EXTRACTING PROTOSTRONGYLID NEMATODE LARVAE FROM UNGULATE FECES

‘f ttiIlli

Junuil

EXTRACTING

PROTOSTRONGYLID

UNGULATE Sean

LARVAE

FROM

FECES

G. Forrester

Department

NEMATODE

/)msea.w,. :i:t:1 P197. PP .51 1-516 \VmhIImf,’ I)m,’as,’ ASSIKmatm,nm 1997

and

of Biology,

Murray

Lakehead

W. Lankester

University,

Thunder

Bay,

Ontario,

Canada

P7B

5E1

T: A major weakness of the Baermann funnel technique for extracting nematode larvae from feces is the funnel. As umny as 67% of Parclaphostrongylus temiis first-stage larvae lodged on the sloping surface of glass Baermann funnels. The number of larvae collected after 24 hr was not significantly correlated with total numbers in the samples, whether feces were supported over tissue paper or over window screening. Instead, we collected about 8 times as many larvae and achieved a significant relationship between larvae collected and the total niminhers present when pelleted fecal material was submerged over screening in vertical-sided beakers. The methodology’ of this more efficient and more accurate way of estimating numbers of protostrongylid larvae is described. Most larvae were located on and in the muCOuS layer covering fecal pellets and readily left fresh pellets emersed in water; 72% of these larvae left after 6 ruin and only 11% remained after 1 hr. Larvae in water at room temperature sank as fast as 6 cm/mm, hut those close to a vertical glass surface sank more slowly (97% sank 18.5 cm in 105 mm). Key ivords: Baermann funnel technique, fecal examination, nematode larval collection, Parelaphostrotzgilus teiuis. ABSTIIA(

INTRODUCTION

numbers

of

from The

Baermann

originally

larvae

from

has

since

used

funnel

employed

1917),

been

variously

modified

to

1961; al.,

detect

and the

feces

Forrester 1970;

and

Uhazy

the

et

al.;

1964;

1973;

Beane

vorsen

and

Wissler,

1994).

The

technique

involves

supporting

filter

ces

over

a

screening must

in leave

the

filter,

funnel

erage

number

data

the

stem

they

are

drained

mean

individual).

The

is limited

is

used data,

larvae

mean

intensity

of

identified Baermann

the Fecal P. tetiuis sunimer

passing

data

(ax’-

per

infect-

of prevalence

sensitivity

of the when being

aponly

passed

1991).

require

protostrongylid

tested.

In this of

using

ungulate

shortcomings

paper,

the

Baer-

feces

from

vi rgin janus)

larvae

of the

Parelaphostrongylus

describe

fun-

a new

tenu-

method

feces

that

of

menin-

the

for

ex-

eliminates

convential

technique.

Reliathat

AND METHODS

pellets containing first-stage larvae of were collected during the spring and of 1996 from experimentally infected captive white-tailed deer and from wild whitetailed deer wintering near Grand Marais, Mmnesota (USA; 47#{176}4l ‘N, 90#{176}35’W). Pellets were fresh or stored at 1 C 2 1110 before use. All pellets IISC(l for a particular test were collected and stored in the same manner. The location of first-stage larvae iIS deer pellets was investigated by submerging 10 replicate pellets, impaled individually on a pin, for :3, 3, 3, 10, 10 and 31 ruin (total I hr) in a series of six standard Petri dishes filled with water at rooni temperature. A grid (0.8 X 0.8 cm) was

to the

of infection

accuracy

we

this

Baermann

(Odocoileus

MATERIALS

an(l

commonly especially

passed

first-stage

sam-

that

through

of animals

by the

or

hr.

intensity of

pass

containing and

the

technique

nematode,

in fecal

reliability

deer

amining

Larvae

into

paratus in detecting infections loxv numbers of larvae are (Mason, 1989; Welch et al., ble

tissue

material,

(proportion and

of

the

drained

proportional

assumption

extract

been

white-tailed

is,

fe-

to

funnel

he

are

using

never

evaluate

geal

al.,

placing

sink

technique quantitative

larvae)

et

funnel.

