixed post-mortem material is often readily accomplished by those techniques, they may be excellent for retrospective studies. Thus, enzyme immunohistochemistry is increasingly used in work on infectious fish diseases; for example, viral diseases and bacterial infections. Among the various methods employed, non-conjugate methods, such as the avidin-biotin (ABC) and the peroxidase-antiperoxidase (PAP) techniques, in general offer the highest sensitivity. A problem with using enzyme techniques on fish tissues is the presence of melanin in macrophages, since the brown colour of melanin may be difficult or impossible to differentiate from that of the oxidation products of the chromogens commonly employed. This is particularly disturbing when, as often is the case, microbial antigens are lodged within the macrophage cytoplasm as a result of phagocytosis. The present authors used the PAP technique to detect immunoreactivity in melanin-containing macrophages of the spleens and kidneys in spontaneous Renibacterium salmoninarum and Yersinia ruckeri infections of rainbow trout, Oncorhynchus mykiss (Walbaum), and Baltic Salmon, Salmon salar L. They also studied the use of intense bleaching in order to eliminate confusion between melanin and chromogen end products.