Vol. 89: 199–207, 2010 DISEASES OF AQUATIC ORGANISMS Published April 9 doi: 10.3354/dao02204 Dis Aquat Org Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis Esteban Soto1, Kimberly Bowles2, Denise Fernandez1, John P. Hawke1,* 1Department of Pathobiological Sciences and 2Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Skip Bertman Dr., Baton Rouge, Louisiana 70803, USA ABSTRACT: Members of the genus Francisella are small Gram-negative facultative intracellular bac- teria that cause francisellosis in a wide variety of fish species worldwide. F. noatunensis subsp. orien- talis has been recently described as a warm-water pathogen of tilapia Oreochromis spp. In this study, a quantitative real-time polymerase chain reaction (qPCR) TaqMan probe assay was developed to rapidly and accurately detect and quantify F. noatunensis subsp. orientalis from fish tissue. The tar- get region of the assay was the F. tularensis iglC gene homologue previously found in F. noatunensis subsp. orientalis. Probe specificity was confirmed by the lack of signal and cross-reactivity with 12 common fish pathogens, 2 subspecies of F. tularensis, F. noatunensis subsp. noatunensis, and tilapia tissue. The range of linearity was determined to be 50 fg to 1.4 mg, and the lower limit of detection was 50 fg of DNA (equivalent to ~25 genome equivalents) per reaction. A similar sensitivity was observed with DNA extracted from a mixture of F. noatunensis subsp. orientalis and fish tissue. The assay was also able to detect and quantify F. noatunensis subsp. orientalis from the spleens of exper- imentally infected tilapia. No signal was observed in the control groups. In conclusion, we have developed a highly sensitive and specific assay that can be used for the specific identification and quantification of F. noatunensis subsp. orientalis. KEY WORDS: Francisella · Tilapia · qPCR Resale or republication not permitted without written consent of the publisher INTRODUCTION During the past 5 yr, bacteria of the genus Francisella have caused significant mortalities in cultured tilapia Francisellosis is an emergent disease in fish caused Oreochromis spp., Atlantic cod Gadus morhua, Atlantic by Gram-negative facultative intracellular bacteria salmon Salmo salar L., hybrid striped bass Morone that are members of the genus Francisella. In tilapia chrysops × M. saxatilis, three-line grunt Parapristipoma the disease can be observed as an acute syndrome trilineatum, and ornamental cichlids, both in warm- and with few clinical signs and high mortality, or as a sub- cold-water environments (Kamaishi et al. 2005, Nylund acute to chronic syndrome with non-specific clinical et al. 2006, Olsen et al. 2006, Ostland et al. 2006, Birk- signs like anorexia, exophthalmia, and anemia. Upon beck et al. 2007, Hsieh et al. 2007, Mauel et al. 2007, macroscopic and microscopic examination, internal Mikalsen et al. 2007, Ottem et al. 2007, Soto et al. 2009a). organs are enlarged and contain widespread multifo- The identification and taxonomic characterization of cal white nodules. Histological examination often the Francisella spp. identified as worldwide emerging reveals the presence of multifocal granulomatous pathogens of fish have been difficult owing to the fas- lesions, with the presence of numerous small, pleomor- tidious nature of the bacteria and the small number of phic, cocco-bacilli (Hsieh et al. 2006, Mauel et al. 2007, isolates recovered from fish (Soto et al. 2009a). In the Soto et al. 2009a). majority of the cases, PCR and sequence comparison of *Corresponding author: [email protected] © Inter-Research 2010 · www.int-res.com 200 Dis Aquat Org 89: 199–207, 2010 the 16S rRNA have made it possible to place the organ- Panangala et al. 2007, Suzuki & Sakai 2007, Griffin et isms at 97% similarity to F. tularensis, 98% similarity to al. 2008, Ottem et al. 2008). The high sensitivity, high F. philomiragia, and 99% to other Francisella spp. specificity, and short turnaround time for results make strains isolated from fish species (Kamaishi et al. 2005, this technique an attractive replacement method for Hsieh et al. 2006, Ostland et al. 2006, Mauel et al. 2007, conventional diagnostic techniques (Espy et al. 2006). Mikalsen et al. 2007, Ottem et al. 2007, Soto et al. The genes of the intracellular growth locus (iglA, 2009a). The Francisella strain utilized in this research iglB, iglC, and iglD) are some of the most interesting project at the Louisiana Aquatic Diagnostic Laboratory, genes identified in the genus Francisella. These genes LSU School of Veterinary Medicine, LADL 07-285A, are present as part of a 30 kb pathogenicity island isolated from tilapia Oreochromis spp. from Costa Rica, described by Nano et al. (2004) and Barker & Klose was confirmed by molecular analysis as F. noatunensis (2007) in Francisella tularensis. The Francisella patho- subsp. orientalis (Soto et al. 2009a) and exhibited 99% genicity island (FPI), a cluster of 16 to 19 genes, has identity with F. noatunensis subsp. noatunensis