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...

An outbreak of mycobacteriosis was investigated in milkfish Chanos chanos, which had a cumulative mortality of up to 66.7% over the course of 1 yr. Gross reddish-or greyish-white nodules appeared on the peritoneal surface, spleen, kidney, liver and gastrointestinal (GI) tract. Epithelioid granulomas with the formation of Langhan's type giant cells were the prominent histopathological changes. Despite large numbers of acid-fast bacilli in the granulomas, neither caseous necrosis nor dystrophic calcification were observed. Using degenerate primers that targeted the heat shock protein 65 kDa gene of Mycobacterium spp., a 441 bp product was amplified. When compared with published sequences, our products were identical to those of Mycobacterium abscessus Type II (GenBank accession number AY603554). This is the first report of M. abscessus infection in milkfish. KEY WORDS: Mycobacteriosis · Mycobacterium abscessus · Visceral granuloma · PCR · Milkfish Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [147][148][149][150][151] 2006 MATERIALS AND METHODS Samples. In April 2001, approximately 55 000 milkfish were cultured in 2 brackish-cultured ponds and subsequently became infected by Mycobacterium abscessus. By November, affected fish showed signs of emaciation and reddish discoloration at the base of fins and anus. Cumulative mortality reached 66.7%. Five moribund fish were submitted to the Southern Taiwan Aquatic Animal Disease Diagnostic Center (STAADDC) for necropsy in March 2002.Pathology. Organs including brain, heart, gills, liver, spleen, kidney, gastrointestinal (GI) tract, mesentery, eyes and body wall were processed. Fish tissues were fixed in 10% neutral buffered formalin for at least 24 h prior to processing, except the gills and body wall, which were decalcified in 5% trichloroacetic acid for 24 h. The sampled tissues were dehydrated through a graded ethanol series, then embedded in paraffin, and sectioned at 5 µm. All sections were stained with haematoxylin and eosin (H&E), and selected sections were also treated with Ziehl-Neelsen acid-fast stain.DNA extraction and PCR amplification. Bacterial DNA was extracted from formalin-fixed, paraffinembedded tissue sections (Marchetti et al. 1998). For detection of Mycobacterium spp., PCR assays targeting the hsp65 gene were used to amplify a 441 bp of expectant product as previously described (Shinnick 1987, Ringuet et al. 1999.Cloning of PCR products. After purification (QIAquick Spin columns, Qiagen), PCR products were cloned into a T-vector using a TA cloning kit (YT&A; Yeastern Biotech), according to the manufacturer's instructions. They were then sequenced using a 373A automatic sequencer and a BigDye Terminator cycle sequencing kit (Mission Biotech).Analysis of sequence data. Sequence similarities in percentage were calculated with the PAM250 residue weight table by using MEGALIGN/DNASTAR software (DNASTAR, Madison). Molecular phylogenetic trees were constructed from nucleotide sequences to avoid ...