Sarconema eurycerca
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Sarconema eurycerca (Wehr): the heartworm of swans and the role of Trinoton anserinum (F) as an intermediate host This item was submitted to Loughborough University's Institutional Repository by the/an author.
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•
A Doctoral Thesis.
Submitted in partial fulfillment of the requirements
for the award of Doctor of Philosophy of Loughborough University.
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c
Sharon Cohen
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Sarconema of
(Wehr)
eurycerca
swans and the
role
: the heartworm
of Trinoton
as an intermediate
anserinum
(F)
host
by Cohen
Sharon
A Doctoral
Thesis
in partial fulfilment Submitted of the requirements for the award of Doctor of Philosophy of the University Loughborough of Technology
April,
Oc
by Sharon
Cohen,
1988
1988
ABSTRACT All
filarial
nematodes
vertebrates
and require
life-cycles. but
few of
Sarconema A recent
aim
the
A total Britain,
new of
1128
Denmark
blood
study
host
of
and
for
samples
sub-periodic
between
11.00
intermediate
insect
to be capable
feeding
on blood. T. anserinum
transmitting
the
Examinations logical
Significantly haematocrit with
and the
nematode
from
were made of
higher and red
heart
lymphocyte blood
larval
louse
the
of
one swan to
counts
corpuscle
S. eurycerca.
i
of
required
an has
T. anserinum
T. anserinum
swan.
S. eurycerca S. eurycerca
mobile
and capable
its
and blood
percentages
occurring
whilst were of
another.
the nematode, tissue
a
supply.
of
stages
juvenile
counts
attributes
the
with
a
incidence
exhibited
ectoparasite,
was very
examining
of
maximum
in
sites
An overall
blood
microfilarie
developing
on both
effects
relationship
is
using
proportion
with
all
by
eurycerca
peripheral
satisfy
ingesting
of All
to
the
from
(microfilariae) study.
host,
As an obligate
and temporal
appears
in
hours
The main
T. anserinum
detected
were
S.
of
a
between
examined
higher
the
within
host.
spatial
in
the
a significantly
was found
a close
found
eurycerca
The microfilariae
19.00
T. anserinum
S.
in
is
country.
swans
of
c.
host
1977).
whether
were
species)
developed
with
this
Infected
stages
rhythm
and
all
Iceland.
technique
diurnal
in
parasite
(of
swans
infected.
being
swans
the
insect
relationship
swans and to determine
of
recorded
the
(Cygnus
Swans
(Seegar,
investigate
birds
swans and geese.
Whistling
(Fabricius)
to
of
intermediate
the
infect
known to
have been elucidated.
nematode
American
that
was
larval
sedimentation 15.0% was
in
their
of
completion
are
hosts
a filarial
anserinum
and British
intermediate
is
for
family
this
of
are parasites
hosts
intermediate
demonstrated
present
insect
within
eurycerca
Trinoton
S. eurycerca the
S.
) has
louse,
of
(Wehr)
of
columbianus
genera
associated
eurycerca study
feather
the
Onchocercidae
intermediate
Eighteen
very
family
the
of
components
and lower were
and patho-
morphology
the
of
eosinophil
recorded
in
swan. percentages,
infected swans
CONTENTS Page i
Abstract ii
Contents List
of
tables
List
of
figures
viii x
Acknowledgements
xiii
I
GENERAL INTRODUCTION
1
II
INTRODUCTION
6
I.
DISTRIBUTION
OF SWANS AND METHODS OF CAPTURE
8
MATERIALS AND METHODS
8
1.1
Methods
of
8
1.2
Captive
swans
1.3
Processing
2.
capture
10
captured
10
swans
RESULTS
II
DIAGNOSIS, DISTRIBUTION AND INCIDENCE OF S. eurycerca IN SWANS
15
MATERIALS AND METHODS
15
2.1
Laboratory
2.1.1
Established
2.1.2
Sedimentation
enumeration 2.1.2.1
swan
diagnosis
filariasis
15
techniques
16
of
diagnostic
for
technique
the
and
18
in swan blood
S. eurycerca
of
diagnosis
Efficiency technique of sedimentation when compared with three established methods of filariasis diagnosing
2.1.2.2
Preservation
2.1.2.3
Estimation
of of
19 19
blood
sample
in
variation
sedimentation
technique 2.2
incidence and
Distribution
in
of S. eurycerca
swans
20
RESULTS
21
2.3
Assessment
2.4
Sedimentation technique with three established
2.4.1
Effects
2.4.2
Estimation technique
2.5
19
Distribution swans
of
of
tests
for
- efficiency techniques of
preservation of
sample
of S. eurycerca
diagnosis
11
of
21 23
blood
variation
and incidence
compared
21
in
sedimentation
S. eurycerca
24
in 25
Page 2.5.1
Mute Swans
28
2.5.2
Whooper Swans
28
2.5.3
Bewick's
Swans
28
2.6
Level
infection
2.7
Long-term
2.7.1
Changes
2.8
Effect
3.
SEASONAL PERIODICITY
of
of
in
incidence
in
changes infection
of
S. eurycerca
individual
of
S. eurycerca
of
28
S. eurycerca
31
swans
34
on swan weights OF S. eurycerca
31
MICROFILARIAE
35
MATERIALS AND METHODS
35
3.1
Daily
35
3.2
Seasonal
periodicity
36
periodicity
RESULTS
36
3.3
Daily
36
3.4
Seasonal
periodicity
36
periodicity
DISCUSSION
43
Detection
43
methods
Differential S. eurycerca
incidence related
and infection to host age,
Differential incidence of to host status and health
levels
of sex and weight
S. eurycerca
45
related 47
III 48
INTRODUCTION 1.
: ITS TAXONOMICSTATUS, COLLECTION AND
T. anserinum ADAPTATION
TO A PARASITIC
MODE OF LIFE
50
1.1
Taxonomic
status
1.1.1
MATERIALS
AND METHODS
1.1.2
RESULTS
1.1.2.1
Arrangement
1.1.2.2
Sternum
1.1.2.3
Shape
1.1.2.4
Species
1.2
Collection
1.2.1
MATERIALS AND METHODS
58
1.2.1.1
Water
58
1.2.1.2
Carbon
1.2.1.3 1.2.2
of
T.
51
anserinum
52 52
of
shape of
male
bristles and
on the
bristle
gular
arrangement
storage
genitalia
54
Hand-search
samples
ectoparasite
container
dioxide
54 54
differentiation
of
region
52
58
58
chamber
59
method
59
RESULTS
111
Page 59
1.2.2.1
Water
1.2.2.2
Carbon
1.2.2.3
Hand-search
1.2.2.4
Determination
of
1.3
Adaptation
T. anserinum
1.3.1
MATERIALS
1.3.2
RESULTS
63
DISCUSSION
63
storage
container
dioxide
2.1
chamber
59
method
of
time
59
to parasitism
60
searching
optimal
60
AND METHODS
Ectoparasite 2.
59
sampling BETWEEN T.
SPATIAL AND TEMPORAL RELATIONSHIP AND THE SWAN
Identification on swans
of
life
64
T. anserinum
of
stages
anserinum
64
2.1.1
MATERIALS AND METHODS
65
2.1.2
RESULTS
65
2.2
Incidence on swans
and degree
infestation
of
of
T. anserinum 70
2.2.1
MATERIALS AND METHODS
70
2.2.2
RESULTS
70
2.2.2.1
Incidence
of
T. anserinum
2.2.2.2
Incidence
of
both
70
on swans
T. anserinum
and S. eurycerca 73
on swans 2.2.2.3
levels
Infestation
of
T. anserinum
on swans
73 77
DISCUSSION Spatial and temporal relationship T. anserinum and swans
between 77 80
3.