6 to 24

Hal-

Gajadhar

a water-filled

which

and

1983;

fecal

prevalence

ed

1983;

we

et

when

has

can

present the

technique

mann

Samuel

Hobbs,

the

after

The generate

and

achieved

larvae -

Todd

and from

counted

nem

numbers

Apparently,

nd

of

(Pillmore,

Senger,

1982;

is

and

larvae

ungulates

Gray

it

that

consistently

total

pies.

hut

trichostrongylid of

to the

hookworm

(Baermann,

protostrongylid in

was

extract

soil

particularly

atodes

technique

to

larvae

funnels,

the 511

512

JOURNAL

etched

OF WILDLIFE

on the

dissecting

DISEASES,

VOL.

33, NO. 3, JULY

1997

bottom of the Petn dish using a The number of larvae that came

pin.

off the pellet in each dish was counted. Thereafter, the intact pellet was broken up finely and shaken in 100 ml of water, and a 25 ml subsampie quickly pippetted into a gndded Petn dish and examined for any remaining larvae using a stereoscopic microscope at a magnification of 16 to 25X.

The

rate

at which

larvae

sink

in water

was

first estimated ing a measured tically in front

by directly observing larvae falldistance in a test tube held verof an horizontally positioned stereomicroscope head. Because larvae fell at varying rates, the following procedure was performed. Parelaphostrongylus tennis larvae were gently inoculated into the top of each of eight glass graduated cylinders (18.5 cm high) filled with 100 ml of water. At 15 mm intervals (for a total of 120 mm) four 25 ml aliquots were pippetted

sequentially,

from

top

to

bottom,

from one of the cylinders and the number larvae in each aliquot was counted. An experiment was designed to compare efficiency of two techniques for detecting tostrongylid larvae in feces. These were

Baermann or vinyl larvae, beakers

funnel screening

tissue

berly-Clarke, irregular

The

method submerged

paper

used

Mississatiga, pore sizes

with

using over

was

window screening ing of 1.0 x 1.4 mm. Glass Baermann test had a top

Kimwipes

had

(KimCanada)

a mesh

mm. open-

used

for the

funnels

0.6

X

diameter

of 15 cm,

at 30#{176} to the vertical, and ml of tap water at room temperature. The funnel stern (10 cm long, inside diameter 11 mm) was fitted with a 4 cm length of neoprene tubing stoppered with a Mohr pinchcock clamp (Fig. 1). The new beaker technique involved placing fecal pellets into an “envelope” formed by folding a piece of vinyl window screening (12 X 12 cm) and stapling the open edges to contain the pellets (Fig. 2a). The envelope was then submerged in a 250 ml beaker filled with tap water for 24 hi (Fig. 2b). A fecal sample of 21 to 30 pellets was divided equally into thirds with 7 to 10 (1 .9 to 2.75 grams dry) used in each method. After 24 hr,

were

the

sides

vertical-sided screening.

Ontario, to 0.4

up

vinyl

straight

the prothe

filled

inner

angled

with

glass

400

surface

of the

beakers was examined adhering to the glass surface tally positioned stereomicroscope and

30 ml of solution mann and

the

FIGURE

1.

collecting

The

Baermann

nematode

larvae

funnel from

technique

for

feces.

technique, using tissue paper to support feces and filter

and a new with feces

The

of

were

drained

Baermann directly

using

funnels for

head.

from

larvae

a horizoneach

Then,

Baer-

funnel and larvae counted. The pellets filters were removed from each funnel and remaining solution swirled to free any lax-

vae

then settle using larvae

stuck

to the

glass.

