Enzootics of visceral granulomas associated with Francisella-like organism infection in tilapia (Oreochromis spp.)

Aquaculture 254 (2006) 129 – 138 www.elsevier.com/locate/aqua-online

Enzootics of visceral granulomas associated with Francisella-like organism infection in tilapia (Oreochromis spp.) C.Y. Hsieh a , M.C. Tung a , C. Tu b , C.D. Chang a , S.S. Tsai a,⁎ a

Department of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shen-hu Rd., Neipu, Pingtung 912, Taiwan b National Animal Health Institute, Taipei, Taiwan Received 18 June 2005; received in revised form 29 March 2006; accepted 29 March 2006

Abstract Between 2001 and 2004, 10 strains of Francisella species were isolated from visceral granulomas of diseased tilapia in Taiwan. In a comparison of nucleotide sequences for whole 16S rRNA gene with those of reference bacteria, the isolated strains had a high sequence similarity to Francisella philomiragia (98.6%), Francisella tularensis subsp. novicida (97.4%) and F. tularensis subsp. tularensis (96.1%). On the basis of electron microscopic examination, phenotypic characteristics and PCR assays for the 16S rRNA gene sequence, the causative agent clearly belongs to the genus Francisella. © 2006 Published by Elsevier B.V. Keywords: Tilapia; Granuloma; Intracellular organism; Polymorphic cocco-bacillus; Francisella sp.

1. Introduction In Taiwan, tilapias infected with an unknown intracellular organism were initially found in October 1992. Now, the disease is widespread in fresh-, brackish- and salt pond-cultured tilapia, involving six species and causing high mortality, up to 95% in some cases (Chen et al., 1994; Chern and Chao, 1994). Prominent features are skin ulceration and the presence of multiple whitish nodules on most visceral organs, especially the kidney and spleen. The agent is a coccobacillus with a polymorphous shape, which can be cultured in CHSE-214 and TO2 cells, but does not grow on artificial synthetic media. Therefore, it was provisionally called a ‘rickettsia-like organism’ (RLO) (Chen et al., 1994; Chern and Chao, 1994). ⁎ Corresponding author. Fax: +886 8 7740295. E-mail address: [email protected] (S.S. Tsai). 0044-8486/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2006.03.044

Francisella spp. are facultative intracellular organisms, capable of penetrating intact skin or penetrating via skin injury to elicit a local inflammatory response with formation of papules and ulcers in situ. Following lymphohematogenous dispersion, the agents can invade parenchymal tissue resulting in the formation of granulomas and abscesses. Francisella tularensis and Francisella philomiragia are the most prevalent agents associated with chronic and necrotizing granulomas (Quan et al., 1956; Mörner, 1992; Sicherer et al., 1997; Polack et al., 1998). Waterborne transmission of Francisella spp. is well known for causing disease in both humans and aquatic animals (Mignani et al., 1988; Hoel et al., 1991; Polack et al., 1998). These organisms have been identified by both culture and PCR assay from crayfish (Anda et al., 2001), muskrats (Jensen et al., 1969) and water (Forsman et al., 1995). Previous research on outbreaks of RLO in Taiwan had been limited to southern Taiwan (Chen et al., 1994;

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Chern and Chao, 1994). In the present study, we collected 120 diseased tilapia from northern, central and southern Taiwan. These fish had similar pathology to those with RLO infections, but these were found to be associated with a different agent, a Francisella species, based on electron microscopic, bacteriological and molecular examinations.

tions. Biochemical tests were performed by conventional methods (Hollis et al., 1989; Clarridge et al., 1996) and using RapID NH and RapID ANA (Remel, Lenexa, KS, USA) commercial identification kits, according to the manufacturer's directions.