INGESTION AND TRANSMISSION OF THE FILARIAL
3.1
Structure
3.1.1
MATERIALS AND METHODS
81
3.1.2
RESULTS
81
Structure Structure 3.2 3.2.1 3.2.2 3.3 3.3.1 3.3.1.1
Morphology
of
of of
of mouthparts of
80
the mouthparts
of mouthparts
the
alimentary
PARASITE
Amblycera T. anserinum canal
81 81 87
MATERIALS AND METHODS
88
RESULTS
88
Crop contents
88
and feeding
methods
91
MATERIALS AND METHODS
91
Blood-feeding
iv
Page
3.3.1.2
Feather-feeding
93
3.3.2
RESULTS
93
Blood-feeding
93
3.3.2.2
Feather-feeding
98
3.4
Location and timing on Mute Swans
3.3.2.1
T. anserinum
of
Technetium-99M
3.4.1.2
Methods of marking
105
Effect
3.4.2
Behavioural studies with Tc 99M
of
Measurement
of
106
Tc 99M
with
label
radioisotope
T. anserinum
on
T. anserinumlabelled
of
temperatures
surface
105
marker
as a radioisotope
3.4.1.3
of
swans
Suitability
3.4.5
The location
3.4.5.1
Location
of of
T. anserinum
on swans from
Locations
lice
of
related
to
different
starting
111
surface
temperatures 113
swans
113
lice
3.4.6
Feeding
3.4.6.1
between Distances moved by T. anserinum 12.00 - 24.00 hours and 15.30 - 06.30 hours
3.4.6.2
Location of T. anserinum 12.00 - 06.30 hours
times
of
in
the
period
4.1.1
118
4.1.1.1 4.1.1.2 4.1.2
DEVELOPMENTOF S. eurycerca T. anserinum
LARVAE WITHIN 120 121
MATERIALS AND METHODS
Laboratory Preliminary Maintenance
rearing
121
of T. anserinum
121
experiments of
T. anserinum
by regular
blood-feeding
RESULTS
4.2.1 4.2.2
Dissection of larval stages
125 T. anserinum to observe of S. eurycerca
MATERIALS AND METHODS RESULTS DISCUSSION
122 122
DISCUSSION 4.2
113 116
DISCUSSION
4.1
110 111
on Mute Swans
lice
lice
of
for
Tc 99M as a marker
locations
4.
109 110
3.4.4
of
109 109
RESULTS
3.4.5.2
103 105
T. anserinum
Marking
3.4.1.1
3.4.3
feeding
AND METHODS
MATERIALS
3.4.1
of
developing 126 126 126 129
Page 5.
TRANSMISSION OF FILARIAL
5.1
MATERIALS AND METHODS
130
5.2
RESULTS
131
5.2.1
General
activities
5.2.2
Activity
from
5.2.3
Distances
5.2.4
Observations of handling swans
PARASITES BY T. anserinum
131
T. anserinum
of
different
131
locations
starting
134
moved by T. anserinum T. anserinum
of
activity
130
whilst 134
DISCUSSION 135
CHAPTER DISCUSSION
IV INTRODUCTION
136
1.
TAXONOMYAND MORPHOLOGYOF
139
1.1
Taxonomy of S. eurycerca
139
1.2
Morphology
139
1.2.1
MATERIALS AND METHODS
141
1.2.1.1
Examination
141
1.2.1.2
Examination
1.3
RESULTS
1.3.1
Morphology
S. eurycerca of
1.3.2
Morphology
of S.
2.
PATHOLOGICAL EFFECTS OF S. eurycerca
2.1
MATERIALS AND METHODS
152
2.1.1
Post-mortem
152
of S. eurycerca
of
nematody
adult
of microfilariae
morphology
141
morphology
144
2.1.2
Dissection
2.1.3
Preparation
eurycerca
of
examinations of
adults
144
microfilariae
151
ON SWANS
swans
152
swan hearts
of tissue
sections
for
histological
examination 2.2 2.2.1 2.2.2
3.1 3.1.1 3.1.2 3.1.3
152
RESULTS
153
Incidence
153
Location
of of
adult adult
S. eurycerca S. eurycerca
in
swan hearts
in heart
tissue
HAEMATOLOGYAND CLINICAL
CHEMISTRY OF SWAN BLOOD
Haematological Clinical
and Clinical analysis
Chemistry
analysis
163 165
MATERIALS AND METHODS Haematology
153 161
DISCUSSION 3.
151
Chemistry
of
swan blood
165 165 166
Page 3.2
RESULTS
3.2.1
Haematological
3.2.2
Clinical
167 167
analysis
Chemistry
170
analysis
DISCUSSION
173
CHAPTER DISCUSSION
175
V
GENERAL DISCUSSION
176
BIBLIOGRAPHY
183
APPENDICES Appendix
I:
May-Grünwald
Appendix
2:
Z scores for statistical comparison of infected and non-infected swans
and Giemsa staining
Appendix
3:
References to of Mallophaga
Appendix
4:
Chromagen
Appendix
5:
Z scores contents
Appendix
6:
Rapid adult
test for of
studies
for
iron
statistical age-classes
concerned
7:
Procedure
Appendix
8:
Occurrence of the mite, on T. anserinum
for
preparation
Vii
with
of weights
feeding
195 196
in blood
197
comparison of crop of T. anserinum
198
Fixation Technique for nematodes for Scanning
Appendix
194
procedure
of preparation Electron Microscopy
of heart Myialges
tissue
section
199 200
trinotoni202
LIST
OF TABLES
Page
Table
I
:
Location
Table
2
:
Suitability
each swan species
and number of for
tests
of
detecting
13
captured.
S. eurycerca
21
microfilariae. Table
Table
3
:
4
Efficiency microfilariae Counts
of
Table
5
Table
6
Table
7
:
. :
in
Incidence
S.
of sex.
and
the
effects
the
using
in
of S. eurycerca in
eurycerca
swans
8
:
Levels
Table
9
:
Incidence
Table
10
:
Incidence
Table
11
.
Incidence
Table
12
:
Long-term
Table
13
:
Changes
Table
14
:
Variations
in microfilariae
Table
15
Percentage
of maximum number of microfilariae
Table
16
Table
17
Table
18
Table
19
Table
20
Table
21
Table
22
Table
23
Table
24
Table
25
.
of
of
29
29
swan species.
of
S. eurycerca
in Whooper
30
of
S. eurycerca
in Bewick's
in
changes in
incidence
infection
in
counts
from swans.
:
Swans with
:
Opportunistic
.
Total
.
Crop contents
body
Measurements Radio-isotopes
S.
optimal
searching
Swans.
30
eurycerca.
32 33
Swans.
Whooper
37
over 24 hours.
in
S. eurycerca
of
Swans.
time
during
24 hours. 38
two Mute Swans over
for
removal
of
T. anserinum
69
samples searches of of
of
of
method).
76
dead Mute Swans for age-classes particles
used to mark
ii
75
T. anserinum.
different feather
search
74
by hand.
on swans searched (hand
T. anserinum
41
60
T. anserinum.
of of
of
individual
T. anserinum
.
species
30
of
.
26
swans.
in Mute Swans.
Determination
:
25
S. eurycerca
:
Incidence
sedimentation
of
.
:
in
S. eurycerca
Levels of infection a 30 week period.
Biometrics
of
different
of
Table
infection
23
24
counts
and incidence
Location
determine
S. eurycerca
(2%).
formalin
in microfilariae
Variation method.
age
to
microfilariae
preservation
detecting
four methods for in swan blood.
of
insects.
from
of
76
T. anserinum.
94
T. anserinum.
crops
of
T. anserinum.
99
104
Page Table
26
:
Times of experimental on swans.
Table
27
:
for all Percentage survival of temperature and humidity.
Table
28
:
Percentage temperature
Table 29
:
Measurements of lengths from
T. anserinum
Table
30
Table
31
:
Measurements
Table
32
:
Swans dissected adult nematodes.
:
Description
Table
33
.
References
S.
Table
34
Table
35
Table
lice
lice of survival and humidity.
to
specific
from
to determine
conditions
of
larvae
dissected in
different
infected
swans and swan hearts
containing
with
.
Haematological Mute Swans.
36
:
Haematological
Table
37
:
Clinical chemistry Mute Swans.
and Haematological
Table
38
.
Clinical swans.
analysis
Table
39
of
adult
summary for
data
analysis
chemistry
Summary of statistically the clinical chemistry infected swans.
lY
of
of
Whooper and
and non-infected
infected
155
157
non-infected
infected
137
156
in hearts.
S. eurycerca
127
140
hosts.
eurycerca.
Location
123
124
swans.
number of hearts
the
:
:
conditions
S. eurycerca
recording
108
Swans.
Mute
S. eurycerca
of
different
under
S. eurycerca
infesting
studies
of
of
at
T. anserinum
of
observations
values
of
swans.
captive
and non-infected
between differences significant and nonand haematology of infected
164 168 169
»1
172
LIST
OF FIGURES
Page
Figure
1:
Capturing
and processing
Figure
2:
Proportions the total
of each species, number trapped.