The

drained into a 400 for 1 hr, decanted gentle (Fig.

od, the screen moved and the

contents

were

allowed

to

to a volume of 50 ml suction, and examined for

vacuum

2c).

entire

ml beaker,

Similarly, envelope solution

for the beaker methand pellets were reallowed

to sit

for

1 hr

before decanting to a volume of 50 ml and examining for larvae. Upon removal of the fecal solution, the filters used in both the Baermann and beaker methods were inverted and placed in an 18 cm diameter petri dish filled with water for 24 hr to recover any larvae stuck to the filter material. To estimate the number of laxvae remaining on or in feces, pellets were macerated in a 500 ml beaker filled with water and three 25 ml aliquots of solution were removed and examined. Each technique was replicated 15 times. All glassware was cleaned between trials following Lankester (1995).

the

method

of

Whitlaw

and

Data were analyzed using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, Illinois, USA). Larval counts were either log or square root transformed to achieve normal distribution. Counts from each technique were compared using a one-way analysis of

variance.

The

relationship

between

collected in 24 hr and the total fecal sample, for each technique, ing

fe((S.

lodged was by

present utilizing

(1970)

of was

83%

using

larvae as 87%

7 cm

8

ex-

in samples. the Baer-

nematode

recovering as high

using 30 cm funnels. Although no explanation for this difference was offered by Todd et al. (1970), our results suggest that 27%

almost

its

ef-

collected comin a sample). third-stage Haefrom herbage but varied with

funnels,

weight Efficiency diameter; but

one reason for the reduced recovery rates probably was a greater proportion of larvae being

retained

surface

area

and size

larvae,

determined

(number of larvae to that actually present COfltOIIUS

the

consistently

to collect al.

slopthese standard

the new methover screening in and

of those studies et

on the 67%;

the type of filter, temperature, herbage samples and soil type. was inversely related to funnel it

(P

different

2

4391

hr.

yielded

larvae

method Todd

ficiency pared

significantly

9

error.

cal pellets that became ing sides of glass funnels could not be collected

only

not

0

2

filter.

it,

r(IrhtiIliI)g

are

84

190

NIlrhIce.

to glass

t ra1)1x(l

Stainlard

letter

off in 24

a(lhenng

Itr’u

sanie

64

311

of

only

of

on

the

the

larger

Gray (1982) also inversely affected

increased

sloping

funnels.

Samuel

reported that the number

funnel of Par-

elaphostrongylus odocoilei larvae collected using the Baermann technique, as did Beane and Hobbs (1983) for Protostrongylus spp. The latter studies did not determine efficiency, but it was that obtained by Todd to 3 times first-stage As

of H.

larvae

fective

well

P. tenuis

contortus

larger and protostrongylid as larvae,

being

the

probably less than et al. (1970) for inmore

which

are

mobile larvae.

inefficient

in

Baermann

funnel

than

extracting

tech-

2

FORRESTER

AND

5-

LANKESTER-EXTRACTION

A

when

funnels

4-

ml

3

from between vae and the ‘C.

2

#{149} ro.29; _____________________

-

I

1

4

0 ‘a

5

6

7

8

to

6

5)

0 V

I’

-

4

0 5)

3

i. .

-

r20.l2;P0.21

6

8

7

8

6 -

4

-

.z,..so 4

and the

I

I

6

(LN) FIGuRE

3.

nematode

Liisear

number

of

larvae

Baermann mann

in

funnel funnel

with

of

after the

vinyl

the

number

sample.

paper.

A.

B. Using

screening.

C.

Using

of

the

lets

in

the

variability

Using

a

a Baera beaker

screening.

collected a variable larvae present and

reliable

estimate

of actual

vae.

A major

source

test

probably

resulted

pellets freeing center

floating in themselves of the

funnel

proportion produced

of the an un-

numbers

of lar-

of this

variation iii our the position of

from

the funnel. More larvae from pellets near the will

and lets

enter the funnel stem situated more towards

the

funnel.

fall

straight

down

compared to pelthe periphery of

of

from pellets at

be

detectability Mason,

a

these

the newly fecal pel-

greatly

reduces

due

Therefore, method

to

collected

since

of fecal material vary. The number

fold

-

weight of the

and

be the

expressed the wild of dried collected

weight

of the

of the subsample subsample)]; (2)

staple

15 pellets screening the

edges

a

content

of larvae [fresh

as

on

moisture

collected in of larvae/gm

number

pellets

pellets

in order to provide factor. Numbers of

should

basis

the

5 additional

to be dried conversion

weight

sample

of 10 to 15 fecal and

a subsample a dry-weight

the

densities 1991).