2. Materials and methods

For ultrastructural examination, samples of kidney and spleen were fixed in 2.5% glutaraldehyde, postfixed in osmium tetroxide, dehydrated in a series of graded alcohols and embedded in Supper's resin. Thin sections were stained with uranyl acetate and lead citrate, and observed with a Hitachi H-600 electron microscope. For scanning electron microscopy, a Francisella-like organism (FLO) was collected from Thayer–Martin agar plates and centrifuged at 2000×g for 15 min at 4 °C, washed with PBS, fixed in 3% glutaraldehyde for 2 h at room temperature and small drops of the samples were placed on a presterilized 0.22μm membrane of a vacuum-driven disposable bottle-top filter (Millipore, Bedford, MA, USA) and allowed to adhere. Thereafter, the samples were post-fixed with 1% osmium tetroxide plus 1% tannic acid, dehydrated in graded ethanol, critical point-dried in CO2, coated with a 3.5-nm-thick chromium layer using a penning sputter system in a high-vacuum chamber and viewed with a Hitachi S3000N scanning electron microscope. Images were obtained using secondary and backscattered electrons.

2.1. Cytological and pathological examinations Duplications of organ smears were made from the spleen and kidney of the diseased fish. The smears were stained with Liu's and Gram methods, respectively. Fish tissues were fixed in 10% neutral buffered formalin for at least 16 h and processed for routine paraffin histology, sectioned at 3–4 μm, and stained with haematoxylin and eosin. 2.2. Isolation and bacteriological examinations Both kidney and spleen were homogenized in a phosphate buffer solution using a Ten-Broeck tissue grinder and centrifuged at 500×g for 2 min at 20 °C. The upper layer was collected and spread over Thayer– Martin agar (Anda et al., 2001). Blood agar, brain–heart infusion agar (containing 5% sheep red blood cells) and blood–cystine–glucose agar were used simultaneously for bacterial isolation. Inoculated media were incubated at 23, 30 and 35 °C with 5% CO2 for a minimum of 10 days, respectively. At the same time, the kidney and spleen emulsions were inoculated on CHSE-214 (chinook salmon embryo) cells according to the method described previously (Fournier et al., 1998), and incubated with 5% CO2 at 23 °C for observation of cytopathic effect (CPE). The infected cells were harvested for light and electron microscopic examina-

2.3. Electron microscopic examinations

2.4. DNA extraction The isolates used for molecular assay included AF01-2, AF-01-6, AF-01-22, AF-01-23, AF-01-27, AF-0128, AF-03-27, AF-03-28, AF-04-15 and AF-04-405 (Table 1). A loop of bacterium was transferred into an Eppendorf tube containing 200 μl of lysozyme reaction

Table 1 Location for sampling, 10 clinical cases Samples no.

Exposure date

Location

Fish species

Water

1. AF-01-02 2. AF-01-06 3. AF-01-22 4. AF-01-23 5. AF-01-27 6. AF-01-28 7. AF-03-27 8. AF-03-28 9. AF-04-15 10. AF-04-405

01/04/2001 01/25/2001 02/14/2001 02/25/2001 02/26/2001 03/15/2001 03/17/2003 03/17/2003 03/01/2004 12/05/2004

Kaohsiung City Kaohsiung City Kaohsiung City Kaohsiung County Pingtung County Hsinchu County Chia-I County Chia-I County Pingtung County Pingtung City

O. mossambica O. mossambica O. niloticus O. niloticus O. mossambica O. mossambica O. mossambica O. mossambica O. niloticus O. niloticus

B B B B F F F F F F

F = fresh water; B = brackish.

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solution (20 mM Tris–HCl (pH 8.0), 2 mM EDTA, 20 μg of lysozyme/ml). It was then suspended through vigorous vortexing and incubated at 37 °C for 30 min. The cells were lysed by 2.5 mg of proteinase K (Clontech) per ml and incubated at 60 °C for 1 h. The lysate was subjected to DNA extraction with a commercial kit (Blood and Tissue Genomic DNA Extraction Miniprep System; Viogene, Taipei, Taiwan). DNA was finally eluted with 100 μl of TE (Tris–EDTA) buffer and stored at − 4 °C before use. 2.5. Polymerase chain reaction (PCR) assay and nucleotide sequencing of whole 16S rRNA gene Eubacteria universal primers (forward, 27f, 5′-AGA GTT TGA TCM TGG CTC AG-3′ and reverse, 1525r, 5′-AAG GAG GTG WTC CAR CC-3′) were specifically designed for the amplification of most eubacterial

