672
experimentally infected fi sh (Yu et al. 2013 ). Other systems have crossed the genus
boundary by recognizing pathogens from two or more genera. For example:
• Del Cerro et al. ( 2002a , b ) detected simultaneously Aer. salmonicida, Fla. psychrophilum and Y. ruckeri in fi sh tissues , recognising the equivalent of 6, 0.6 and
27 CFU, respectively, using multiplex PCR.
• 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 oligonucleotide 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 amplifi ed by PCR, hybridised, and the specifi c signals were
produced with alkaline phosphatase-conjugated anti- digoxigenin labelled PCR
products (Matsuyama et al. 2006 ).
• González et al. ( 2004 ) used a multiplex PCR and DNA microarray, and achieved
the simultaneous and differential diagnosis of Aer. salmonicida, Ph. damselae
subsp. damselae, V. anguillarum, V. parahaemolyticus and V. vulnifi cus, with a
minimum detection limit of <20 fg per reaction, which equates to 4–5 bacterial
cells.
• A multiplex PCR has reportedly been developed, and successfully recognised
from culture and fi sh tissues the fi sh pathogenic lactococci-streptococci, i.e.
Lactococcus garvieae, Str. diffi cilis, Str. iniae and Str. parauberis with a sensitivity for the purifi ed DNA of 30 pg, 12.5 pg, 25 pg and 50 pg, respectively (Mata
et al. 2004 ).
• A PCR based on a reverse line blot and involving a set of primers for the specifi c
amplifi cation of the 16S rRNA gene resulted in amplifi cation products of 241 bp,
and rapidly detected and differentiated Lactococcus garvieae, Str. agalactiae,
Str. iniae and Str. parauberis (Soltani et al. 2012 ).
• A multiplex PCR detected and differentiated Lactococcus garvieae, Str. agalactiae and Str. iniae with sensitivities of 9.76–39.06 pg of DNA (Itsaro et al. 2013 ).
• A highly specifi c multiplex PCR protocol was developed to simultaneously
detect Edw. tarda, Str. iniae and Str. parauberis , with sensitivities of 0.01 ng,
1.0 ng and 0.1 ng of genomic DNA, respectively (Park et al . 2014 ).
• A LAMP was developed that permitted the recognition and differentiation of for
pathogens of relevance to ornamental fi sh, i.e. Aer. hydrophila, Str. agalactiae,
Koi herpes virus and Iridovirus, within 65 min with a detection limit of 65 copies
(Chang et al. 2013 ).
• DNA microarrays have been used to detect fi sh pathogens, including Aer.
hydrophila, Noc. seriolae, Str. iniae, V. alginolyticus, V. anguillarum and V.
harveyi , and demonstrated congruence with other methods, i.e. culturing and 16S
rRNA gene sequencing (Shi et al. 2012 ).
• Lukkana et al. ( 2014 ) differentiated simultaneously Aeromonas from
Streptococcus using a multiplex-PCR, and distinguished the pathogens from
other waterborne bacteria. DNA fragments. were amplifi ed using genus specifi c
13 Diagnosis
experimentally infected fi sh (Yu et al. 2013 ). Other systems have crossed the genus
boundary by recognizing pathogens from two or more genera. For example:
• Del Cerro et al. ( 2002a , b ) detected simultaneously Aer. salmonicida, Fla. psychrophilum and Y. ruckeri in fi sh tissues , recognising the equivalent of 6, 0.6 and
27 CFU, respectively, using multiplex PCR.
• 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 oligonucleotide 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 amplifi ed by PCR, hybridised, and the specifi c signals were
produced with alkaline phosphatase-conjugated anti- digoxigenin labelled PCR
products (Matsuyama et al. 2006 ).
• González et al. ( 2004 ) used a multiplex PCR and DNA microarray, and achieved
the simultaneous and differential diagnosis of Aer. salmonicida, Ph. damselae
subsp. damselae, V. anguillarum, V. parahaemolyticus and V. vulnifi cus, with a
minimum detection limit of <20 fg per reaction, which equates to 4–5 bacterial
cells.
• A multiplex PCR has reportedly been developed, and successfully recognised
from culture and fi sh tissues the fi sh pathogenic lactococci-streptococci, i.e.
Lactococcus garvieae, Str. diffi cilis, Str. iniae and Str. parauberis with a sensitivity for the purifi ed DNA of 30 pg, 12.5 pg, 25 pg and 50 pg, respectively (Mata
et al. 2004 ).
• A PCR based on a reverse line blot and involving a set of primers for the specifi c
amplifi cation of the 16S rRNA gene resulted in amplifi cation products of 241 bp,
and rapidly detected and differentiated Lactococcus garvieae, Str. agalactiae,
Str. iniae and Str. parauberis (Soltani et al. 2012 ).
• A multiplex PCR detected and differentiated Lactococcus garvieae, Str. agalactiae and Str. iniae with sensitivities of 9.76–39.06 pg of DNA (Itsaro et al. 2013 ).
• A highly specifi c multiplex PCR protocol was developed to simultaneously
detect Edw. tarda, Str. iniae and Str. parauberis , with sensitivities of 0.01 ng,
1.0 ng and 0.1 ng of genomic DNA, respectively (Park et al . 2014 ).
• A LAMP was developed that permitted the recognition and differentiation of for
pathogens of relevance to ornamental fi sh, i.e. Aer. hydrophila, Str. agalactiae,
Koi herpes virus and Iridovirus, within 65 min with a detection limit of 65 copies
(Chang et al. 2013 ).
• DNA microarrays have been used to detect fi sh pathogens, including Aer.
hydrophila, Noc. seriolae, Str. iniae, V. alginolyticus, V. anguillarum and V.
harveyi , and demonstrated congruence with other methods, i.e. culturing and 16S
rRNA gene sequencing (Shi et al. 2012 ).
• Lukkana et al. ( 2014 ) differentiated simultaneously Aeromonas from
Streptococcus using a multiplex-PCR, and distinguished the pathogens from
other waterborne bacteria. DNA fragments. were amplifi ed using genus specifi c
13 Diagnosis