We studied amoebae associated with nodular gill disease (NGD) outbreaks in rainbow trout Oncorhynchus mykiss (Walbaum) in fish farms in South-Western Germany. Gills of 12 diseased rainbow trout were examined in fresh, by isolation attempts, histologically and using in situ hybridisation (ISH). A total of nine amoeba strains of the genera Acanthamoeba (1), Hartmannella (2), Naegleria (1), Protacanthamoeba (1) and Vannella (4) were isolated and determined using light microscopical, ultrastructural and molecular methods. Specific molecular probes designed from the SSU rDNA sequences of individual amoeba strains were used for non-radioactive ISH in histological sections. Association of Naegleria sp. with NGD and a direct ISH proof of Naegleria trophozoites attached to hyperplastic gill epithelium are novel findings, expanding the number of possible agents of NGD and supporting the hypothesis on multicausal aetiology of this disease.
Echinactinomyxon-type actinospores were found in a mixed-species oligochaete culture originating from the Temperate Water Fish Hatchery near Budapest, Hungary. On the basis of DNA sequence analysis, the actinospores were identified as Myxobolus pavlovskii (Akhmerov, 1954), the 18S rDNA sequence from myxospores of which is available in GenBank. Silver carp Hypophthalmichthys molitrix (Valenciennes) fry specimens were successfully infected by cohabitation with the echinactinomyxon-releasing oligochaetes, which confirmed the molecular data congruence. The echinactinomyxons and the myxospores that developed in the gills of exposed fish fry were analysed morphologically and on DNA basis. The infected gill tissue was examined histologically. As typical characters of M. pavlovskii, numerous small plasmodia were observed in the epithelia of gill lamellae. Plasmodia contained thousands of myxospores with polar capsules unequal in size and with large intercapsular processes. The 18S rDNA sequence from actinospores and those from myxospores originating from the experimentally infected fish were identical. The oligochaete species releasing actinospores was morphologically determined as Limnodrilus sp. This is the first record of an echinactinomyxon as an alternate stage within the genus Myxobolus.