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16S rRNA genes (Lane, 1991). Specific primers (forward, F11, 5′-TAC CAG TTG GAA ACG ACT GT-3′ and reverse, F5, 5′-CCT TTT TGA GTT TCG CTC C-3′) were used for the detection of Francisella genus (Forsman et al., 1994). The PCR buffer (pH 8.5) contained 60 mM Tris–HCl, 15 mM (NH4)2SO4, and 1.5 mM MgCl2. Control reactions without template were included. After purification of PCR products (QIAquick spin columns; Qiagen, Hilden, Germany), they were cloned into T vectors using a TA cloning kit (YT&A; Yeastern Biotech, Taipei, Taiwan), according to the manufacturer's instructions, and sequenced, using a 373A automatic sequencer and a BigDye Terminator cycle sequencing kit (Mission Biotech, Taipei, Taiwan). Both strands were sequenced as a crosscheck. The sequences determined (1520 bp) were aligned and compared with the sequences in GenBank by using the multiple alignment algorithms in the MegAlign package

Fig. 1. Pathological, cytological and ultrastructural examinations of Francisella-like organism (FLO)-infected tilapia. (A) Grossly, infected tilapia shows white nodules of varying sizes in the enlarged spleen (S), kidney (K) and gill (G). (B) Many Gram-negative organisms (arrows) are observed within the cytoplasmic vacuoles of a phagocyte on the smear made from kidney (Gram stain). (C) Histopathological lesions of the kidney reveal multiple granulomatous formations with a necrotic center, encapsulated by multiple layers of epithelioid or foamy cells and fibrous tissues (arrows) (H&E stain). (D) Ultrastructural examination of the kidney reveals that the intracellular organisms (⁎) are extremely irregular and pleomorphic in shape, of 1.45 ± 0.35 × 0.35 ± 0.15 μm in size, and appear in the cytoplasmic vacuoles of a phagocyte. The organisms are separated from the cytosol only by a narrow electron-lucent space (arrows).

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(Windows version 5.0.221; DNASTAR, Madison, WI, USA). 2.6. Preparation of nucleic acid probe from 16S rRNA gene of FLO The DIG-labeled FLO 16S rRNA gene probe was generated with the PCR DIG Probe Synthesis kit (Roche Molecular Biochemicals, Mannheim, Germany), following the manufacturer's instructions. The DNA probe was synthesized by incorporation of digoxigenein-11dUTP during PCR using Francisella-specific primers, F5/F11 (Forsman et al., 1994). Briefly, a 200-μl reaction tube was added to the following final concentration of reagents: 1× PCR buffer (10 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2), 200 μM PCR DIG Probe Synthesis Mix, 200 pM each of forward and reverse primers, 1.5 U Taq DNA polymerase, 100 pg plasmid templates and distilled water with a final volume of 100 μl. The PCR was carried out following cycling conditions of 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. The resulting DIG-labeled FLO 16S rRNA gene probe was quantified further with DIG Quantitation Teststrips, following the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany).

0.4% BSA, Ficoll, PVP360, respectively, in dd H2O), 5% Dextran sulfate and 0.5 mg/ml sheared salmon sperm DNA. Hybridization was done overnight at 42 °C. The digoxigenin-labeled probe (1 ng μl in the standard hybridization buffer) and DNA in the tissue slides (tissues were covered with the standard hybridization buffer) were heated for 6 min at 95 °C on a heating block and quenched on ice for 1 min. The tissue slides were then replaced with 1 ml fresh standard hybridization buffer containing approximately 50 ng of the digoxigenin-labeled probe, and hybridization was performed at 42 °C overnight in a humid chamber. Subsequently, sections were thoroughly washed twice in 2× SSC for 5 min at 20 °C, once in 0.1× SSC for 10 min at 42 °C, and once in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) for 15 min at 20 °C. For detection of hybridization, sections were incubated with anti-digoxigenin conjugated with alkaline phosphatase (Roche Molecular Biochemicals) diluted 1:500 in 0.1 M

