97
Which Method Is Best? Confusion surrounds which of the available methods is
most suitable for detecting and thus diagnosing the presence of BKD. Essentially,
the opposing opinions include those who favour serology or molecular methods and
those who recommend cultivation. Certainly molecular methods are now becoming
accepted for their reliability (Etchegaray et al. 1991 ). However, it is generally diffi cult for most microbiologists to determine which of the alternative approaches is
best, but comparative work on the various methods has been conducted (Bruno et al.
2007 ).
Comparing kidney and ovarian fl uid from broodstock Atlantic salmon, the selective medium, SKDM (see Austin et al. 1983 ) detected a higher number of positive
BKD samples than iFAT, which in turn was more sensitive than ELISA or western
blots (Griffi ths et al. 1996 ). Interestingly, renibacterium were found in either kidney
or ovarian fl uid, but not both. However, the use of culturing in SKDM broth followed by western blotting increased the sensitivity beyond the maximum level
recorded by SKDM alone (Griffi ths et al. 1996 ). This is interesting because the
benefi t of this broth stage in increasing the detection rate for renibacterium paralleled an observation with peptone water during the 1970s. Here, we found that preincubation of kidney, spleen and more importantly heart tissue in peptone water
enhanced the level of resulting colonies, and therefore positivity, on solid medium.
Another study concluded that ELISA, using a polyclonal rather than a monoclonal
[this was less sensitive] antiserum, was more sensitive than SKDM. Here, SKDM
detected Ren. salmoninarum in 45 % of kidney samples, compared to ELISA, which
found that 50 % of the kidneys were positive (Jansson et al. 1996 ).
Pascho et al. ( 1987 ) compared fi ve techniques for the detection of Ren. salmoninarum in coho salmon. The conclusion was that the ELISA (Dixon 1987 ) was most
sensitive, followed by FAT, fi ltration-FAT, culturing, counterimmuno- electrophoresis
and immunodiffusion. This view was echoed by results from Meyers et al. ( 1993 ),
who regarded ELISA as more sensitive than FAT. Specifi cally, FAT did not detect
Ren. salmoninarum in 80 % of the samples positive by ELISA. However, a complication was that in the same study FAT detected Ren. salmoninarum in 28 % of the
samples negative by ELISA (Meyers et al. 1993 ). The comparative benefi t of a
membrane-fi ltration FAT over ELISA was illustrated when the former detected Ren.
salmoninarum in 66/103 (= 64 %) ovarian samples compared to 40/103 (= 39 %) of
positives with the latter (Pascho et al. 1998 ). The membrane-fi ltration FAT was
capable of detecting ≥25 renibacterial cells/ml of ovarian fl uid, whereas the ELISA
was not consistent in detection at levels of ≤1.3 × 10
4 cells/ml (Pascho et al. 1998 ).
Yet both methods were inferior to a nested PCR amplifying a 320 bp fragment of the
p57 antigen. This PCR detected Ren. salmoninarum in all of the ovarian samples
(Pascho et al. 1998 ). The method of McIntosh et al. ( 1996 ) for the detection of the
p57 antigen by PCR was modifi ed by using an improved DNA isolation procedure
and by redesigning the forward primers and the conditions for carrying out the procedure to prevent false positives (Chambers et al. 2009 ). The modifi cations led to
the minimum detection limit of only 5–72 CFU/mg of head kidney. Okuda et al.
( 2008 ) opined that there was not any difference in the detection levels between iFAT
and PCR.
Renibacterium salmoninarum
Which Method Is Best? Confusion surrounds which of the available methods is
most suitable for detecting and thus diagnosing the presence of BKD. Essentially,
the opposing opinions include those who favour serology or molecular methods and
those who recommend cultivation. Certainly molecular methods are now becoming
accepted for their reliability (Etchegaray et al. 1991 ). However, it is generally diffi cult for most microbiologists to determine which of the alternative approaches is
best, but comparative work on the various methods has been conducted (Bruno et al.
2007 ).
Comparing kidney and ovarian fl uid from broodstock Atlantic salmon, the selective medium, SKDM (see Austin et al. 1983 ) detected a higher number of positive
BKD samples than iFAT, which in turn was more sensitive than ELISA or western
blots (Griffi ths et al. 1996 ). Interestingly, renibacterium were found in either kidney
or ovarian fl uid, but not both. However, the use of culturing in SKDM broth followed by western blotting increased the sensitivity beyond the maximum level
recorded by SKDM alone (Griffi ths et al. 1996 ). This is interesting because the
benefi t of this broth stage in increasing the detection rate for renibacterium paralleled an observation with peptone water during the 1970s. Here, we found that preincubation of kidney, spleen and more importantly heart tissue in peptone water
enhanced the level of resulting colonies, and therefore positivity, on solid medium.
Another study concluded that ELISA, using a polyclonal rather than a monoclonal
[this was less sensitive] antiserum, was more sensitive than SKDM. Here, SKDM
detected Ren. salmoninarum in 45 % of kidney samples, compared to ELISA, which
found that 50 % of the kidneys were positive (Jansson et al. 1996 ).
Pascho et al. ( 1987 ) compared fi ve techniques for the detection of Ren. salmoninarum in coho salmon. The conclusion was that the ELISA (Dixon 1987 ) was most
sensitive, followed by FAT, fi ltration-FAT, culturing, counterimmuno- electrophoresis
and immunodiffusion. This view was echoed by results from Meyers et al. ( 1993 ),
who regarded ELISA as more sensitive than FAT. Specifi cally, FAT did not detect
Ren. salmoninarum in 80 % of the samples positive by ELISA. However, a complication was that in the same study FAT detected Ren. salmoninarum in 28 % of the
samples negative by ELISA (Meyers et al. 1993 ). The comparative benefi t of a
membrane-fi ltration FAT over ELISA was illustrated when the former detected Ren.
salmoninarum in 66/103 (= 64 %) ovarian samples compared to 40/103 (= 39 %) of
positives with the latter (Pascho et al. 1998 ). The membrane-fi ltration FAT was
capable of detecting ≥25 renibacterial cells/ml of ovarian fl uid, whereas the ELISA
was not consistent in detection at levels of ≤1.3 × 10
4 cells/ml (Pascho et al. 1998 ).
Yet both methods were inferior to a nested PCR amplifying a 320 bp fragment of the
p57 antigen. This PCR detected Ren. salmoninarum in all of the ovarian samples
(Pascho et al. 1998 ). The method of McIntosh et al. ( 1996 ) for the detection of the
p57 antigen by PCR was modifi ed by using an improved DNA isolation procedure
and by redesigning the forward primers and the conditions for carrying out the procedure to prevent false positives (Chambers et al. 2009 ). The modifi cations led to
the minimum detection limit of only 5–72 CFU/mg of head kidney. Okuda et al.
( 2008 ) opined that there was not any difference in the detection levels between iFAT
and PCR.
Renibacterium salmoninarum