Figure
3:
Key to
Figure
4:
Comparison with three microfilariae.
locations
swans.
Geographical microfilariae
Figure
6:
Daily periodicity in circulating
Figure
7:
Daily periodicity Swans, represented filariae occurring
Figure
9.
Arrangements
Figure
10
:
Shape of
Figure
11
:
Shape of sternum and bristle from specimens collected in
Figure
12
:
Shape of posterior
Figure
13
:
Adaptation
Figure
14
:
Adaptation
Figure
15A :
Locations
Figure
15B :
Developmental
Figure
16
:
Mean head width
Figure
17
:
Head width separation
Figure
18
of
sternum
bristles
on gular
plate
of of
T. anserinum biometric stages
for
values
T. anserinum location
Figure
21
:
Structure of the mouthparts light microscope. micrographs
of
57
to parasitism. on T. anserinum
taken in
each size
the
class
from
swan's
and incidence of
sp.
56
62
removed
distribution
sp, 55
parasitism.
to
of
of
T. anserinum
egg. T. anserinum.
of
the mouthparts
show
on swans.
of
71 72
on swans. viewed
67 68
plumage.
T. anserinum
T. anserinum
66 66
body length to of T. anserinum and total into different of specimens age-classes.
Frequency
Electron
of
53
sP
Trinoton
42
61
T. anserinum
of
two
of Trinoton
bristles
genitalia
40
sp.
arrangements of Trinoton the present study.
measurements
:
:
27
in
Trinoton
of
22
39
microfilariae
of
of male
20
22
S. eurycerca
of
region
and arrangements
Figure
Figure
Britain.
microfilariae of Mute Swans.
S. eurycerca
of
T. anserinum
of
Geographical
14
in Mute of S. eurycerca microfilariae of the maximum microas percentages 24 hours for each swan. during
Seasonal periodicity Mute Swans.
:
in Great
of S. eurycerca vessels peripheral
8
19
12
and incidence
distribution in swans.
Figure
Figure
of
swans out
technique of the sedimentation of the efficiency S. eurycerca established methods of detecting
5:
Eggs of
age and sex of
swan capture
of
Figure
:
9
through
T. anserinum.
82 83
Page Figure
23
Figure
24
Figure
:
Light
:
Light
25
:
Electron
Figure
26
:
Diagrammatic Mallophaga.
Figure
27
:
Alimentary microscope.
Figure
28
:
Crop contents
Figure
29
:
Sequence of meal.
Figure
30
:
Feather
Figure
31
:
Strucutre
Figure
32
:
Body T.
microscope microscope
34
:
Skin
Figure
35
:
Distances
Figure
36
:
Location of 1200 - 0630
Figure
37
:
Developing
38
Figure
39
Figure
40
(A Figure
& B) 41
Figure
42
Figure
43
Figure
44
Figure
45
Figure
46
surface
(after
feathers
bird
89
light
90
(N = 259). a blood-
to
record
movement
of
107
T. anserinum
on Mute Swans.
112
temperatures
of Mute Swans.
114
T. anserinum hours.
(N = 20) on Mute
on Mute
Swans between
larvae
of S. eurycerca
stages
Swans (cm). 115
the
period
from
dissected
anserinum.
: .
Mean distances locations. Adult
moved by T. anserinum (S.
nematode
from
three
starting
) drawn using
eurycerca
a light
microscope. :
Developmental S. eurycerca
:
Electron micrograph S. eurycerca.
stages female
:
Electron
.
Electron micrograph S. eurycerca.
:
Electron of adult
:
Microfilariae
of microfilariae viewed through of
of vulva
micrograph
of
of micrographs S. eurycerca. of
external
oral
xi
of
female
adult of
opening
adult
striations
viewed
with
128
133
142
of uteri microscope.
145
adult
146
of
morphology
cuticular
S. eurycerca
within a light
117
132
on Mute Swans.
T. anserinum
of
patterns
female
97
101
1981).
Marshall,
95
100
T. anserinum.
of
crop
of
ingesting
T. anserinum
of
the
through
of T. anserinum
each age-class
in
canals
viewed
moved by T. anserinum
Activity
.
of T. anserinum
of
86
of T. anserinum.
alimentary
of
of a swan divided time. over
regions anserinum
Figure
85
photographs
of
Movement of
of mouthparts
of T. anserinum.
of mouthparts
components
:
of mouthparts
84
representation
canal
33
Figure
views
micrographs
Figure
T.
views
of T. anserinum.
S. eurycerca. female
148
and annulations
a light
147
microscope.
149 150
Page Figure
Figure
Figure
47
48
49
:
:
:
Mute Swan heart with in pericardial sac. Cross section of tissue myocardial microscope. Histological
by adult Figure
50
:
adult
female
nematode
S. eurycerca 157
female nematode S. eurycerca adult of Mute Swan heart viewed through
tissue
sections
of Mute Swan heart
parasitised
nematode S. eurycerca.
Comparisons
of
parasitised
xii
with
non-parasitised
in a light
heart
158
159
tissue-160
ACKNOWLEDGEMENTS
Malcolm
Greenwood
source
inspiration,
of
I am indebted
to
thank
friends
-
several
Alan
Dr Ali
them both
Spray
I
my gratitude
at
Loughborough
with
Study to
staff the
of
members
particularly
University by the
given
Wildfowl
Trust
Coleman.
Dr A. Ashton
and
are
Virus
Research
for
Trust
and
Swan Study
blood
with
(Steve
help
Thanks
Case,
Marjorie
specialists
Natural also
are
and History); due to
Dr T. Ladner,
- particularly
for
ornithology
Salsbury, providing
the
and
Leicestershire to
a grant
help
with
research.
Finally, support vegetable
I sincerely during garden
this
thank study
my family - particularly five
to accommodate
Xlii
and Ernest
for
my father Mute Swans.
was
and Zyllah
Medicine
Museum of
Institute).
their
several
of Tropical
(British
sampling
Support
acknowledged.
of
am
Edward Grey Institute,
the
expertise
closely I
and
crew
Dr M. Ogilvie,
Swan Rescue Centres
School
worked
Group
assistance
greatly
A. V. S.
the
I have
of
my activities
Thornback,
Dr J.
Sears
in
assisted
Mobbs.
Brian
to
for
plc
and Mick
due
Rutland
this
is
and Mrs Harris
Pharmaceuticals
Thanks
help
Dr D. Denham (London
Dr Boorman (Animal Fisons
Further
The singular
Dr C. Lyal
Bonington),
G. V. T. Matthews,
RSPCA and members of
appreciated: Hygiene);
and
and Dr J.
and their
Cook and Dave Lewis).
duty
of
matters),
Department.
Professor
by Dr C. Perrins
was given
who have
people
Dr M. Ball,
-
to
Dr P. Bacon and Bert
Oxford
many
Ecology
grateful
call
sincerely
Group).
the
of
the
on veterinary
my studies.
I also
Sutton
Centre,
adviser
University
technical
and
me beyond
helped
Investigation
(Swan
Dr Chris
have done.
they
all
who have
(independent
Zubaidy
extend
for
have been a constant
throughout
and expertise
guidance
(Veterinary
Hunt
(my supervisors)
and Dr Jim Fowler
their for
unrelenting
giving
up his
is
CHAPTER I
GENERAL INTRODUCTION
Parasitism parasite,
(Baer,
actions on hosts
do not
without
(Waage,
reasonable
1979).
The relationship the
of
in which
definitive
For be capable
usually
involving
cycle
outside
the in
of
inter-
of
may feed
without
sometimes on host
is
over
this
and Nematodirus
most
example,
is
known as the
developmental
within
stages or
secondary
the
hosts
parasite
stage
sp..
Other
in
ensure
in
conditions
incorporate
for
incorporate
and overcome
several life-
climatic
adverse
life-cycle
the
its
of
some parasites
parasites
dispersal
new ones to
some stage
To overcome process,
resting
must
can be accomplished
spending
host.
parasites
all
and locating
dispersal
transmission
to achieve
life-cycle
example,
adverse
Ascaris or
vectors
an
inter-
environmental
conditions. An intermediate both
a means of
stages from is
one host
adopted,
climatic dispersal of
the
of
the
a
physio-
For
maturity
applied
fitness
extends
life.
way of
injury
causing
morphological,
other
the
of
their
This
definitive
protected
hosts
from
species.
ways,
encountered
completion
escaping
the
of
mediate
diversity
(facultative
them
in one or a number of
place
other,
Insects
usually
sexual
1984) although
successful
of
inactive,
reaches
the
of
hosts.
the
survival
this
the
one,
a number
effect
may exhibit
parasite
the
therefore
and host
to
Within
on hosts is
adaptations the
may take
intermediate
and it
(Wakelin,
host
life-cycle
sp.
parasite
and behavioural
species
the
between life-cycle
parasite's
logical the
1979).
body
upon
a negative
(Waage,
the
exist
dependent
where
which
definition.
above
Parasitism
forms
assumption
from
there
may be dependent
haematophagous
to
the
with being
insects
or
phoresy)
only
conform
in
species
1971).
