WHIRLING DISEASE Myxosoma cerebralis SPORE CONCENTRATION USING THE CONTINUOUS PLANKTON CENTRIFUGE Author(s): JOSEPH J. O'GRODNICK Source: Journal of Wildlife Diseases, 11(1):54-57. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/0090-3558-11.1.54 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-11.1.54
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
54
Journal
of
WHIRLING DISEASE Myxosoma SPORE CONCENTRATION USING PLANKTON CENTRIFUGE JOSEPH RD.
J. 1,
Box
O’GRODNICK, 200-C,
Pennsylvania Bellefonte,
Fish
Wildlife
Benner
16823,
Abstract: A method for concentrating whirling disease (Myxosoma cerebra/is) volves homogenization of head skeletons, ing with a continuous plankton centrifuge.
Vol.
11,
January,
1975
cerebra/is THE CONTINUOUS
Commission,
Pennsylvania
Diseases
Spring
Fish
Research
Station,
USA.
and detecting of salmonids screening out
the spores is described. tissue shreds,
of the cause of The method inand concentrat-
INTRODUCTION
The detection of whirling disease spores in carrier fish has always presented the biologist with diagnostic problems.’ Since new disease regulations may require the diagnosis of whirling disease in large lots of trout,’ practical methods for concentrating spores should be available to the fishery pathobiologist. At the Benner Spring Fish Research Station a procedure using the continuous plankton centrifuge LU (Figure 1) has been developed to concentrate whirling disease spores from fish in research experiments and production lots. This method has been used for 4 years and has given satisfactory results in the quantitative studies carried out in our whirling disease research projects. Rainbow trout, for example, which do not exhibit signs of the disease, have been shown by this method to harbor relatively large numbers of spores. FIGURE MATERIALS
AND
METHODS
excess
the
centrifugal
drum. The
blender, ger),
needed
materials blender membrane
bottle filter
are: (175
Waring
ml
or
apparatus,
drum.
wire
the
wall
centrifuge
LU Plankton Nanuet, LU For
more e.g.,
centrifuge New
manufactured York 10954.
critical 520-260-120
work
one may nm mesh.
by
G.
screen
M.
Mfg. the
and
homogenate
The
plankton
water
This
through lar-
1.
the
from
force excess
created water
centrifuge the
the
is
carried
rubber
tube
situated
Most
solid
particles
are
the
revolving
through
by
a of
removes
filtrate
revolving away
below
the
deposited
drum
in
on
the
first
Box
326,
run.
Instrument through
Corporation, graded
P.O. mesh-size
screens,
Journal
of
Wildlife
Diseases
Vol.
11,
January,
55
1975
mesh prefilter screen-mesh size 725 mm,LU continuous plankton centrifuge, separatory funnel and ring stand, 27 ml specimen bottle, hemocytometer. The fish are decapitated near the base
of led mm sue or
the skull. Larger fish are best handby heating the heads at 45 C for 2-3 and then removing as much soft tisas possible. Large pieces may be split cut into smaller pieces.
STEPS
1.
Take five to ten heads of 8-15 cm fish or one to five blend for 5 mm in 175-200 ml of water. If spores experiments, chlorinated water should be avoided.
heads from are intended
larger for
2.
Remove the blended material and vacuum filter the entire volume through a membrane bacteriological filter using a wire mesh prefilter. The filter may clog and should be cleaned with water. The rinse water becomes part of the filtrate to be saved. The procedure removes fish scales and other coarse material which is discarded.
3.
Place the entire amount plankton centrifuge. Set fuge and run centrifuge
of filtrate in a separatory funnel discharging a low flow from the separatory funnel into at high speed.
4.
Centrifuge until all water is removed. revolving drum will contain spores the walls of the plankton centrifuge Be sure to get all material.
5.
Place the residue and suspended and fill to the top with distilled until the material is uniformly
6.
Place a small a spore count four chambers,
The residue adhering and some fish tissue. revolving drum with
material water. suspended
the
into the centri-
to the walls of the Scrape residue from a rubber policeman.
from the drum into Place the cap on the (2 mm).
amount of suspension under a coverslip is desired.#{176}Count the spores under four counting a total of 16 squares.
fish and infection
the 27 ml bottle and
bottle shake
of the hemocytometer 1 mm’ squares. We
if use
Formula:
number
of
27 ml spores. 7.
spores
1 ml (Suspension
=
total
Volume)
Equipment should be scrubbed bleach (NaOC1) for 10 mm and
PLANKTON
CENTRIFUGE
number
number of x number
yule,
G. L. 1974. West Virginia
EFFECTIVENESS
Personal 25430.
spores
counted
x 10’
1 mm’ areas counted of spores in 1 ml =
total
thoroughly, soaked in half-strength rinsed thoroughly after each use.