2.7. In situ hybridization By using PCR assay, 10 FLO-infected fish and three FLO-free fish were selected for in situ hybridization. The organs of the fish were placed in Bouin's fixative solution (10% formalin and 5% glacial acetic acid) at a 10:1 (fixative/tissue by volume) for 16 h. The fixed samples were dehydrated through an alcohol series and embedded in paraffin, according to standard laboratory procedures. Sections were cut into 3-μm-thick serial sections, floated on a water bath and mounted on silanecoated slides (Muto Pure Chemicals, Tokyo, Japan) for in situ hybridization. Sections were heated at 60 °C for 30 min, deparaffinized in xylene and subsequently rehydrated in PBS (pH 7.4 and 0.01 M) for 5 min. Deproteinization was carried out in 0.2 N HCl for 20 min at 37 °C in proteinase K (Clontech) 100 μg/ml in PBS in a humid chamber. After digestion, tissues were fixed in 0.4% cold formaldehyde in PBS for 5 min. After rinsing twice with a 2× saline sodium citrate (SSC) (1× SSC contains 50 mM NaCl and 15 mM sodium citrate, pH 7.0), the slides were allowed to equilibrate for 60 min at 42 °C in a humid chamber in a standard hybridization buffer consisting of: 4× SSC with 50% deionized formamide, 1× Denhardt's (20× Denhardt's contains

Fig. 2. Francisella-like organisms inoculated on CHSE-214 cell line and Thayer–Martin agar. (A) The agents induce CPE in the CHSE-214 cell culture at approximately 7 days post-inoculation. These are frequently seen within the cytoplasmic vacuoles of CHSE-214 cell. (B) Francisella-like organisms collected from Thayer–Martin agar plates and examined by scanning electron microscope. The morphology of the agents is extremely irregular and pleomorphic.

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Tris–HCl (pH 7.4) and 0.1 M NaCl, with 1% blocking reagent (Roche Molecular Biochemicals). After three washes in maleic acid buffer, the substrate consisting of nitroblue tetrazolium (NBT) and 5-bromocresyl-3indolyl phosphate (BCIP) was layered over the sections, covered with a coverslip and left overnight in the dark. The slides were then washed with distilled water for 1 min and stained a few seconds with 0.5% methyl green (Sigma-Aldrich, St. Louis, MO, USA). The slides were mounted with Eukitt mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA).

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zone (Fig. 1C). It was very interesting to find that large foamy cells were frequently observed in the necrotizing or granulomatous lesions, except for very old lesions. 3.2. Ultrastructural study

3. Results

Transmission electron microscopic examination revealed that the intracellular organisms were extremely irregular and pleomorphic in shape, with a size of 1.45 ± 0.35 × 0.35 ± 0.15 μm, and observed within the cytoplasmic vacuoles of phagocytes. At high magnifications, some organisms were separated from the cytosol only by a narrow electron-lucent space (Fig. 1D).

3.1. Pathological and cytological examination

3.3. Isolation and bacteriological examination

Grossly, the fish had or had no skin ulcerations. Whitish nodules with multifocal distribution were found on most organs, including the kidney, spleen and liver (Fig. 1A). Large amounts of Gram-negative coccobacilli with polymorphic shapes were found intracellularly on organ smears stained with the Liu's and Gram stain methods (Fig. 1B). Based on histopathological examination of 120 tilapia, granulomas were found in the spleen (100%), kidneys (100%), liver (85.8%), gills (72.5%), gonads (63.3%), gastrointestine (56.7%), heart (47.5%), swim bladder (40%) and eyes (30.8%). The initial lesions were multi-focal aggregations of large foamy cells containing cocco-bacilli in their cytoplasm. Necrosis gradually developed in the central area and, finally, fibrous encapsulation formed in the outermost