Askew,
however,
necessarily
haematophagy) (e. g.
1962;
Olsen,
associations
which
two
requirements
nutritional
1951;
insect/vertebrate
between
a relationship its
obtains
host
the
is
host
dispersal
to the
activities
the
is
parasite
but
parasite of
a vector
(Wakelin,
another
fluctuations, of
and an environment develop;
parasite
both
from
can be differentiated
simply
1984).
removed
to
definitive
Whichever
host
also
range in intermediate and the
a vector
or juvenile
larval the
transmits
some extent
an intermediate by extending
in which
by providing
parasite
dispersal from
the
dangers in
assists space
strategy
the
as a result
hosts.
of
When transfer larval
location
stages,
upon the
between
activities
Nematodirus
spathiger
summer, but
are
cold
N.
spathiger
that
the
lambs but
is
nematode
do not
1963).
to
only
into
the
host
(Croll
in
soil
long-lived
and
Matthews, can
and
remain
spring
only
develop
by
1977).
The
eggs
in
for
years
susceptible free-living
bides
are
released
ingested
by
a suitable and
resistant
development
without
is
host
next
notoriously
are
periods
ensures
are
the
if
further
the
allows
when
stages
lumbric
Ascaris
eggs
long
conditions,
ingestion
of
eggs of
in sheep
of
which
suitable in
their
soil
out
passed
When resting
larvae,
faeces
and
under
infection.
Thus,
For example,
The stimulus
free-living
and
active
fortuitous.
entirely
until
dependent
extent
have experienced
they
(Crofton,
for
produce
are
sheep nematode,
emerge
to a large
themselves.
parasites
active
available
is
host
free-living
by active
accomplished
next
to hatch
unable
larvae
are
the ,a
temperatures
at
the
of
of
is
hosts
or
deterioration.
The inclusion of
the
the
of
depends
When myxomatosis agent
transmitted
Onchocercidae,
was regarded
solely
arthropod
hosts.
filariae,
which in
accumulate
stage
the
definitive
host
of
the
infection
elephantiasis
to
the
provides
as seen in elephantiasis
of
part
host
1878). is
cuniculi
live
larvae,
definitive
host
liberate
of
the
host
arthropod
the
microor
takes
in which
up micro-
development
the
reintroduces
to
into
parasite
meal.
are
responsible
began with
characteristic
for
Our knowledge
by Wuchereria
2
Spilopsyllus
intermediate
Inflammation also
being
the myxoma virus
by blood-feeding
of man.
fatigans
the
conveyed.
blood
nematodes
1953,
of vertebrates,
and then
diseases
filarial
(Manson,
in
an environment
such as these
of Culex
Kingdom
nematodes
blood
a subsequent
parasitic
as hosts
being
nematodes
can occur, at
on the intermediate
flea,
rabbit
The intermediate
infections
important
insects
in
feeds,
infective
most
the
achieved
female
skin.
the
Filarial
is
circulate the
dispersal.
between
as a vector;
tissue-invading
of
Adult
when it
of
the United
in any way whilst
dispersal
of
on any activity
relationship
through
family
method
life-cycle
the
host.
on the mouthparts
In a major
filariae
upon the
in
or a vector
dependent
swept
developing
and not
host
a more reliable
not
definitive
and the
dispersal
is
host
next
but
parasite
or vector
to provide
appears
a parasite
Location
an intermediate
of
some of
of
Manson's
of Brugia
of
role
observations
bancrofti
and blockage
the
the
which of
lymph
malayi
causes nodes also ,
Jordan,
1962; Wharton,
Onchocerca
the
enter
filariae
(Nelson,
Grove,
1982).
stages
demonstrate
hosts
1953; is
of
is
Ctenocephalides Echindophaga
D.
immitis
D.
reconditum
felis,
C.
fleas
review
vectors
the
that
an effective observations,
Filaria
too
when the
the
Nelson
of
Eighteen genera (Anderson, 1956) but
also
of very
the
(1964)
.
remarked
hosts.
The
on the studies Nelson (1964) also
have been made for
may have been the more accessible
statement,
Nelson
have been
may well
(1962) Nelson D. reconditum of . (Amblycera) also acts as spiniger
hosts
for
the the
hirundis
the African
of of
microfilariae
Nelson
intensive
searches
vectors
confirmed
Dennysus
the
1956;
Wright, development
the
as intermediate
due to
Heterodoxus host
and
report
W. bancrofti
this
louse,
intermediate
the mallophagan cypseli
of
intermediate
other
in
irritans,
Pulex
Onchocercidae,
the
fruitless
real
intermediate
the
1956).
of mosquitoes
often
tissues.
develops
mis-identified
was undoubtedly
In making
to
discovered
hosts
filarioid
connective
(Newton
(1921)
and Breinl
have
workers
another ,
reconditum
wickhami
first
the
species
except
cheopis,
were
were
mosquito
immitis
Orchopeas
probably
of
(1892)
these
then,
sub-cutaneous
D.
immitis,
(1900)
Since
Xenopsylla
Wright,
the
in
mosquitoes.
have
vertebrates
presumed
reconditum
D.
human parasite
ectoparasites". referring
of
predominance
"all
that
out
that
canis,
and
of
mosquito-transmitted
that
damnosum
Calandruccio
Noe
and
a number
found
(1954)
but
Grassi
is
and
Stueben
and
a mosquito
in
other
1972).
Dipetalonema
to than
he explained,
mosquito
Simulium
heartwormDirofilaria
experimentally.
and
rather
apparent
of but
1970).
(Newton
In his
points
micro-
volvulus
infect
(Bradley,
develop
can
gallinacae,
in
gut
immitis,
similar
1962).
reason,
in man and some-
nodules
by blackflies,
Onchocercidae
dogs
development
Otto,
fleas
upon the
in
non-pathogenic
are
Nelson,
skin
is
host
Onchocerca
The nematode
the
D.
immitis
life-cycle
The
in
this D.
that
dogs
in
live
transmitted
the
disease
larvae
(Kartman, of
within
filarial
shown
an intermediate
blindness.
and are
diseases
a pathogenic
developing to
.
within
1964;
noted
1961;
1926).
Filarial
causes
(Laing,
species)
and other
uses
The nematodes
eye and cause
develop
(Blacklock,
that
parasite
volvulus
fatigans
1962).
filarial
Another
times
(Culex
by mosquitoes
transmitted
original is
the
his
During
dog filarioid. findings
Dutton
of
host
intermediate
(1905)
of
swift.
family
few of
Onchocercidae the
3
intermediate
are
known to
hosts
of
infect
these
birds
nematodes
have
been life-cycle
the this
of
nematode
filariae Developing have
larvae
been
are
unsuccessful mavis
had
of
filarioids
but
is
in
several
is
required
under
species
of
habits
of
little
allows
(1956)
insect
life-cycle
these
of
in
hosts
to
simulids
mosquitoes.
crawl
ducks.
and wild
that
is
some caution
developed they
a large
feathers
under
feeding
the
of
laboratory, lack
a
necessarily
fallisensis
both
The
develops
fallisensis
species
0.
filarioid
Ornithofilaria
emphasized
the
because
(Corvidae
an avian
that
0.
and although
1000
unidentified
had to be taken
note
made
cardinals
the
of
in domestic
and S. parnassum
ornithophilic
any
conditions,
a blackfly
over
family
with
who demonstrate
Special
fed
) and
infected
the
filariae.
avian of
of
crow
albicoilis
each of
other
attempts
of
Ornithofilaria
(1955)
in
feed.