Using this technique we have recovered 3.4 x 10’ to 9 x 10#{176} from our pooled samples during the past 4 years. G. L. Hoffman recovered 4.2 x 10’ to 4.3 x 10#{176} spores per fish from heavily infected fish using this technique.LU The late W. Tidd also obtained good results with the plankton centrifuge.
LU Hoffman,
of
communication.
number
of
household
To confirm the effectiveness of the plankton centrifuge in spore concentrating, a test was devised to determine the relative loss of spores in the water which is removed from the centrifuge. Five heavily infected rainbow trout heads were run through the spore concentration procedure which consisted of three trial runs, reusing the same suspension.
Eastern
Fish
Disease
Laboratory,
Kearneys-
56
Journal
Spores from the first run were concentrated and the number per ml estimated. The water from the first run was collected and recentrifuged, and the number of spores per ml of suspension was estimated for the second time. The water from the second run was collected, recentrifuged, and again the spores per ml of suspension was estimated. By this time the spore count was very low and further concentration was believed unnecessary.
TABLE
1. Spores
recovered
from
the
same
of
Wildlife
Diseases
Vol.
11,
January,
The estimated spore number each run was added and total spore obtained (Table 1). To
determine
the
absolute
1975
from count loss
of
spores after the first centrifuge run, a seed population of 10,250,000 spores was added at step three of the procedure. An estimated recovery of 8,370,000 spores was achieved, which indicates an 83 % efficiency of recovery.
suspension
in
three
runs.
Estimated Spores Per
Percentage ml
of Total Recovered
Spores
Run
#1
458,750
96.2
Run
#2
17,500
3.7
Run
#3
630 TOTAL
0.1
476,880
DISCUSSION
Because the spores are trapped in skeletal structures and granulomas they are difficult to detect in carrier fish. Recent studies on spore concentration methods include blending, filter paper filtration and centrifugation,#{176}’ and fish digestion.’4’7 The plankton centrifuge
concentrates whirling disease spores effectively for diagnostic purposes. It has the advantage of speed and simplicity and the final suspension is fairly debrisfree. Relatively few spores are lost after the first centrifuge run, thus a reasonable quantitative estimate can be made and the disease level of the population determined.
Acknowledgment The
author
aid
in
the
Fish
Research Restoration
LITERATURE
1.
2.
wishes
testing on
of
to the
thank method
whirling disease (Dingell-Johnson)
Dr.
G.
L.
and
in
the
is
being Act.
Hoffman,
Eastern
preparation
of
supported
by
Fish the
funds
Disease
Laboratory,
for
the
Aid
his
manuscript. issued
under
Federal
in
CITED
CONTOS, N. and and purification Cult. In Press.
H. ROTHENBACKER. method for spores
1973. of Myxosoma
An
efficient cerebra/is.
HOFFMAN, G. L., C. E. DUNBAR and A. BRADFORD. disease of trouts caused by Myxosoma cerebra/is in the Department of the Interior. Special Scientific Report, (reprinted with additions), 15 pp.
concentration Progr. Fish-
1969. Whirling United States. U.S. Fisheries No. 427
Journal
of
Wildlife
Diseases
Vol.
11,
January,
1975
57
3.
HOFFMAN, G. L., S. F. SNIESKO and K. WOLF. for determining absence of viral hemorrhagic disease in certain fish and fish products. Fish Fish and Wildlife Service, 7 pp.
4.
LANDOLT, M. 1973. Myxosoma cerebra/is: isolation and concentration from fish skeletal elements-trypsin digestion method. J. Fish Res. Bd. Canada 30: 1713-1716. MARKIW, M. E. and K. WOLF. 1974. Myxosoma cerebra/is: isolation and concentration from fish skeletal elements-sequential enzymatic digestions and purification by differential centrifugation. J. Fish Res. Bd. Canada 31: 15-20.
5.
1968. Approved procedure septicemia and whirling Disease Leaflet No. 9, U.S.
6.
PRASHER, J. B., W. M. TIDD and R. A. TUBB. 1971. Techniques tracting an dquantitatively studying the spore stage of the protozoan site Myxosoma cerebra/is. Progr. Fish-Cult. 33: 193-196.
7.
RYDLO, M. 1971. Evidence of Myxosoma cerebra/is of rainbow trout afflicted with whirling disease. 97-99 (in German).
8.
TIDD,
WILBER Myxosoma 227-228.
M.,
R.
cerebra/is
Received
A. TUBB and V. spore extraction
for
publication
18
WRIGHT. technique.
