of Wildlife

Journal

INFLAMMATORY

CELL

SPHAERIDIOTREMA MALLARD K. Head 1

GLOBULUS

DUCKS

Mucha

Department

and

(ANAS

Jane

of Biological

East StroudsbUrg, 2Author to whom

STIMULATION

AND

27(3),1991,

Diseases, © Wildlife

WOUND

HEALING

EXPERIMENTALLY

428-434 1991

pp.

Association

Disease

IN

INFECTED

PLATYRHYNCHOS)

E. Huftman2

Sciences,

Pennsylvania correspondence

East

18301, and

StroUdsburg

USA reprint

University,

requests

should

be

sent

Thirty laboratory-reared mallard ducks (Anas platyrhynchos) were experimentally with Sphaerldiotrema globulus. Host cell-mediated immunity and wound healing in S. globulus infected ducks were evaluated by gross and histological examination. Establishment, location, and life span of S. globulus differed from previous reports of sphaeridiotremiasis in both naturally and experimentally infected waterfowl. No worms were recovered from the ceca, and worm migration occurred anterior to the ileo-cecal valve with greater dispersion (less worm crowding) at higher rates of infectivity. Parasite death and host lesion resolution were evident at days 8 to 10 postinfection (P1) in ducks fed a moderate dose (200 metacercariae, group A) with a 5% mean parasite recovery rate. Host death occurred at days S to 6 in ducks fed a high dose (550 metacercaniae, group B) with a 16% mean parasite recovery rate. Mast cells increased significantly (P < 0.005) in group A ducks from days 4 to 10 P1. Eosinophil proliferation was greater in group B than in group A on day 4 P1, but comparatively fewer eosinophils were identifiable in group B ducks on day 6 P1. Key words: Sphaerldiotrema globulus, trematode, mallard duck, Anas platyrhynchos, mast ABSTRACT:

infected

cell,

eosinophil,

wound

healing,

experimental

infections.

INTRODUCTION

Sphaeridiotrema

globulus

is a

genic digenetic trematode stomatidae) inhabiting the of

waterfowl.

This

and pect

Huff man, etiologic

several an,

1934;

Trainer

1972; and and

Jackson,

and

1977).

lethal

of

cysts

from

man

and

trointestinal ported; discharge,

pathologies anorexia, ballooning presenting

munology,

Eu1969;

rhagic

of by

of mast

or

as

entenitis

Wild March

a

1934; et

a!.,

to

(1)

sphaenidievaluating (2) inves-

cells

and

experimentally intestinal tissue,

potentiators

in host

MATERIALS

die

and host

of

eoinand eoim-

hemor-

immunopathology.

AND METHODS

mallard

ducks

through

April,

eggs 1989.

were

collected Eggs

from

were

incu-

and hatched at the New Jersey Fish, Game and Wildlife facility in Rockport (New Jersey 07840, USA). Ducklings were provided food (Country Egg Producer, Agway, Inc., Syracuse, New York, 13221, USA) and water ad libitum.

bated

(Huff-

Clinical

and

gas-

have

been

re-

hemorrhagic,

frequency

in S. globulus mallard duck

and

hosts

(Price,

a!., 1972; Huffman and Roscoe, 1989). of this study were

(3) evaluate the role of mast cells sinophils as effector cells in avian

Huff-

ingesting

desquamation, death

the pathogenesis in mallard ducks serial histopathology,

the

sinophils fected

metacercania!

bloody of the

and

tigate

1963;

waterfowl

snail 1989).

Cow-

and

after

intermediate

and

(Campbell

S. globulus

Roscoe,

North

Scott, 1988), Manfredini,

Susceptible

postinfection

dose

Roscoe

susof

in

epithelial

congestion

describe otremiasis gross and

at Lake (Roscoe

Fischer,

Australia

3 to 8 days

junum

Cornwe!l and

et al.,

1962),

enzootic

olor) (USA)

waterfowl

1983; Hoeve (Francalanci

Testi,

is

and has been the in a fatal entenitis

of

(Price, 1963;

Speckmann man, rope

1982), agent

species

America

helminth

mucosa

vascular

Speckmann et 1984; Huff man The objectives

(family Psilosmall intestine

swans (Cygnus New Jersey

among mute Musconetcong,

patho-

extensive

penicloacal ileum and

je-

Ducks

(i2-wk-old)

groups,

A,

200

necrotic

and

Metacercarial

428

B, 550

were and

C.

Groups

metacercarial cysts

suspended

divided

into

A and cysts,

three

B were

fed

respectively.