(1937)
Robinson the
and micro-
lice
the
of
adults
GJnnert
).
of
bird
contents
gut
host
But Anderson
as natural
of
arthropod
of
the
on
A number
the
by simulids
that
of
venustum
significance
which
study
host.
each species
the
The
lice.
duck.
were
experimental
in concluding
in Simulium
in
).
when
hosts
was observed
simulids.
intermediate
natural
of
birds
all
transmitted
shows that
bodies
(Zonotrichia
by Anderson
fallisensis
fat
on members
comprehensive
was performed
blood
musicus
);
of
than
determine
no development
The first
tissues
a domestic
species
cardinalis
cypseli
intermediate
from
sparrows
(Richmondena
(Filaria
microfilaria
fed
several
provided
rather
the
the
(Turdus
on white-throated
study
in
to
a thrush
mosquitoes
found
one
attempts
in
lymph
information
first
the
sub-cutaneous
from
observed
he presumed
which
in
determine
to
made
(1931)
Thomas
located
ingested
are
filarioid
an avian
are
(1905)
Dutton
elucidated.
are
of
basal
to
claw blood
take
meals. The only was undertaken
of
study
avian
by Seegar
Trinoton
anserinum
nematode
Sarconema
(1976,1977)
(Amblycera)
columbianus
eurycerca ) in columbianus
development
of
been
injected
infective into
development
No close
observations
the
larval
of
feeding stages
infections the
S. eurycerca
within
4
dynamics the
on the
made observations
(Cygnus
which
had then
olor
) and
approximately
habits
and recommendations population
the heartworm
of
T. anserinum
after
feeding
louse,
Swans ( Cygnus
Seegar from
host
Anderson
of
a. feather
that
Mute Swan cygnets
were made of
mechanisms,
the work
of Whistling
dissected
larvae
follow
intermediate
the U. S. A.
of heartworm
of
the
(Wehr)
of T. anserinum
characteristics
to
who demonstrated
is
non-parasitised
reported
studies
filarioids
14 weeks.
and behavioural for made were
further
development the and
intermediate
host.
of
In an attempt on swans, 1)
the
to substantiate
to determine in resident
the
to examine Sarconema
3)
and verify
has three
incidence
main
the
the
to
investigate
role
of
found
Trinoton
levels flocks
of Sarconema
eurycerca
of Whooper and Bewick's
Britain; between
relationships
eurycerca
to
of
role
aims:
infection and
Mute Swans and wintering
Swans in Great 2)
thesis
present
the
the
(nematode) insect
Trinoton
and the
swan with
as an intermediate
the pathological
effects
anserinum specific
(louse), reference
host;
of S. eurycerca
on swans.
CHAPTER II
INTRODUCTION
Until
1950s,
the
Swan.
This
was
birds
wild
due
partly their
and
little
relatively
was
the
the
introduction
absence
of
Another
amongst
for
their
1960,
study Before
fewer
than
had been a great
there that
end of
truly
not
scientific
in
ring
and by the
were
individuals.
By 1965 however,
ringers
the Mute
of
neglecting
marking
longer-lasting
a stronger,
interest
of
for
swans
justify
not
reason
swan ring
1000 Mute Swans had been marked. upsurge
that
did
status
1967).
a suitable
of
beliefs
widespread
semi-domestic
(Ogilvie,
investigation
to
the biology
was known about
14,000
year
swans had been ringed. Ringing
information
provides
including
population
territories
and
information
of
head wires, and oil
(Ogilvie,
parasitism
(intestinal),
Detailed
percentage national
1982),
lead
Sears
Bewick's
Blood in
blood
levels
of Mute
is
poisoning
from
died
localities
particular
lead the
where
even higher
than
the 75%
and Richmond)
oxford
in
Swans wintering
lead in
(Oxford
Grey Institute
Swans on the Thames. incidence
shows the
Interest
area
77%
of
poisoning
lead
The most in
poisoning
has extended
whose lead
Britain,
University)
levels
monitors
recent this
work
area
years.
has therefore During
large
formed
an integral
swan catches
6
at
part
is
to Whooper and are
also
determined
samples.
collection
recent
1981) reported
90% and Stratford-on-Avon
Trent
of
factor
mortality
England
Thames (between
river
river
lead
ingestion
1981).
(1986)
decreasing.
from
by the Edward
research
blood
from
the
in NCC report,
Ongoing
by J.
on the
average;
highlight
studies
and buildings
1982,1983).
from
swans examined
swans dying
of
(Birkhead,
all
over-
with
vehicles
due to
to be a major
in
that
reports
by MAFF (NCC report,
conducted
of
with
poisoning
1981; Birkhead,
NCC report,
investigation
poisoning.
(Hunt
has been found
weights
provide
due to collisions
collisions lead
often
(1963)
was mostly
More recently,
50% (N = 226)
that
of
breeding
movements,
recoveries
Eltringham
swans death
1981;
A national
death.
400 ringed
lead
Ringing
studies.
causes
local
and
Swan biology
Mute
of
aspects
many
regional
life-history
pollution.
anglers'
the
dynamics,
about
a sample
about
of
Caerlaverock,
swan catching Abbotsbury
and Welney
for
it
example,
has been customary
a maximum number of blood variety
of
including
analyses
The blood
samples.
the detection
to work
in
is
portioned
then
lead
of
a team to obtain for
a
and S. eurycerca
microfilariae. In and the
this
chapter,
swan is
the
relationship
investigated
1)
the
distribution
2)
the
diagnosis
3)
the
seasonal
of
between
the
nematode
S. eurycerca
by studying: swans and methods
and incidence and daily
of
swan capture;
of S. eurycerca
periodicity
in
of S. eurycerca
swans; microfilariae.
1.
DISTRIBUTION
The British location
of
OF SWANS AND METHODSOF CAPTURE
Isles
and countries
large
numbers
breeding
sites
winter.
Consequently, for
climes
in
the winter
Mute Swans have swans, which
are mild
Britain,
Mute
of
in winter
enough
movements Swans
are
for
searches
of
for in
to breed
usually
Caerlaverock
Wildfowl
Trust
Swans breed
around
(3,700
Some 5000 - 5,500 large
the Wildfowl
Trust
of
Refuge
their
are
movements
weather
Iceland,
Many are
or
sites
Swans winter
(up to 2000)
to
seen each year
at
the
in in
2,300
and fly
Russia
southern Britain
England
and
each year;
on the Ouse Washes near
collect
(Birkhead
migrate
Dumfries.
near
wintering
at Welney
in
Sea in northern
Bewick's
by one of
A swan hook Each summer, fly.
lakes
on food
extent
to
Seasonal
1986).
and Perrins,
three
methods
by canoe
: by baiting,
round-
swan pipes.
swans usually
unable
Indeed,
area.
Swan Capture
Mute Swans on urban a large
natal
severe
supply,
Reserve
the Kara
numbers
Swans were captured in
Within
AND METHODS
Methods
ups or
areas
resident.
common.
not
winter.
Nature
km) to and from
particular,
I. I.
England
and northern
MATERIALS
the Eurasian
new territories.
for
in
in
to warmer
all
same general
food
to
related
Scotland
Ireland.
of
range
50 km are
than
more
the
territories
miles
to migrate
them to remain
Whooper Swans, whose breeding
Bewick's
to birds
and Whooper Swans, many inhabit
Bewick's
the
breeding
southerly
Mute Swans tend
permanent
suitable
months.
the most
and unlike
forced
are
are
inhospitable and
become cold
many wildfowl
the winter
are
latitudes
Northern
of wildfowl.
summer but
the
Europe
of north-west
come close is
used
for
swans moult Ringers
and rivers
by the
provided enough those all
are
semi-domesticated
which flight
their
and researchers
8
When food
public.
to be captured
birds
and survive
straight
is
from
to
offered, the water.
are more cautious. feathers take
and for
advantage
of
a few weeks are flightless this
FIGURE 1:
Capturing
and processing
swans.
A.
Swan pipe where migrant and resident swans are trapped Dumfries. Trust Reserve, Caed averock, at Wildfowl The two gaps in the wooden fence usually remain open (swan feed the to within area swans and come pipe). Once a year, the pipe is enclosed and swans feeding inside are trapped.
B.
Pens at the end of the are caught individually
C.
Whooper Swans at
D.
Blood
sample
the
taken
swan pipe from where with swan hooks.
blood
from
the
field
collecting
tarsal
9
vein
(leg).
the
birds
station.
period
and use canoes
caught
in canoe
Swan pipes numbers
are
feed
to
encouraged
Once or twice
a year
the
channelled
swans are (Fig.
1.2.
Captive
Within
the
eight
(Fig. swans
the
Trust
grain
gates
at
into
the
pens from
swan pipe
the
end of
either
large
trap
employed
are
Swans
and Whooper Swans.
in
provided
to
Such pipes
IA).
