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

Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis

“…The development of this highly sensitive diagnostic method will enhance the diagnosis of this fastidious organism, which could be present at low levels in fish tissue and may require specialized media in which to grow; this organism may possibly be overgrown by secondary contaminants following attempts at primary isolation. Molecular diagnostic tests have been used to alleviate some of these problems and have been used along with DNA sequencing to give definitive diagnosis in complicated cases (Hsieh et al 2006, Mauel et al 2007, Soto et al 2009a. As with many other diagnostic techniques, conventional PCR has certain limitations.…”

“…Upon macroscopic and microscopic examination, internal organs are enlarged and contain widespread multifocal white nodules. Histological examination often reveals the presence of multifocal granulomatous lesions, with the presence of numerous small, pleomorphic, cocco-bacilli (Hsieh et al 2006, Mauel et al 2007, Soto et al 2009a). …”

“…In the majority of the cases, PCR and sequence comparison of the 16S rRNA have made it possible to place the organisms at 97% similarity to F. tularensis, 98% similarity to F. philomiragia, and 99% to other Francisella spp. strains isolated from fish species (Kamaishi et al 2005, Hsieh et al 2006, Ostland et al 2006, Mauel et al 2007, Mikalsen et al 2007, Ottem et al 2007, Soto et al 2009a. The Francisella strain utilized in this research project at the Louisiana Aquatic Diagnostic Laboratory, LSU School of Veterinary Medicine, LADL 07-285A, isolated from tilapia Oreochromis spp.…”

“…Previous diagnosis of francisellosis in fish species has been made with the aid of histopathology, electron microscopy, conventional culture assays, conventional polymerase chain reaction (PCR) using Francisella sp. specific primers, 16S rRNA sequencing, and in situ hybridization (Kamaishi et al 2005, Hsieh et al 2006, Ostland et al 2006, Mauel et al 2007, Ottem et al 2007, Soto et al 2009a). However, the diagnosis of the pathogen remains a challenge, and some of the current techniques are difficult, time consuming, and expensive; some require specialized personnel and are prone to show false negatives because of low sensitivity, or false positives because of low specificity.…”

General and Specialized Media Routinely Employed for Primary Isolation of Bacterial Pathogens of Fishes

“…from three-line grunt (Parapristipoma trilineatum) on cystine-heart agar (BD Difco) supplemented with 1% hemoglobin after two weeks at 25 C. Eugon agar (BD BBL, Franklin Lakes, New Jersey, USA) with 1% hemoglobin and Eugon broth (BD BBL) with 2 mM FeCl 3 also supported bacterial culture growth; the latter was also good for large-scale cultivation (Kamaishi et al, 2005). Hsieh et al (2006) isolated Francisella sp. from tilapia (Oreochromis spp.)…”