Archived formalin-fixed, paraffin-embedded tissues from 28 diseased ornamental cichlid fish associated with visceral granulomas were examined by polymerase chain reaction (PCR) and in situ hybridization (ISH) for detection of Francisella-like bacteria (FLB). The 16S rDNA FLB-specific primer pair 180f/465r was used on naturally infected ornamental cichlids, resulting in 11 positive cases (39%). Using DNA probes, all 28 cases (100%) showed a positive reaction, and most labeled cells were observed in the visceral granulomas of infected individuals. FLB was detected in cells morphologically resembling epithelioid and endothelioid macrophages. ISH was more sensitive than PCR or routine histopathological examination, based on the examination of archived formalin-fixed, paraffin-embedded tissues in this study. Furthermore, this technique located a new fish pathogen, FLB, in ornamental cichlids. The causative agent was similar to the pathogen inducing systemic granulomas in tilapia.
KEY WORDS: Francisella-like bacterium · FLB · PCR · In situ hybridization · Cichlid · Ornamental fish
Resale or republication not permitted without written consent of the publisherDis Aquat Org 75: [29][30][31][32][33][34][35][36] 2007 Disease Diagnostic Center [STAADDC] and collected between 1998 and 2002) of ornamental fish with visceral granulomas for investigation of the causative agent. A DIG-labeled DNA probe specific for a novel FLB partial 16S rRNA gene (base pairs 180 to 485) was developed for localization of the agent in the infected tissues of ornamental fish. This is the first report concerning granulomatous disease in ornamental fish due to infection with FLB, and comparing the sensitivities of ISH, PCR and histopathological assays.
MATERIALS AND METHODSSampling. Twenty-eight diseased cichlid ornamental fish, consisting of 11 species: firebird Aulonocara rubescens, elegans Pseudotropheus elegans, zebra Pseudotropheus zebra, Rhodes's chilo Chilotilapia rhoadesii, Malawi eyebiter Dimidiochromis compressiceps, brown discus Symphysodon aequifasciatus, deep-water hap Haplochromis electra, electric blue hap Sciaenochromis fryeri, blue-white labido Labidochromis caeruleus, Placidochromis milomo and Frontosa cichlid Cyphotilapia frontosa were used as samples for study by PCR and ISH (Table 1). The disease had been tentatively diagnosed as FLB infection by cytological and histopathological examinations. Samples from 3 FLBfree firebird A. rubescens, as diagnosed by PCR and ISH, were selected as negative controls.Tissue processing. Brain, eye, gill arch, kidney, spleen, liver, heart and gastrointestinal (GI) tract were removed and cut into small pieces of ~5 mm 3 , and placed into neutral buffered formalin solution (20:1 [v/v] ratio of fixative to tissue) for 16 h. The fixed samples were dehydrated through an alcohol series and embedded in paraffin according to standard laboratory procedures. Serial sections 4 µm thick were cut, floated on the surface of a water-bath and mounted on positively charged slides (Muto Pure Chemic...