Bewick's
to capture
from
and in Leicester.
by the Wildfowl
and resident
Swans were
of Mute Swans.
Abbotsbury
at Alvecote,
and Welney
flocks
whole
have been constructed
of migratory
examined
round-up
round-ups
Caerlaverock
at
to
they
where
are
pipe are
day.
every closed
captured
and
and
IB).
Swans Wildfowl
Trust
of
species
there
collections
swans.
All
species
were
over
the
incidence
of
representatives
are
examined
of
Peakirk
at
all
and
Washington. Increasing
concern
prompted
many people
prominent
of
it
staff
to establish
and RSPCA centres
also
to
obtain
was possible
care
in
poisoning swan rescue
in Norwich,
are based
services
rescue
Veterinary
in Britain
lead
for
Three
centres.
Cheltenham
swans,
and Windsor. the
and with
cooperation
and injured
from many sick
samples
swans has
Mute
Swans. Five
Mute Swans were maintained
easy
access
on three
secured remaining located
side
from
prevailing
The ring
BTO ring
conspicuous
by the Wildfowl by field
Darvic
Trust.
inspection.
(Vitalin).
(2.5
Swans were Bread
and fresh
the
protected mx0.7
mx1.5 fed
15 m
5mx
of
was an area
on wheat green
mixed
m) was with
vegetables
swans
each captured
was placed
plastic
winds.
A pool
within
station
foodstuffs.
captured
number of
at
a field
Wooden boards
wire.
enclosure.
complex basic
the
Processing
the
captivity
The enclosure
by chicken
sides
vitamin
supplemented
metal
laboratory.
at one end of
a balanced
1.3.
the
of
in
on the ring This
Mute Swan was noted, leg.
In addition
was placed enables
if
to a metal
a
unringed ring,
a
Swans Bewick's and identified be swans to
on Whooper
individual
or
A timed
blood
heparinised
(3.0
sample
by being
As far
the
as possible
distinguished head. Females
tend
fleshy
were
knob at
the
with
ID). from
and suspended
white
of
heads
base of
are
a pair
than
the
in
comparable
the
of
appearance this
way.
and the male has a large,
males,
bill.
the
plumage
bill.
Whooper and Bewick's
(first
Cygnets and
dull
coloured
plumage
and
pale
orange
of this study as "juveniles".
Adults
characteristics.
plumage
*For the purpose to collectively
in
differences
obvious
Mute Swans were
Swans
examination.
on their
aged
grey
were
to have smaller
coloured
their
with
(Fig.
coated)
bandages
a 5.0 cm'
in
vein
lithium-heparin
restrained
the male and female
sexed by cloacal
a full
tarsal
each swan was determined.
sex of
when there
Usually
Swans
the
balance.
a spring
were
cm')
(Sarstedt-Monovette
syringe
Each swan was weighed
black
from
was taken
birds)
winter bill.
all
by
immature
were
with
white
identified
were
Juveniles
birds
*
bills.
both
are
and juvenile
cygnets
swans are
referred
1128 swans were processed.
From
RESULTS During the of
the
course
total
this
of
swans sampled,
unknown age.
was almost
(Fig. 2B).
equal
Three
main
total
number caught.
species
the
2A).
Mute
Swans (N = 718) were
four
Wildfowl
42.5% male
Swans,
Swans (31.1%)
to male swans caught
and for
17.2% was not
at
63.6% of
constituting
Swans (3.1%).
and Bewick's from Wildfowl
processed
captured
of
(N = 219),
50 sites
in
Great
Trust
351 Whooper Swans were caught; (N = 123) and from in Iceland
Thirty-five
Bewick's from
others
(N = 4)
Swans were
Caerlaverock
(N = 6),
trapped,
they
were
the Wildfowl
(Fig.
Trust
including
(Fig.
3).
in Caerlaverock collections
from Welney
(N = 2),
Peakirk
(N = 25),
I summarises
examined
in
this
the study.
locations
and numbers
at
of
each species
and
(N = 1) and
(N = 1).
Iceland
The
3).
mostly
Washington
sampled
the
collections.
Britain,
Trust
(N = 5) and Washington
Peakirk
1.5% were
2C). Mute
species
female
of
and
(N = 4) reserves and six rescue centres Swans (N = 149) were also captured in Denmark (1985).
A total
Table
female,
(Fig. sex
Whooper
(Fig.
Mute
The proportion
were examined:
2.2% were other
remaining
27.0% juveniles
71.5% were adults,
: 40.3% were
to determine
possible
investigation,
of
swan
FIGURE
2:
Proportions swans out
age and sex of of each species, number trapped. of the total
A.
Proportion the course of each swan species trapped during (Total investigation the number of swans trapped of 1128).
B.
Proportion (Percentage
of adult and of juvenile
Proportion
female and of male
C.
juvenile swans
12
swans includes
trapped cygnets)
swans trapped.
Coscoroba 0.4 Black-necked
Block 0.5% Trumpeter 0.47 `Nhistling 0.4 ? ewick's 1 17 .
A
Unknown 1.= ä
B
C
TABLE 1: SPECIES
LOCATION AND NUMBER OF EACH SWAN SPECIES CAPTURED LOCATION CODE
MUTE
NUMBER
LOCATION
1.
Montrose
81
2.
Lothians
18
3. 5.
Caerlaverock Nottingham
77 5
6.
Alvecote
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
149 51 2 1 31 20 41 1 25 28 1 38
Leicester Peakirk St Neots Welney Cheltenham Oxford Hemel Hempstead Thames Reading Lymington Abbotsbury
149
Denmark
718 WHOOPERS
3. 4.
Caerlaverock Washington
219 4
8.
Peakirk Iceland
5 123 351
BEWICK'S
3. 4. 8.
10.
6 2 1
Caerlaverock Washington Peakirk
25
Welney
1
Iceland
35 4.
Washington
2
8.
Peakirk
2
TRUMPETER
4. 8.
Washington Peakirk
2 2
BLACK
4. 8.
Washington Peakirk
4 2
BLACK-NECKED
4.
Washington
4
8.
Peakirk
2
4. 8.
Washington Peakirk
2 2
WHISTLING
COSCOROBA
---------------------GRAND TOTAL
--------------------------
-------------
24 ------1128
FIGURE
3:
KEY TO LOCATIONS OF SWANCAPTURE IN GREAT BRITAIN
1.
Montrose
2.
Lothians
3.
Caerlaverock
4.
Washington
(Wildfowl
5.
Nottingham
region*
6.
Alvecote,
Staffs.
7.
Leicester
region*
8.
Peakirk
9.
St Neots,
region* (Wildfowl
(Wildfowl
Trust Trust
Trust
Dumfries
reserve),
Tyne
collection),
and Wear
Cambs.
collection),
Cambs.
10.
Welney
(Wildfowl
11.
Wildlife
12.
Oxford
13.
Hemel Hempstead
14.
Thames region*
15.
Reading
16.
Lymington,
17.
Abbotsbury,
(*
swans were caught
Trust
Hospital,
Cambs.
reserve),
Cheltenham,
Glos.
region*
region* Southampton Dorset
at a number of
14
sites
within
the
region)
d
2.
DIAGNOSIS, DISTRIBUTION
To date,
The most
Swans.
USA and Alaska
the
determine
to
(1977)
of
results
adult
postin
nematodes in
of S. eurycerca
study
live
Swans in
795 Whistling
who surveyed
incidence
the
of
presence
comprehensive
by Seegar
swans was performed
have presented
eurycerca
by reporting
mortem examinations Whistling
of S.
studies
most
IN SWANS
AND INCIDENCE OF S. eurycerca
in blood
of microfilariae
samples. is
There adults
or microfilariae
present
where
the
1979a).
There
is
Thames with this
adult
the in
applicability to determine
in
type
of
incidence
the
for
available study
are
and Bewick's
detecting
in
the
in (Seegar,
17.1%
Swan from
filariae
the
and their is
One technique
used
Mute Swans and in
in British
Swans wintering
Until
1965).
examined.
of S. eurycerca
either
sites
was
a Mute
of
(Boughton,
the heart
three
nematode
literature
of
Britain.
only
at
the
of
the
techniques
this
Whooper
migrant
in
report
nematodes
section,
surveyed
incidence
overall one
been
swans in
in
S. eurycerca
Swans have
and incidence
distribution
the
of
of
Mute
study,
Britain,
In
record
no national
Britain.
