Potential for cross-contamination of in vitro explant cultures initiated from Ichthyophonus-infected rainbow trout, Oncorhynchus mykiss (Walbaum).

In vitro culture of pathogens has been used for over a century to identify the causative organisms responsible for a variety of diseases (Koch 1881; Bass & Johns 1912; Dobell & Laidlaw 1926; Diamond 1960; Kocan 1969). For in vitro culture to correctly identify pathogenic organisms and/or determine infection prevalence, the parasite should be isolated in pure culture with a high degree of certainty that cross-contamination of cultures has not occurred. Recently, extensive field studies were conducted on the prevalence of Ichthyophonus in several species of fish using in vitro explant culture as a method for evaluating infection prevalence (Hershberger, Stick, Bui, Carroll, Fall, Mork, Perry, Sweeney, Wittouck, Winton & Kocan 2002; Kocan, Hershberger & Winton 2004). During the course of these studies, culture medium was sterilized, instruments were disinfected between fish and the investigators had extensive experience with in vitro culture techniques. However, because of the high infection prevalences detected, concern was expressed that cross-contamination of cultures may have occurred. Likewise, cross-contamination of cultures has been invoked to explain spurious data obtained in other field studies (Whipps, Burton, Watral, St-Hilaire & Kent 2006). Ichthyophonus is not difficult to culture, but unlike the laboratory environment, field conditions do not have many of the built-in safeguards that reduce the probability of cross-contamination. To evaluate the probability of cross-contamination under worst-case scenarios, we conducted two studies: a simulated field study and a controlled laboratory study, each designed to evaluate the potential for cross-contamination of cultures. Conditions encountered during field sampling of wild fish were simulated by initiating explant cultures from heart tissue derived from 48 oneyear-old rainbow trout, Oncorhynchus mykiss (Walbaum), sampled from three different raceways known to contain Ichthyophonus-infected fish. The infection prevalence for each raceway was not known at the time of sampling. To evaluate the probability of cross-contamination, heart tissue was excised from each fish and placed into Eagle s minimal essential medium, supplemented with 5% foetal bovine serum and antibiotics (Hershberger et al. 2002) by two experienced technicians using disinfected and contaminated instruments. Technician 1 disinfected instruments between fish by dipping scissors and forceps in 70% alcohol and wiping them with sterile gauze. Technician 2 used similar instruments for all cultures but did nothing to disinfect them between fish, i.e. no attempt was made to remove body fluids and host tissue from the instruments between fish. Journal of Fish Diseases 2008, 31, 317–320 doi: 10.1111/j.1365-2761.2007.00892.x