FLO induced CPE in CHSE-214 cells at approximately 7 days post-inoculation. The organism was frequently seen within the cytoplasmic vacuoles of CHSE-214 cell (Fig. 2A). Bacterial colonies developed slowly, requiring 3–6 day incubation at 23 °C on Thayer–Martin agar. Smooth colonies with a grayish pigment were observed; however, they showed little or no growth at 30 °C and none at 35 °C. Under the scanning electron microscope, the morphology of FLO was extremely irregular and pleomorphic (Fig. 2B). None of the isolates grew on blood agar, MacConkey agar, brain–heart infusion agar containing 5% sheep red blood cells, blood–cystine–glucose agar or in 6% NaCl. The organisms were strict aerobes, non-motile and had negative reactions for reduction of nitrate, beta-

Table 2 Comparison of distinguishing phenotypic features of all FLO isolates with F. tularensis, F. novicida, and F. philomiragia Test system

Rapid NH

Rapid ANA

Miscellaneous test

Test category or substrate

ID no. Hydrolysis of amides (proline) Hydrolysis of glucosides (ONPG) Utilization of carbohydrates (sucrose) Reduction of resazurin ID no. Hydrolysis of L-arabinoside and ONPG Hydrolysis of amides (leucine, glycine, and proline) Growth on SB-TSA at 2 days Growth in 6% NaCl Growth at 23 °C Growth at 30 °C Growth at 35 °C Oxidase (tetramethyl-p-PDD) Catalase

Result for strains All FLO isolates

F. tularensis

F. novicida

F. philomiragia

3730 + − + − 000110 −, − −, −, + − − + +/wk − − +/wk

2710 − − − −/+ 000763 −, − −, −, +/wk − − + + + − +/wk

3710 + − + + 000773 −, − +, +, + + + + + + − +/wk

7710 + + + + 014773 −, + +, +, + + + + + + + +

Source: Data for known species cited from Hollis et al. (1989) and Clarridge et al. (1996). + = positive; − = negative; wk = weak; +/wk = weakly positive.

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Fig. 3. Results of PCR amplification. (A) The expected PCR products of all FLO isolates are obtained using 27f/1525r primers complementary to conserved regions in all eubacterial 16S rRNA gene, and (B) using a Francisella-specific F5/F11 primer pair. Lane M: 100 bp DNA molecular weight marker. Lanes 1–10, DNA extraction from isolates: AF-01-2, AF-01-6, AF-01-22, AF-01-23, AF-01-27, AF-01-28, AF-03-27, AF-03-28, AF-04-15 and AF-04-405, respectively. Lanes 11 and 12, DNA extraction from F. tularensis subsp. tularensis (NCTC 10857) and F. tularensis subsp. novicida (CIP 56.12), respectively. Lanes 13 and 14, F. philomiragia (ATCC 25015) and F. philomiragia (ATCC 25017), respectively. Lane 15, tissues genomic DNA extraction from FLO-free tilapia. Lane 16, no DNA template; negative control.

Fig. 4. Level of sequence similarities and evolutionary distances, based on alignment of 1439 nucleotides of 16S rRNAs from Francisella species, and some reference species belonging to the γ subclass of the Proteobacteria. Construction of the evolutionary distance tree is carried out using the multiple alignment algorithms in the MegAlign package (Windows version 5.0.221; DNASTAR), and shows the relationships among the organisms used in this study and members of the γ subclass of the Proteobacteria that exhibit the highest levels of similarity to members of the genus Francisella. Piscireckettsia salmonis is included as an outgroup. The unrooted tree is constructed by the Clustal algorithm after evolutionary distances have been calculated from nucleotide substitution values. The NCBI accession numbers for sequences used in the alignment are shown as follows: Francisella sp. CYH-2002, AF385857; Francisella philomiragia, AY928394; Francisella novicida, AY928396; Francisella tularensis, Z21931; Wolbachia persica, M21292; Ornithodoros moubata symbiote B, AB001522; Piscireckettsia salmonis EM-90, U36940; Piscireckettsia salmonis NOR-92, U36942; and E. coli, J01695.