June
1974
for
expara-
spores in various organs Osterreichs Fisch. 24: 1973. Progr.
Modification Fish-Cult.
of 35:
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
54
Journal
of
WHIRLING DISEASE Myxosoma SPORE CONCENTRATION USING PLANKTON CENTRIFUGE JOSEPH RD.
J. 1,
Box
O’GRODNICK, 200-C,
Pennsylvania Bellefonte,
Fish
Wildlife
Benner
16823,
Abstract: A method for concentrating whirling disease (Myxosoma cerebra/is) volves homogenization of head skeletons, ing with a continuous plankton centrifuge.
Vol.
11,
January,
1975
cerebra/is THE CONTINUOUS
Commission,
Pennsylvania
Diseases
Spring
Fish
Research
Station,
USA.
and detecting of salmonids screening out
the spores is described. tissue shreds,
of the cause of The method inand concentrat-
INTRODUCTION
The detection of whirling disease spores in carrier fish has always presented the biologist with diagnostic problems.’ Since new disease regulations may require the diagnosis of whirling disease in large lots of trout,’ practical methods for concentrating spores should be available to the fishery pathobiologist. At the Benner Spring Fish Research Station a procedure using the continuous plankton centrifuge LU (Figure 1) has been developed to concentrate whirling disease spores from fish in research experiments and production lots. This method has been used for 4 years and has given satisfactory results in the quantitative studies carried out in our whirling disease research projects. Rainbow trout, for example, which do not exhibit signs of the disease, have been shown by this method to harbor relatively large numbers of spores. FIGURE MATERIALS
AND
METHODS
excess
the
centrifugal
drum. The
blender, ger),
needed
materials blender membrane
bottle filter
are: (175
Waring
ml
or
apparatus,
drum.
wire
the
wall
centrifuge
LU Plankton Nanuet, LU For
more e.g.,
centrifuge New
manufactured York 10954.
critical 520-260-120
work
one may nm mesh.
by
G.
screen
M.
Mfg. the
and
homogenate
The
plankton
water
This
through lar-
1.
the
from
force excess
created water
centrifuge the
the
is
carried
rubber
tube
situated
Most
solid
particles
are
the
revolving
through
by
a of
removes
filtrate
revolving away
below
the
deposited
drum
in
on
the
first
Box
326,
run.
Instrument through
Corporation, graded
P.O. mesh-size
screens,
Journal
of
Wildlife
Diseases
Vol.
11,
January,
55
1975
mesh prefilter screen-mesh size 725 mm,LU continuous plankton centrifuge, separatory funnel and ring stand, 27 ml specimen bottle, hemocytometer. The fish are decapitated near the base
of led mm sue or
the skull. Larger fish are best handby heating the heads at 45 C for 2-3 and then removing as much soft tisas possible. Large pieces may be split cut into smaller pieces.
STEPS
1.
Take five to ten heads of 8-15 cm fish or one to five blend for 5 mm in 175-200 ml of water. If spores experiments, chlorinated water should be avoided.
heads from are intended
larger for
2.
Remove the blended material and vacuum filter the entire volume through a membrane bacteriological filter using a wire mesh prefilter. The filter may clog and should be cleaned with water. The rinse water becomes part of the filtrate to be saved. The procedure removes fish scales and other coarse material which is discarded.
3.
Place the entire amount plankton centrifuge. Set fuge and run centrifuge
of filtrate in a separatory funnel discharging a low flow from the separatory funnel into at high speed.
4.
Centrifuge until all water is removed. revolving drum will contain spores the walls of the plankton centrifuge Be sure to get all material.
5.
Place the residue and suspended and fill to the top with distilled until the material is uniformly
6.
Place a small a spore count four chambers,
The residue adhering and some fish tissue. revolving drum with
material water. suspended
the
into the centri-
to the walls of the Scrape residue from a rubber policeman.
from the drum into Place the cap on the (2 mm).
amount of suspension under a coverslip is desired.#{176}Count the spores under four counting a total of 16 squares.
fish and infection
the 27 ml bottle and
bottle shake
of the hemocytometer 1 mm’ squares. We
if use
Formula:
number
of
27 ml spores. 7.
spores
1 ml (Suspension
=
total
Volume)
Equipment should be scrubbed bleach (NaOC1) for 10 mm and
PLANKTON
CENTRIFUGE
number
number of x number
yule,
G. L. 1974. West Virginia
EFFECTIVENESS
Personal 25430.
spores
counted
x 10’
1 mm’ areas counted of spores in 1 ml =
total
thoroughly, soaked in half-strength rinsed thoroughly after each use.