in water

were

ad-

AND HUFFMAN-SPHAERIDIOTREMIASIS

MUCHA

were obtained from naturally infected Goniobasis virginica snails from Lake Musconetcong (Netcong, New Jersey; USA 40#{176}45’N,74#{176}42’W) (Huffman and Roscoe, 1989). Group C were controls and were not infected. Twenty ducks comprised group A. Four birds were killed by cervical dislocation at 2 day intervals, from days 2 to 10 postinfection (P1). The worm location was recorded in cm anterior to the ileo-cecal valve and reported as a percentage of individual duck intestinal length. The number of parasites recovered were recorded for each duck. Tissue samples of each ulcerated region from 10 ducks were fixed immediately in 10% buffered neutral formalin for 24 hr, then dehydrated in graded alcohols, cleared in xylene, embedded in Paraplast, and sectioned at 6 tim. Ten ducks comprised group B. Two birds were killed by cervical dislocation and necropsied at each 2 day interval from day 2 to 6 P1, or necropsied immediately if the animal succumbed to the infection. Two ducks from group C were killed by cervical dislocation and uninfected tissue samples were processed as previously described. Infected tissue sections were stained in Giemsa and Pollack’s Tnchrome solution using Toren’s mast cell stain (Toren, 1963). Giemsa staining time was modified for avian tissue from 75 mm to 25 mm. Tnichrome staining time also was modified from 10 to 15 sec to 5 to 8 sec. Cells (mast cells, globule leukocytes, and eosinophils) were counted per 10 villus/crypt units (Miller and Jarrett, 1971), adjacent to parasites attached to intestinal tissue and at 20 VCU- and 40 VCU-increments from the parasites. A total of 50 VCU’s from five separate segments of intestine were counted per necropsied duck. Histological serial counts were taken from 10 group A ducks killed at 2 day intervals (days 2 to 10 P1), and 8 group B ducks (days 2 to 6), or upon immediate necropsy if a duck succumbed to the infection. Logarithmic (to base 10) transformation of cell counts (x + 1) was performed. This transformation tends to be variance stabilizing and to normalize such data (Handlinger and Rothwell, 1981). Cell counts were presented as geometric means (anti-log of mean log of data). Intensity variation was reported as standard error. RESULTS

Data cluded (four Worms

from from ducks) were

day 2 P1 ducks group A (four in the difficult

infectivity to observe

were ducks)

not or

inB

results. and had

not

yet

not

produce

IN MALLARD

aggregated

into

visible

DUCKS

clusters

lesions.

429

and

did

Parasites

ne-

covered after day 2 P1 were found in clusters usually adjacent to one another. Lesions containing one parasite were uncommon. Sixty-three percent of the ducks in group A were

infected

of

43

0 to

covery

rate

All group range of asite

(10

16)

was

5.0

3.7%

±

B ducks 67 to 121

recovery

a range

parasite (160

re-

of 3,200).

were infected, with a helminths. Mean par-

rate

was

16.0

of

A ducks

with

Mean

of 4,400). The prevalence in group

of

helminths.

6.4%

±

infection

from

(696

decreased

75%

(three

of four)

at day 4 P1, to 50% (two of four) at day 8 P1, to 25% (one of four) at day 10 P1. The mean each

parasite 2 day

decreased to 11.3 individuals spectively.

intensity necropsy

of four

ducks

days

4 to

on

from 10

P1,

from 22.5 ± 13.5 individuals, ± 5.5, 3.75 ± 3 and 2.8 ± 3.2 at days 4, 6, 8 and 10 P1, reLesion site resolution was evi-

dent

after day 8 and 10 P1. Parasites recovered from group A ducks were located 18.7 to 36.8% distance anterior

to the

covered 56.1% valve.

from

ileo-cecal

valve.

group

distance anterior In the high dose

migrated with less

anterior crowding area

Parasites

B were

located

to group

the ileo-ceca! (B), parasites

to the ileo-cecal per 2.5% distance

The

depicts locations parasites

comparative worm frequencies and in % distance of group A and B recovered.

signs and symptoms infected waterfowl. death,

a penicloacal

steady acteristic

gait,

ducks.

Four

shown

globulus ducks

in

valve in-

terval.

Clinically, S. infected mallard

chart

re14.1-

as found in naturally Six to 12 hr prior bloody

discharge,

B ducks

death.

The

minimum

to un-

were char(group B)

died

infection on day 5 to 6 P1. One on day 3 P1 with 121 parasites at necropsy. A single group B vived 6 days P1, although it was near

1

experimentally presented similar

and wing droop signs of high dose group

Figure

lethal

from

the

duck died recovered duck surataxic and number

430

JOURNAL

OF WiLDLIFE

DISEASES,

VOL. 27, NO. 3, JULY

1991

asites penetrated in vessel congestion Resolution

of

moderate to 10 P1. blasts,

the

dose Scar

musculanis, resulting and hemorrhage.

the

lesion

(group tissue

occurred

A) ducks composed

polymorphonuclear

bnin,

and

leucocytes,

collagen,

aged villi parasites

and from

formed

over

dead worms, surrounding

A mast

cell

fi-

the

dam-

walling tissues.

mal mucosal architecture in group A ducks by 8 to Group

in

by days 8 of fibro-

off the A nor-

was generated 10 days P1.

frequency

increased

from day 4 P1 through day 10 P1 (P < 0.05) using Duncan’s Multiple Range Test compared to (SAS Institute, Inc., 1987) group C control values for uninfected mallard #{149}Paale

1.

FIGURE

locations

Comparative

of parasites

S. globulus

to the

sy

in

Gross revealed

at

of

worm

lesions

ulceration.

The on

present amination

in the ulcer. showed

posed

of fibnin,

cosal cells.

epithelial

The vanced

lesion of

forming sion was

consisted

of

and out

a raised evident group

grouped ad-

lesion.

The

a focal

lesion by day

a

necrotic

lesion

ulceration.

vessel

focus

site. The early le4 Pt in both group present

congestion, infiltration Frequently,

by days and accomthe

pan-

analysis Inc.,

1) of

1.90

of variance ANOVA 1987) compared to

control ducks. was observed.

to 100

6 P1, using Inc., 1987). ence and

was

+

and ducks ±

VCU’s, with a total of VCU’s. Group A ducks mean range of 3.9 ± 0.84

a

(P