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galactosidase and resazurin, and hydrolysis of glucoside, ONPG, L-arabinoside, leucine and glycine. They were weakly positive for catalase, but strongly positive for β-lactamase, proline and sucrose (Table 2).

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3.4. PCR assays In our isolates, the sequences of PCR products amplified by eubacterial universal primers 27f/1525r for

Table 3 Sequence signatures for the genus Francisella and FLO Position a

140 141 143 189 207 208 210 211 213 214 215 219 220 271 383 421 460 461 462 463 473 475 476 477 480 489 503 546 548 648 661 673 679 748 840 853 866 867 875 999 1033 1035 1036 1037 1053 1292 1488 1524 a b

Nucleotide in: Francisella strains b All FLO isolates

F. philomiragia

F. novicida

F. tularensis

P. salmonis

E. coli

C U U C C U U G C G C G C U A U G U G A U G C A A G G C G A U A C A U A G C U A C U C G G G A G

U U A U – – U A G G C G C U A U A U A/G A U G U A A A G C G G U G U A U A G C U A C U C G G G G G

U U A U U U C G G G C G U C C C C A A G C U G G G A C G A G C G U G G C G C A G C U C G G A G G

U U A U U U C G G C G G U C C C C A A G C U G G G A C G A G C G U G G C C G A A U C – – – A G –

U A U A U A A U A G C C U U A/G C G C U A U G C U A A C G G G U G U A U G G C A A C U C G G G G G

U G A A C U C G G C C C U C C C U A A A U U G C A A C G A G U G U A U G G C U A C U C G G U G G

Escherichia coli numbering. Bases found in Francisella species, including P. salmonis.

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16S rRNA gene were 1520 nt long. All isolates yielded appropriate amplified PCR products (∼1140 bp) using Francisella genus-specific primers F11/F5 (Fig. 3). These sequences were deposited in GenBank and were given accession numbers from AY928388–AY928393, and from DQ007453–DQ007456. Blast searches of GenBank yielded high sequence similarities of 98.6% for F. philomiragia, 97.4% for Francisella novicida, and 96.1% for F. tularensis (Fig. 4). Compared to reference strains, the nucleotide differences are shown in Table 3. Based on the high sequence similarities of whole 16S rRNA genes and phenotypic characteristics, all our isolates certainly belonged to the genus Francisella. 3.5. In situ hybridization In order to investigate the relationship between granulomas and FLO, FLO-infected and FLO-free tilapia were processed for in situ hybridization using

Fig. 5. In situ hybridization of tilapia (Oreochromis spp.) kidney with digoxigenin-labeled oligonucleotides complementary for Francisellalike organisms 16S rRNA sequences. (A) Positive reactions using DIG-labeled probe are localized in the granulomas of tilapia kidney with natural FLO infection; brown pigment signals (arrows) are observed mainly in the foamy cells. (B) The intracellular organisms, FLO, (arrowheads) appear in the cytoplasmic vacuoles of phagocytes.