MATERIALS AND METHODS 2.1.
Laboratory is
Filariasis Many methods essential
diagnosis
by identifying
diagnosed have
filariasis
of
been developed
requirements
of
microfilariae
for
a method
in blood
this for
There
purpose.
diagnosing
are
S. eurycerca
samples. three in
swan
blood. The method has to be:
Eight
1)
quantitative;
2)
applicable
3)
inexpensive
widely
suitability
used for
to both for
tests
diagnosing
fresh
routine
and preserved use.
and one new technique the
blood;
presence
15
were
assessed
of S. eurycerca.
as to
their
2.1.1.
Established
At
simplest
the
A thick
diagnostic level,
under their
active
whole blood was placed on a glass
(x10)
low power
the
wriggling
smear was prepared
end of
a slide
Microfilariae
were
were detected
of blood
(
0.02
the blood
is
spread
a drop
another
and examined by
1972).
by placing
and by using
layer.
(Bradley,
microfilariae.
slide
Microfilariae
magnification.
motion
A thin
thin
filariasis
smear can detect
wet blood
a direct
mount of fresh
for
techniques
slide,
stained
May-Grünwald
using
cm')
at one in a
out
and Giemsa
(Appendix Counting (0.02
into
cm3) was placed was designed
The cell a slide of
have been employed
chambers
settled
rapidly
to
(1971)
Denham
the
excluding
The most
this
addition
of
the
concentrate is
blood for
for
one end with
in
putty.
were visible
Five
or
six
tubes
capillary
a glass
The Knott A blood
slide
test sample
and thoroughly The supernatant drops
of
glass
slides
(1.0
cm3) was placed
was discarded
0.1% methylene
in
(Knott,
a tube
sediment
The stained 1939).
16
with for
(x10). fraction.
cell
by fixing
them at
of
stained (9.0 2% formalin 5 minutes with
was placed
one
1966).
and Jain,
volume
at
sealed
cm')
blood
resuspended
sediment
this
was centrifuged
with
a small
which
1962).
the microscope
(Schalm
was centrifuged
and the
blue.
and examined
in
larvae
concentrates
uses
(0.075
the white
plasticine
which
those
tube
together
examined
also
mixed.
are
One method
was examined
of
The tube
by
syringe
were
in a piece
microfilariae
in a heparinised
above
suspension
1944).
(Bennett,
just
cm'
cell.
a microcapillary
microfilariae
1.0
holds
Lawton,
technique
The plasma
of
Microfilariae
microfilariae
volumes. tube
Live
end of
detecting
small
collected
counting
the
and
live
it
area;
applied.
with
cell.
and consists
added,
(Brady
use
the
to
acid
into
5 minutes
for
method
cm in
M) was
chamber
counting
in water
2x5
Blood
samples.
a Sedgwick-Rafter
a coverslip
the
of
techniques larvae
and
the microcapillary
Anticoagulated rpm)
bottom
and
cm3 0.1
needle
the
of
organisms
cm deep (1.0
acid
modified
accurate
principle
enumerating
a dissecting
with
chamber
0.1
Hydrochloric
stirred
(1500
for
a depression
with
fluid.
the
infected
to diagnose
at
blood. cm3)
1500 rpm. several
on several
In
the
petri-dish in
and deposited diameter). initial
hours
later
dish,
was drawn
then
the
(50.0
and washed
was washed twice
into
the
holder
same syringe
syringe
was passed
plunger.
with
technique
expendable
The anion-exchange
was later
depends
consisting blood
technique
used to elute
cm3) to
to dry
and allowed
a low power
under
time
technical
and
from
of
on differences of
a
ionic the
less
strength parasites,
blood
from
parasites in
column
negatively of
the
varies
of
rats
and mice
blood
absorbs charged
cells
and platelets
The anionmore negatively flagellates
phosphate-saline-glucose for
and
(Lanham and mammals
charge.
surface
DEAE-cellulose
whilst
the
by
devised
was originally
in man and other
trypanosomes
components
The optimal
the
through
membrane was removed
and scanned
considerable
trypanosomes
The separation
fundamentally
exchanger
eluted.
to
on
pressure
supplies.
isolate
to
was
The membrane was washed
was rinsed
slide
requires
centrifugation
applied 1970).
Godfrey,
It
was
5 pm porosity).
(10.0
saline
The Millipore
cm3).
on a microscope
placed
Lanham (1968)
(10.0
cm3) was passed
sodium
by a Swinnex
by a steady
filter.
on the
(1973).
blood
the
until
by
described
saline
and replaced
by formal
Giemsa or Haemalum.
This
objective. expensive,
water
with
being
followed
collected
distilled
and stained
charged
saline
(5.0
to wash the membrane,
any microfilariae
filter
the
was possible
method.
(25 membrane mm diam.,
through
Normal
it
found
containing
shaken
or
was removed
Millipore
The final
cm3) in normal
9.0
10 minutes.
and Southgate
in a syringe
were
microfilariae
based on the method
(10%,
the
of
water.
by Desowitz
overnight,
serum samples
containing
by this
the
Twelve
200 g for
at
blood
and rotated
The needle
containing
blood
Haemolysed
from
Teepol
cm3).
haemolysed.
completely
before
saline.
angle
portion
Obeck (1973)
cm3) was collected
(2.0
clear
distilled
with
developed
solution
twice
with
twice
After
position.
Pooled
sediment
has been further
citrate
syringe
the
technique,
sample
the
cm3) and centrifuged
membrane filter
(1.0
in
dish.
a slight
lowest
the
and needle.
50-85% of microfilariae
(1967),
filter
plate
a syringe
mixed
A blood
buffer
with
supernatant,
The Millipore
fix
off
in
(15 x 150 mm
dish
the
of at
was stored
cm3) was collected
petri
two-thirds
had collected
serum which
the
recover
the
of
discarding
sediment
drawn
the
tubes
was thoroughly
Bell
third
(3.0
blood plastic
over
plate
in
placed
After
the
1973),
a sterile
of
was spread
had formed,
the bloodless
with
to
the bottom
The blood clot
(Obeck,
technique
different
species
are (PSG) (Lanham,
1985). in
The technique
the
During
to
these
field
perstans
also
1980). this
centrifuged
2.1.2.
This
freshwater (50.0
(50.0
cm3,0.1
was then
filled
for
24 hours
sample,
base
releasing
sedimented
suggested
2.5
and error
cm3 of
preserved
that
ensured filariae
whether
were
0.25 ratio of
1: 9).
of
make the
1: 9.
Therefore, and 2.25
blood It
sedimented count
preserved
the
blood
that
up to
0.25cm'
blood.
)
cm3 of
The chamber
The chamber
slip.
deposited
on
chamber
was
the
bottom
was
the
of
from
removed detected
were
fresh
blood
2.5
in
cm3 of
cm3
9.0
18
to
and
the counted
blood
Preserved formalin blood
microfilariae of
comparable
solution
would to
according
micro-
all
was sedimented,
factor
was
sediment
These dilutions
blood.
preserved
when counting
and therefore
blood
cm3.2%
2% formalin
a correction
of
debris.
sedimented
blood
was necessary
was mixed
microfilariae.
all
volume
or fresh
1.0 cm3 fresh
as
cm3 fresh
fresh
from
of
and 0.1
amongst
acid
Zeiss).
an optimal
preserved
visible
was collected (a ratio
blood
The
pipettes.
glass
Microfilariae
(1M35
counting
chamber
(*)
Blood
of
the
for
hydrochloric
a cover
were
liquid.
(1958)
sedimented.
cells.
with
material,
(*
of
enumeration
al
with
sedimentation
x100
was
microfilariae.
and
material
disposable
and sealed
at
Trial
red blood
the
on an invertescope
the
concentration
a cylindrical
of
which
microfilariae
extraneous
find
method.
by Lund et
base and filled
the
to ensure
To examine
into
base
acid
for
diagnosis
consisted
pre-calibrated
with
using
P04 : glucose;
each buffer
by this
the
devised
lyse
using
an infected
base.
for
The apparatus
M) to
(ratio
and examined
swan blood
used to
was
al,
blood
swan
onto
from
extracted
was originally
was placed
acid
In
in
algae.
the
left
were
cm3) and a separate
chamber
into
10 minutes
from
S, eurycerca
The eluate
technique
eurycerca
technique
isolate
1979).
al,
(Lumsden et
column
eluting
no microfilariae
S.
the anion-exchange
use
of Dipetalonema
microfilariae
S. eurycerca
9).