feces:

a sample

ces

al.,

et

we recommend the following collecting protostrongylid lar-

examined

larvae

also

it

improving

at low

Welch

ungulate

(1) Weigh

among

counts,

test,

of larvae

for

from

animals,

larval

1989;

and

diameter

of

recovery

sensitive

P.

cm

in

all

same animal, and bea group. Being more

estimating

a more

(see

to be

water

the within

sample of 10 to fiber glass window

15

only is sub-

submerged

between

funnel stem. This may explain why Samuel and Gray (1982) maximized the number of from

that

larval

seasons,

(dried weight

larvae

to leave

we believe that of submerging

Smaller funnels and those with less water will position more pellets closer to the center and over the opening of the

odocoilei

able

avoids

in

the

dry

pellets present

and provides a new tool to more assess variation in larval counts

efficient

vae

nique total

deer

surface

pellets

a beaker

would

total

once subfirst-stage

1968;

the

beaker

24 hr test

of variation.

samples tween

larvae

24 hr versus

fecal

tissue

with

with

vinyl

regression

collected

larvae

of

number

Total

of

are of

Keeping

between

8

7

surface

probably

some before evapora-

the

Anderson,

portion

technique accurately

r20.98;

5

the

laxrel-

Finally,

and over

are

and

100

result

content, periods

larvae

In conclusion, proposed method

7

funnel.

submerged

on

only

in total of pellets

the

in funnels

vertical-sided

C

5

of

515

will

may expose parts of the pellets. Most P. tennis

sources

I

center

totally

merged. 5

4

with

period merged

from

1

differences position

of water

study)

C

filled variation

on the moisture float for varying

(Lankester

2

LARVAE

pellet random

the

becoming

B

5

were

Additional

depending pellets will tion

5)

they

of water.

ative

P=0.04

I

-

a ‘0

OF PROTOSTRONGYLID

will fefrom

sample initial

±

Place

the

on a piece of (12 X 12 cm) of the

screen-

516

JOURNAL

OF WILDLIFE

DISEASES,

VOL.

33, NO. 3, JULY

ing to form an envelope in which the pellets are uniformly distributed side-by-side in a monolayer (wrapping the screen envelope in a single layer of tissue will reduce the amount of debris sedimenting from some pellets and produce a cleaner sample of larvae, but the proportion of total larvae, and

therefore

sensitivity,

(3) Submerge beaker filled

(to prevent may have

clamps

such

requiring

er

as clothes larger

beaker

can

be

remove containing

and

larvae Decant

and

genfle

pour

used);

28:

(4)

discard feces and

the

all but

vacuum

suction

remaining

50

for

ameter

Petri

dish

and

examine

at 16 to 25X the observer present), but used,

decant

all

and

10

(Samuel

etched

solution

but

at

and

to

100

centrifuge

1,100

Gray,

50 ml of solution in an etched 9 cm

Petn

glassware

dish;

(6)

Wash

soapy

water, then rinse (Whitlaw and Lankester

with

with 95% 1995).

for

operating

funds

LITERATURE C.

Auffindung

Larven voor

1917. von

in

Erdproben.

57:

E.

undig

R.

the

of

1961.

C.

(Nematoda,

Symposium Goat Council N.

evaluation

using

infective

for

larvae

J.

J.

1991.

Sheep

AND

F. L.

and

ANDERSEN.

Baermann

technique contortus.

Helminthological

HOLMES,

AND

in

Rocky

the

PYBUS,

W.

Society

J.

of

G.

in

elk.

big-

Journal

SAMUEL,

of fecal

C.

AND

examination

of meningeal

tennis)

STELFOX.

Mountain

Canadian M.

Reliability

infections 19:

from

Biennial

57-63.

C.

M.

larvae the

Wild

of western Canada. 51: 817-824.

detecting

Evaluation of lung-

of Haemonchv.s

the

eiaphostrongyius

Bulletin

Northwest

Aid

75-80.

