There is evidence that molecular techniques are finding use for the

There is evidence that molecular techniques are

This preview shows page 236 - 238 out of 594 pages.

There is evidence that molecular techniques are finding use for the identification of infections caused by Y. ruckeri (e.g. Argenton et ai, 1996; Taylor and Winton, 2002; Sakai et al, 2006). A PCR was successful in detecting Y. ruckeri in artificially and naturally diseased trout tissues, with a sensitivity of 60-65 cells/PCR tube (Gibello et al, 1999). The value of PCR was echoed by Altinok et al (2001), who detected the pathogen in the blood of rainbow trout within 1 h of immersion in a suspension containing 4.5 x 10^ CFU of Y. ruckerijX. Indeed, the approach was more rehable than culturing at detecting the organism (Altinok et al, 2001). Detection of the yruRjyrul genes involved with quorum-sensing has been regarded as sensitive (i.e. 1 pg; 12 CFU) specific for the 6 isolates of Y. ruckeri tested, but not to representatives of 5 other Yersinia species (Temprano et al., 2001). Others have proposed a PCR and RFLP targeting the aroA gene (Yugueros et al., 2001). A nested PCR had a detection limit of 1.4 X 10^ CFU/reaction. However, use of species-specific primers improved detection to < 14 CFU/sample (Taylor and Winton, 2002). Real time PCR, which produces a result in 4-5 h, has been developed for Edw. ictaluri, and detected the equivalent of 2.5 cells using DNA samples from cultures and fish blood (Bilodeau et al., 2003). Sequencing of the 16S rDNA is becoming an accepted procedure for the identification of bacteria, including fish pathogens. The technique has been instru- mental in the recognition of new pathogens, including Str. dysgalactiae (Nomoto et al., 2004), and confirmed the presence of Lactococcus garvieae in Taiwan (Chen et al., 2002). An ideal situation would involve techniques that could recognise and differen- tiate between multiple diseases, and this has been achieved with multiplex PCR. Del Cerro et al. (2002) detected simultaneously Aer. salmonicida, Fla. psychrophilum and Y. ruckeri in fish tissues, recognising the equivalent of 6, 0.6 and 27 CFU, respectively. Similarly, Gonzalez et al. (2004) used a multiplex PCR and DNA microarray, and achieved the simultaneous and differential diagnosis of Aer. sal- monicida. Ph. damselae subsp. damselae, V. anguillarum, V. parahaemolyticus and V. vulnificus, with a minimum detection limit of <20 fg per reaction, which equates to 4-5 bacterial cells. Matsuyama et al. (2006) developed a low-density oligonucleotide DNA array for the detection and discrimination of multiple Photobacterium and Vibrio spp. within a day, albeit with some cross-hybridisation reported. These workers designed a low-density oHgonucleotide DNA array between the 16S and 23S ribosomal DNA leading to the development of three oligonucleotide probes, which were immobilized on nylon membranes. The low-density oligonucleotide DNA arrays were amplified by PCR, hybridised and the specific signals produced with alkaline phosphatase-conjugated anti-digoxigenin-labelled PCR products (Matsuyama et al., 2006).
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Diagnosis 215 PHENOTYPIC TESTS For many pathogens emphasis has been placed on conventional phenotypic tests for diagnosis. For example, Boulanger et al. (1977) highUghted the value of confirming
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