Sedimentation
that
concentrations
700 g for
at
However,
8,1:
7,2:
in man
of buffer
for
strength
6,3:
was found
was made to A range
ionic
it
for
miniaturised (Lumsden et
apparatus
trypanosomaemias
through
passed
technique.
5: 5,4:
low
surveys
An attempt
optimal
was modified
detect
field
and the
contain
the
original
from
0.1
2.5 was applied with
counts
made
cm3 to
2.1.2.1
Efficiency
of
established
the
efficiency
tests
of blood
In the
the
Wildfowl
13.00 blood
tube,
reserve
samples
were in
were quick
on large
to perform
and easy
smear
Caerlaverock,
at
from
taken
the
box until
a cool
Swans were captured
at
08.00
and
Dumfries. tarsal
they
Between
in heparinised
vein
Capillary
analysed.
were
tube,
12 hours of blood within smear tests were all completed (1.0 cm3) was removed from each sample tube and
Blood in
three
with
the wet mount and thin
Swans and 6 Bewick's
Whooper
wet mount and thin
preserved
The capillary
Trust
collection.
in blood,
samples.
and kept
syringes
method was compared
they
three
with
number of microfilariae
sedimentation
because
1985,88
February
the
when compared
filariasis
diagnosing
estimating
methods.
were chosen
numbers
for
of
established
for
methods
As a new technique
technique
sedimentation
(9.0
2% formalin
cm3) until
by the
examination
sedimentation
method.
2.1.2.2
Preservation
20 blood
When more than impossible
of
to
in
and to ensure
that
significantly
altered
Eight
were
samples
was then
2% (9.0
After
All
Estimation
methods
sampling
of
of
microfilariae
by the
the
Therefore,
effects
infection
up to
three
of
formalin
was some samples It
months.
was
on microfilariae
in each sample
not
were
fresh
preserved
sample
blood
for
in
variation
the
20 and 90 days at
the level
involved
in
organisms
formalin 12°C.
7-
determined. was
of infection
technique
sedimentation
of
abundance
Each
by sedimentation. blood (1.0 cm') in
are
to chance
subject
by
microfilariae
estimating
investigated.
sources
from
periods
by diluting
The errors
Two potential
taken
at
samples.
as fresh
of preservation
were
variations
of
quantified
sedimentation
in
for
it
one time,
by preservation.
estimating
errors.
as fresh
preserving
levels
the
preserved
each period
2.1.2.3
the
Samples were
cm').
were collected
formalin
to determine
essential
sample
samples them all
analyse
had to be preserved
blood
of variation operator
sub-samples original
(operator
counts sample
errors
exist: (that
error) is,
[sub-sample
arising
and,
different error].
from
errors volumes
counting
due to of
blood
Ten blood
samples
1)
operator
2)
sub-sample
2.2
and
-
error
samples Denmark
locations was
error
Distribution
Blood
of
examined
sedimentation
five -
counts
five
determine
capture and
the
technique
the
sites
determined
errors
were
species
eight
distribution
counted
from
in
swans
incidence
of
(Section
20
S.
of of
described
are
in
eurycerca
2.1.2).
by:
same sub-sample;
of S. eurycerca
from
taken
the
of
sub-samples
incidence and were
to
and the
were examined
S.
in
swans
one sample.
Britain,
Iceland Geographic
eurycerca. 1.
Each
blood
assessed
using
the
Section
sample
RESULTS
2.3
Assessment Three
attributes
S. eurycerca these
were
diagnosis required
of S. eurycerca
(Table
for
a technique
of
(p. 15 ).
microfilariae
attributes
TABLE 2:
for
tests
of
Each test
detecting in
was assessed
terms
of
2 ).
SUITABILITY
OF TESTS FOR DETECTING S. eurycerca
MICROFILARIAE
ATTRIBUTES OF TEST TEST
Applicable to fresh blood and preserved
Quantitative
Wet mount
X
Thin
X
smear
Sedgwick
Pf
tube
test
Petri
dish
Millipore
X
membrane X
exchange
None of
the
a test.
X
J
'/
established
techniques
had all
The sedimentation
technique,
however,
eight
and was therefore
requirements
2.4
P/ Pf
X
Sedimentation
of
X
X
Knott
Anion
X
Rafter
Capillary
Inexpensive
Sedimentation
technique
- efficiency
the
all
exhibit
this
compared
required
attributes
did
use in
for
adopted
the
study.
three
with
established
methods 3
Table
is
presented
A comparison
of
technique
ation
in
the
the
results
is
the most
test
detected
same format of
all
four
sensitive
as that methods
by Seeger
adopted shows that
in detecting
the
microfilariae
(1979b).
sedimentin
swan
blood. The thin for
smear
by sedimentation.
microfilariae
and the
capillary
as detecting
5.0% of
100%.
tube
test
45.0%.
(Fig. 4)
21
samples
determined
The wet mount The sedimentation
test
to be positive detected technique
20.0% was taken
FIGURE 4.
Comparison sedimentation of methods microfilariae.
the efficiency of the technique three with established detecting S. eurycerca of
(The sedimentation 100% of detecting
is taken technique infected samples)
22
as
100 90
80
70
60 c
50
U] 40
30 20 10 0
Thin smear
Wet mount
Capillary
Sedimentation
The sedimentation
technique
blood.
preserved
decreased
tests 30 per
0.25
Below
cm3.
recorded
recorded
detected
filaremias
below
0.25
per 3:
TABLE
was less
as detected
samples,
test
capillary Overall
test.
the
in
the swans that I to 46 per
were
0.25
(N = 94)
sampled
cm'.
OF FOUR METHODS FOR DETECTING S. etzrycerca
MICROFILARIAE
NUMBEROF SAMPLES DETECTED AS POSITIVE BY EACH TEST
0-9
Wet mount
smear
Capillary
Sedimentation
0
1
7
26
10 - 19 20 - 29
0
2
6
8
0
2
2
3
30 - 39
2
3
3
3
Total
2
8
18
40
% sensitivity
5
20
45
100
% samples (*) infected
2.1
number of
2.4.1
Effects
Three
counts
(Table
4);
of
number
in
2)
number
in blood
3)
number
in
For
each of
(Table
8.5
samples
preservation
fresh
blood
the
blood
of blood for
were obtained
(per
42.5
0.1
cm3 x 2.5
each of
for
20 days
(per
0.25
cm3);
preserved
for
90 days
(per
0.25
cm3).
counts to
test
from for
4 ). 23
fresh
and preserved
any effects
of
the
eight
samples
factor);
correction
preserved
samples,
statistically
19.1
= 94)
analysed
of microfilariae
1)
compared
new
SWAN BLOOD
Thin
total
and
72% had micro-
swans of which
from
cm3, ranging
MICROFILARIAE PER 0.25 cm3
(*
than
cm'.
infection
EFFICIENCY IN
infected
0.25
The mean microfilarial was 8.0
by the wet mount
60% more 10 per
by the
as negative
cm' of
and wet mount
infection
27 known positive
level,
as negative
technique
tube
capillary
of microfilarial
this
were
the
of
level
when the
by sedimentation, 37 were
estimated
The sensitivity
per 0.25
microfilariae
blood
were
preservation
process
One of blood
the
in preserved
than
1.
Estimation Operator For
of
ten
each of
five
the in 2.
samples,
five-,
during
samples
seven
showed no
in
technique
sedimentation
of microfilariae
were
exactly
(Table
5 ).
There
and the
risk
sample
counting
were made of
counts
this
of
one sedimented same for
the
due to
was no error is
each
ignored
therefore
work.
Sub-sample
error
Variations
in
was applied
TABLE 4:
variation
in each
counts
further
(Table
sample
Counts
operator
There
The other
fresh
error
sub-sample. of
blood.
in
more microfilariae
differences.
significant
2.4.2
(YSH) had significantly
samples
5).
sub-sample
to determine None of
the
was no significant
Counts
of
preservation
SAMPLE
counts
were observed
if
differences
the
sets error
microfilariae in formalin
of
counts
due to
and the in
counts
chi-square were
significant different.
was significantly sub-sampling.
to determine (2')
the
effects
of
COUNTS OF MICROFILARIAE Fresh blood
for Preserved 20 days
Preserved for 90 days
X2
229
13
12
14
0.15
ns
YSH
23
5
5
19.64
p
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