96
mRNA from viable cells of Ren. salmoninarum from fi sh tissues (kidney and ovarian fl uid) with detection limits stated to be 1–10 bacterial cells (Cook and Lynch
1999 ). Subsequently, a nested reverse transcription PCR of 16S rRNA sequences
successfully detected 1–10 renibacterial cells in ovarian fl uid, but was unreliable
with kidney (Magnússen et al. 1994 ). Although sensitive, this system took 1–2 days
to carry out. In a further development, McIntosh et al. ( 1996 ) devised a simplifi ed
PCR invoking the 376-base pair region of the gene encoding the 57 kDa surface
antigen. This system had a minimum detection limit of 5 × 10
3 renibacterial cells/ml
in rainbow trout lymphocytes. Two 24-base oligonucleotide primers used to amplify
a 501 base-pair region of the gene encoding the 57 kDa soluble protein (p57) formed
the basis of a PCR, which was capable of detecting 2 renibacterial cells within individual salmonid eggs (Brown et al. 1994 ). This PCR was considered to have value
for the screening of broodstock for the presence of BKD (Brown et al. 1994 ). A
nested PCR amplifying a 320 bp fragment of the p57 antigen was suitable for detecting Ren. salmoninarum in ovarian samples (Pascho et al. 1998 ). Terminal-RFLP
permitted the detection of ~30 CFU/mg of artifi cially inoculated kidney tissue
(Nilsson and Strom 2002 ). A qPCR was developed to detect the pathogen in chinook salmon, and correlated well with ELISA at high levels of infection (Powell
et al. 2005 ).
A clone, pRS47, of 5.1 kb, was used to develop a specifi c DNA probe (Hariharan
et al. 1995 ). In a dot blot assay, the biotinylated pRS47/ Bam HI insert probe hybridised with only DNA from three strains of Ren. salmoninarum but not with
Arthrobacter protophormiae, Aer. salmonicida, Cor. aquaticum, Car. piscicola,
Micrococcus luteus, Ps. fl uorescens, V. anguillarum, V. ordalii or Y. ruckeri. With
kidney tissue from fi sh challenged with Ren. salmoninarum, the dot blot assay was
regarded as sensitive as culture and FAT. With the latter technique, samples negative
by dot blot assay and culturing revealed the presence of ≤1 fl uorescing object, presumed to be a bacterium/50 microscope fi elds. Consequently, this probe was
regarded as having potential for the diagnosis of BKD (Hariharan et al. 1995 ).
A specifi c, sensitive real-time PCR was developed to recognise BKD in kidney
tissue, and was capable of detecting 1–10 Ren. salmoninarum genomes/reaction
(Jansson et al. 2008 ). The method was deemed to be more sensitive than ELISA
insofar as BKD was detected in 39.9 % of fi sh by real-time PCR, but only 28 % by
ELISA incorporating polyclonal antisera (Jansson et al. 2008 ).
LAMP methods have been proposed as a sensitive [more sensitive than quantitative real time PCR] and specifi c method for the detection of Ren. salmoninarum
(Saleh et al. 2008 ; Gahlawat et al. 2009 ). Saleh et al. ( 2008 ) developed a rapid, i.e.
1 h, system which amplifi ed a fragment of the p57 gene, and was specifi c to Ren.
salmoninarum, and detected 1 pg of genomic DNA. Notwithstanding all the excellent publications, the dilemma remains about which method is best. Sandell and
Jacobson ( 2011 ) compared different qPCRs and compared the value and sensitivity
of msa/ non-fl uorescent quencher qPCR and abc/ non-fl uorescent quencher qPCR to
ELISA.
3 Aerobic Gram-Positive Rods and Cocci
mRNA from viable cells of Ren. salmoninarum from fi sh tissues (kidney and ovarian fl uid) with detection limits stated to be 1–10 bacterial cells (Cook and Lynch
1999 ). Subsequently, a nested reverse transcription PCR of 16S rRNA sequences
successfully detected 1–10 renibacterial cells in ovarian fl uid, but was unreliable
with kidney (Magnússen et al. 1994 ). Although sensitive, this system took 1–2 days
to carry out. In a further development, McIntosh et al. ( 1996 ) devised a simplifi ed
PCR invoking the 376-base pair region of the gene encoding the 57 kDa surface
antigen. This system had a minimum detection limit of 5 × 10
3 renibacterial cells/ml
in rainbow trout lymphocytes. Two 24-base oligonucleotide primers used to amplify
a 501 base-pair region of the gene encoding the 57 kDa soluble protein (p57) formed
the basis of a PCR, which was capable of detecting 2 renibacterial cells within individual salmonid eggs (Brown et al. 1994 ). This PCR was considered to have value
for the screening of broodstock for the presence of BKD (Brown et al. 1994 ). A
nested PCR amplifying a 320 bp fragment of the p57 antigen was suitable for detecting Ren. salmoninarum in ovarian samples (Pascho et al. 1998 ). Terminal-RFLP
permitted the detection of ~30 CFU/mg of artifi cially inoculated kidney tissue
(Nilsson and Strom 2002 ). A qPCR was developed to detect the pathogen in chinook salmon, and correlated well with ELISA at high levels of infection (Powell
et al. 2005 ).
A clone, pRS47, of 5.1 kb, was used to develop a specifi c DNA probe (Hariharan
et al. 1995 ). In a dot blot assay, the biotinylated pRS47/ Bam HI insert probe hybridised with only DNA from three strains of Ren. salmoninarum but not with
Arthrobacter protophormiae, Aer. salmonicida, Cor. aquaticum, Car. piscicola,
Micrococcus luteus, Ps. fl uorescens, V. anguillarum, V. ordalii or Y. ruckeri. With
kidney tissue from fi sh challenged with Ren. salmoninarum, the dot blot assay was
regarded as sensitive as culture and FAT. With the latter technique, samples negative
by dot blot assay and culturing revealed the presence of ≤1 fl uorescing object, presumed to be a bacterium/50 microscope fi elds. Consequently, this probe was
regarded as having potential for the diagnosis of BKD (Hariharan et al. 1995 ).
A specifi c, sensitive real-time PCR was developed to recognise BKD in kidney
tissue, and was capable of detecting 1–10 Ren. salmoninarum genomes/reaction
(Jansson et al. 2008 ). The method was deemed to be more sensitive than ELISA
insofar as BKD was detected in 39.9 % of fi sh by real-time PCR, but only 28 % by
ELISA incorporating polyclonal antisera (Jansson et al. 2008 ).
LAMP methods have been proposed as a sensitive [more sensitive than quantitative real time PCR] and specifi c method for the detection of Ren. salmoninarum
(Saleh et al. 2008 ; Gahlawat et al. 2009 ). Saleh et al. ( 2008 ) developed a rapid, i.e.
1 h, system which amplifi ed a fragment of the p57 gene, and was specifi c to Ren.
salmoninarum, and detected 1 pg of genomic DNA. Notwithstanding all the excellent publications, the dilemma remains about which method is best. Sandell and
Jacobson ( 2011 ) compared different qPCRs and compared the value and sensitivity
of msa/ non-fl uorescent quencher qPCR and abc/ non-fl uorescent quencher qPCR to
ELISA.
3 Aerobic Gram-Positive Rods and Cocci