1982. recovery of

of the

37:

A.,

Federal

Project W-95of Game and

pp.

LEVINE,

Lungworms

D.

animals,

Report, Department

B. GRAY. technic for

D.

of

WILKE.

of

and

232-243.

An

J.

nematodes

of diseases

Protostrongylidae)

S.,

sheep

of lung

Proceedings

1970.

S.,

review.

cervi-a

Colorado,

3:

Washington

Ca-

Dougherty.

46: 373-383.

J.

of the

Proceedings

1968.

of meningeal

tennis,

game

Collins,

ruminants.

L.

ANDERSON.

hosts

Study

M., AND Baermann

wild

ta-

Ra7lgfer

Investigations

In

worm

K.

Metastrongylo-

3-10.

Job Completion 1-b, Colorado

Fort W.

horn

worm Wildlife

(ParSociety

1-12.

H., AND M. W. LANKESTER. 1995. A practical method for cleaning Baermann glassware. Journal of Wildlife Diseases 31: 93-95.

WHITLAW,

Method

13 1-137.

YA-

3: 33-39. AND

sheep.

1973.

to MWL.

Geneesk

(;.

Methods

reindeer

Elaphostrongyius

of Zoology

einfache

D.

1983.

from

affecting

SAMUEL,

UHAzY,

ziir

Ankylostomum-(Nematoden)-

Nederlandsch-Indi#{235}

R.

Projects, R-4, Job

CITED

Eine

W.

(Nematoda,

16:

parasites

ZAR, BAERMANN,

in

of Elaphostrongyius

of Zoology

1989.

bighorn

for assistance in the Brown for statistical Science and Engiof Canada (NSERC)

awarded

A sursheep

Management

WI55LER.

faeces

Journal P.

WELCH, K.

1964.

AND

density

W,

Surveillance

ACKNOWLEDGEMENTS We thank B. Peterson collection of deer feces, advice, and the National neering Research Council

in M.

MASON,

hot

ethanol

K.

the

L. Rangifer

nadian

TODD,

centrate the larvae into so it can be examined

in-

bighorn

of Wildlife

Mitskevich

Fish,

con-

in

Gastropods as intermediate worm, Pnerunostrongylus

larvae

to

SENGER.

S. V. TESSARO,

,

AND

larvae

LANKESTER,

150 ml of r.p.m. for

1982)

M.

Journal

0.,

PILLMORE,

on experience of amount of debris larger than 250 ml

C.

infection

estimating

randus

on the for

A. A.

idea)

(aspirator), and into a 9 cm di-

a grid the

(depending and the if a beaker

solution mm

with

Baer-

1994.

rangferi

the screen enlet the solution

ml

The

Protostronglus

481-491.

for

50 ml of solution

1983.

HOBBS.

sheep: effect of laboratory of Wildlife Diseases 19: 7-9.

AND

The

HALVORSEN,

hr.

estimating

Diagnosis of Elaphostrongylus cervi infection in New Zealand red deer (Cervus ciaphus) quarantined in Canada, and experimental determination of a new extended prepatent period. Canadian Veterinary Journal 35: 433-437.

enby

24

T

for

lungworm

GAJADHAR,

fecal larg-

After

J.,

D. of

TES.

or

large and

N.

fections in bighorn procedures. Journal

Montana.

samples

pellets

of

producing of screening

discard

bottom

is

for

AND

technique

vey

for an additional 1 hr (the slowest sink at a rate of about 11 cm/hr); (5)

settle

by

and

numbers

mann

in a 250 ml at room tem-

pins

R. D.,

BEANE,

FORRESTER,

reduced);

it from sinking, the be held in place

to

of ungulates a larger piece

gently velope

are

the envelope with tap water

perature velope

species pellets,

also

1997

Tijdschr

ift

J.

H.

Prentice

1984.

Hall,

Biostatistical

Englewood

analysis,

Cliffs,

2nd

New

pp. Received

for publication

4 November

1996.

edition,

Jersey,

718