FLO 16S rRNA DIG-labelled DNA probes. Positive reactions, characterized by a brown precipitate, were detected in FLO-infected tilapia and none in FLO-free fish. The brown pigments were localized mainly in the large foamy cells of the granulomas (Fig. 5A and B). 4. Discussion Between 1994 and 2003, a Hawaiian tilapia Piscirickettsia-like organism (HTPLO) was reported as causing significant losses on certain farms (Mauel et al., 2003). Multiple granulomas were observed in the gills, spleen, kidney, choroid gland and testes, but not in the liver. This organism also caused enzootic diseases in Jamaica, Indonesia, Florida and southern California. However, the agent cannot be isolated by cell culture. Pathologically, HTPLO is similar to RLO and our cases described from Taiwan. We also used the conserved primers PS2S and PS2AS of Piscirickettsia salmonis (Mauel et al., 1996) to amplify our samples, but no amplicon was obtained (data not shown). Microbial pathogens of fish are routinely diagnosed via isolation and identification methods. However, a number of fish pathogens could be consistently visualized, but failed to grow. Although the intracellular cocco-bacillus in tilapia was successfully cultivated in CHSE-214 cells and on Thayer–Martin agar in Taiwan, its isolation was difficult and time-consuming, due to the lack of convenient culture systems and the presence of bacterial contamination in the inoculum. All our isolates belonged to a unique group of low-temperature growing organisms. They showed similar morphological and biochemical characteristics to F. tularensis, F. novicida, and F. philomiragia (Table 2). Universal primers for the bacterial 16S rRNA gene have been used to detect the presence or absence of eubacteria (Lane, 1991), Rickettseae (Roux and Raoult, 1995), bacterial endosymbionts (O'Neill et al., 1992; Schröder et al., 1996), P. salmonis (Mauel et al., 1996, 1999), and uncultured bacillus of Whipple's disease (Relman et al., 1990, 1992). We used broad-range eubacterial primers to amplify a novel 16S rRNA gene from both our tissue specimens and bacterial isolates. Sequences obtained were identical. In a comparison of the 16S rRNA gene sequences of our isolates with some reference strains of Proteobacteria associated with intracellular pathogens, very high levels of sequence similarity were found, but it was quite different from those of P. salmonis (Fig. 4). Despite the high sequence similarities between our isolates and reference strains, the nucleotide sequences of our isolates were different from F. philomiragia by

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14 nt (2 insertions and 12 substitutions—one base of the F. philomiragia sequence in GenBank is AY928394), F. novocida by 34 nt (0 insertions, 34 substitutions—one base of the F. novicida sequence in GenBank is AY928396), and F. tularensis sequence by 43 nt (4 insertions, 39 substitutions—one base of the F. tularensis in GenBank is Z21931) (Table 3). By using nonradioactive-labeled DNA probes, the FLO nucleic acids could be detected in formalin-fixed, paraffin-embedded tissue specimens, such as the spleen, kidney, liver and gill of tilapia naturally infected with FLO. Detection of hybridization signals provided scientific evidence that the cells were certainly infected with FLO. The results also indicated that the major cellular sites of infection were mononuclear cells. An exclusive cytoplasmic localization of FLO in mononuclear cells suggested that FLO could survive in their cytoplasm. This finding was similar to Francisella spp. present in the cytoplasm of macrophage cell lines of mouse and human origin (Fortier et al., 1995; Golovliov et al., 2003.). Based on histopathological and cytological examinations, ultrastructural study, the sequences of the 16S rRNA gene, in situ hybridization and phylogenetic analysis, we proposed that our isolates might represent a species of Francisella. According to our data, FLO from diseased tilapia was closely related to F. philomiragia, which was isolated from water (Jensen et al., 1969; Hollis et al., 1989) and induced chronic granulomatous disease in humans (Sicherer et al., 1997; Polack et al., 1998). Whether our FLO isolates also play an important role in zoonotic disease needs further study. Acknowledgments We thank the Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ) Council of Agriculture, Executive Yuan for the financial support of this research (Grant no. 90AS-6.3.1-BQ-B1). We also thank Mr. S.P. Chen (Animal Technology Institute Taiwan) for EM technical assistance. References Anda, P., Segura del Pozo, J., Díaz García, J.M., Escudero, R., García Peña, F.J., López Velasco, M.C., Selleck, R.E., Jiménez Chillarón, M.R., Sañchez Serrano, L.P., Martínez Navarro, J.F., 2001. Waterborne outbreak of tularemia associated with crayfish fishing. Emerging Infect. Dis. 7, 575–582. Chen, S.C., Tung, M.C., Chen, S.P., Tsai, J.F., Wang, P.C., Chen, R.S., Lin, S.C., Adams, A., 1994. Systematic granulomas caused by a

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