isolated been found duplicated in some F. tularensis genomes, from diseased Atlantic cod in Norway (Euzéby 2009, but as a single copy in the F. philomiragia subsp. Ottem et al. 2009), by sequence comparison of the 16S philomiragia ATCC 25015 isolate, a close relative of rRNA. The LADL 07-285A and 07-285B isolates used the tilapia pathogen F. noatunensis subsp. orientalis in this study also belong to the same group described strain LADL 07-285A (Nano & Schmerk 2007, Soto et as F. asiatica (Mikalsen & Colquhoun 2009); however, al. 2009b). this name does not yet have valid taxonomic standing The functions of the conserved proteins correspond- (J. Euzéby pers. comm.). ing to the genes are elusive. Overall, Igl proteins The diagnosis of this highly virulent fish pathogen appear to be essential for the ability of Francisella has many constraints, including the fastidious nature tularensis to survive inside the macrophage and cause of the bacterium and the lack of biochemical, molecu- disease (Golovliov et al. 1997, Lai et al. 2004, Lauriano lar, and serological tests specific for this aquatic animal et al. 2004, Nano et al. 2004, Santic et al. 2005, Brotcke pathogen. Previous diagnosis of francisellosis in fish et al. 2006, de Bruin et al. 2007). Homologues of the species has been made with the aid of histopathology, F. tularensis iglABCD genes in the tilapia pathogenic electron microscopy, conventional culture assays, con- F. noatunensis subsp. orientalis strain LADL 07-285A ventional polymerase chain reaction (PCR) using Fran- were identified and described in a previous study (Soto cisella sp. specific primers, 16S rRNA sequencing, and et al. 2009b). The presence of a single iglC gene in the in situ hybridization (Kamaishi et al. 2005, Hsieh et al. completely sequenced and closely related isolate F. 2006, Ostland et al. 2006, Mauel et al. 2007, Ottem et philomiragia subsp. philomiragia ATCC 25017 sug- al. 2007, Soto et al. 2009a). However, the diagnosis of gests that the gene is also present in single copy in the pathogen remains a challenge, and some of the F. noatunensis subsp. orientalis strain LADL 07-285A. current techniques are difficult, time consuming, and The presence of a single copy of the iglC gene in F. expensive; some require specialized personnel and are noatunensis subsp. orientalis makes the gene an excel- prone to show false negatives because of low sensitiv- lent target for developing a highly specific diagnostic ity, or false positives because of low specificity. More- test and will provide a means to quantify with a high over, studies with F. tularensis have shown that diag- degree of confidence the amount of bacterial DNA pre- nosis based on isolation by culture is prone to show sent in a sample. false-negative results (Fujita et al. 2006). The aim of this study was to develop a quantitative Real-time PCR is a well known molecular technique real-time PCR assay using the previously described that is currently used in many laboratories for diagno- iglC gene of the fish pathogen Francisella noatunensis sis of microbial pathogens, including the fastidious subsp. orientalis as a target. We describe a highly bacteria Mycobacterium spp., Bacillus anthracis, Fran- sensitive, specific, and reliable molecular diagnostic cisella tularensis, and organisms that are non-cultur- technique for identification and quantification of F. able on cell-free media, the Rickettsia spp. and viruses noatunensis subsp. orientalis from diseased fish. (Bode et al. 2004, Kocagoz et al. 2005, Takahashi et al. 2007, Tomaso et al. 2007, Abril et al. 2008, Kidd et al. 2008). In recent years, fish disease diagnosticians have MATERIALS AND METHODS used this technique to identify and quantify bacterial, viral, and parasitic fish pathogens such as Aeromonas Bacterial species and strains. The bacterial strains salmonicida, Flavobacterium columnare, Renibacteri- used in this project were chosen because they repre- um salmoninarum, Henneguya ictaluri, largemouth sent common bacterial fish pathogens, or are mem- bass virus, and recently Francisella piscicida in Norwe- bers of the genus Francisella. Strain LADL 07-285A, gian cod (Balcázar et al. 2007, Getchell et al. 2007, isolated from diseased cultured tilapia Oreochromis Soto et al: PCR assay for Francisella noatunensis subsp. orientalis 201 spp. was chosen as a representative of the warm- & A2) and 16S rDNA sequences, as well as pheno- water strain of fish pathogenic F. noatunensis subsp. typic characteristics, temperature requirements, and orientalis. The majority of the isolates tested were host range analysis, demonstrated that isolated Fran- recovered by the Louisiana Aquatic Diagnostic Labo- cisella sp. 1, Francisella sp. 2, Francisella sp. 3 (Ost- ratory (LADL), in the School of Veterinary Medicine land et al. 2006), and F. victoria (Kay et al. 2006) are (LSU-SVM) at Louisiana State University, from dis- in fact members of the recently described species F. eased fish, while others were acquired from the noatunensis subsp.