Using this technique we have recovered 3.4 x 10’ to 9 x 10#{176} from our pooled samples during the past 4 years. G. L. Hoffman recovered 4.2 x 10’ to 4.3 x 10#{176} spores per fish from heavily infected fish using this technique.LU The late W. Tidd also obtained good results with the plankton centrifuge.
LU Hoffman,
of
communication.
number
of
household
To confirm the effectiveness of the plankton centrifuge in spore concentrating, a test was devised to determine the relative loss of spores in the water which is removed from the centrifuge. Five heavily infected rainbow trout heads were run through the spore concentration procedure which consisted of three trial runs, reusing the same suspension.
Eastern
Fish
Disease
Laboratory,
Kearneys-
56
Journal
Spores from the first run were concentrated and the number per ml estimated. The water from the first run was collected and recentrifuged, and the number of spores per ml of suspension was estimated for the second time. The water from the second run was collected, recentrifuged, and again the spores per ml of suspension was estimated. By this time the spore count was very low and further concentration was believed unnecessary.
TABLE
1. Spores
recovered
from
the
same
of
Wildlife
Diseases
Vol.
11,
January,
The estimated spore number each run was added and total spore obtained (Table 1). To
determine
the
absolute
1975
from count loss
of
spores after the first centrifuge run, a seed population of 10,250,000 spores was added at step three of the procedure. An estimated recovery of 8,370,000 spores was achieved, which indicates an 83 % efficiency of recovery.
suspension
in
three
runs.
Estimated Spores Per
Percentage ml
of Total Recovered
Spores
Run
#1
458,750
96.2
Run
#2
17,500
3.7
Run
#3
630 TOTAL
0.1
476,880
DISCUSSION
Because the spores are trapped in skeletal structures and granulomas they are difficult to detect in carrier fish. Recent studies on spore concentration methods include blending, filter paper filtration and centrifugation,#{176}’ and fish digestion.’4’7 The plankton centrifuge
concentrates whirling disease spores effectively for diagnostic purposes. It has the advantage of speed and simplicity and the final suspension is fairly debrisfree. Relatively few spores are lost after the first centrifuge run, thus a reasonable quantitative estimate can be made and the disease level of the population determined.
Acknowledgment The
author
aid
in
the
Fish
Research Restoration
LITERATURE
1.
2.
wishes
testing on
of
to the
thank method
whirling disease (Dingell-Johnson)
Dr.
G.
L.
and
in
the
is
being Act.
Hoffman,
Eastern
preparation
of
supported
by
Fish the
funds
Disease
Laboratory,
for
the
Aid
his
manuscript. issued
under
Federal
in
CITED
CONTOS, N. and and purification Cult. In Press.
H. ROTHENBACKER. method for spores
1973. of Myxosoma
An
efficient cerebra/is.
HOFFMAN, G. L., C. E. DUNBAR and A. BRADFORD. disease of trouts caused by Myxosoma cerebra/is in the Department of the Interior. Special Scientific Report, (reprinted with additions), 15 pp.
concentration Progr. Fish-
1969. Whirling United States. U.S. Fisheries No. 427
Journal
of
Wildlife
Diseases
Vol.
11,
January,
1975
57
3.
HOFFMAN, G. L., S. F. SNIESKO and K. WOLF. for determining absence of viral hemorrhagic disease in certain fish and fish products. Fish Fish and Wildlife Service, 7 pp.
4.
LANDOLT, M. 1973. Myxosoma cerebra/is: isolation and concentration from fish skeletal elements-trypsin digestion method. J. Fish Res. Bd. Canada 30: 1713-1716. MARKIW, M. E. and K. WOLF. 1974. Myxosoma cerebra/is: isolation and concentration from fish skeletal elements-sequential enzymatic digestions and purification by differential centrifugation. J. Fish Res. Bd. Canada 31: 15-20.
5.
1968. Approved procedure septicemia and whirling Disease Leaflet No. 9, U.S.
6.
PRASHER, J. B., W. M. TIDD and R. A. TUBB. 1971. Techniques tracting an dquantitatively studying the spore stage of the protozoan site Myxosoma cerebra/is. Progr. Fish-Cult. 33: 193-196.
7.
RYDLO, M. 1971. Evidence of Myxosoma cerebra/is of rainbow trout afflicted with whirling disease. 97-99 (in German).
8.
TIDD,
WILBER Myxosoma 227-228.
M.,
R.
cerebra/is
Received
A. TUBB and V. spore extraction
for
publication
18
WRIGHT. technique.
June
1974
for
expara-
spores in various organs Osterreichs Fisch. 24: 1973. Progr.
Modification Fish-Cult.
of 35:
