The role of immunity in inhibited development of Obeliscoides cuniculi (Graybill), a stomach
nematode of rabbits
by Joseph Carl Fox
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF
PHILOSOPHY in Veterinary Science
Montana State University
© Copyright by Joseph Carl Fox (1975)
Abstract:
A study was designed to examine the role of immunity in the inhibited development of Obeliscoides
cuniculi. Rabbits, infected per os with 1-3 doses of third stage larvae (L3), inhibited larvae in
subsequent challenge infections at the 4th stage of development.
Massive infections in rabbits given 200,000-834,700 L3 resulted in almost complete worm inhibition.
In rabbits infected with 100,000 L3, 50-75% of the worms were inhibited at the 4th stage. Rabbits,
pre-infected with 75,000 L3, treated 10 days later with levamasole HCl and subsequently challenged
with 5,000 L3 harbored infections comprised of 18.3% 4th stage larvae (L4). All 5th stage worms (L5)
recovered from these infections were retarded in growth, whereas L5 recovered from controls were
normal. Egg production by worms in actively immunized rabbits was complete suppressed, and in
passively immunized rabbits patency did not occur until 6 days after serum transfers were terminated.
When inhibited L4 were transferred into previously unexposed rabbits, many developed to adults
(28-87%). However, significant numbers failed to develop beyond the 4th stage. Also, the sex ratio of
adult worms which developed from inhibited larvae following transfer shifted toward larger numbers of
females, and in actively immunized recipients the differences were further accentuated. Egg
production, by adult worms which developed from both inhibited and normal L4 after transfer, was
suppressed in actively immunized recipients; fewer eggs were produced by worms of the inhibited
type.
Two immunosuppressant drugs, 9-fluoroprednisolone (FP; corticosteroid) and cyclophosphamide (CY;
alkylating agent), were given to rabbits infected with 100,000 L3 to determine if larval development
would resume. When rabbits were treated with FP on days 0-28 there was an increase in the numbers of
adult worms. However, development did not resume after treatments on days 0-6. Significant numbers
of worms developed to adults when FP treatments were given days 9-15 or 20-26; also normal egg
production occurred in these worms. Non-treated inhibition control animals (100,000 L3) passed only
low number of eggs ( < 100) even though 25-42% of their worms were L5. Treatment of rabbits with
CY inhibited further development of larvae as only 13% of the worms progressed to the 5th stage, and
none were found that contained eggs.
Treatment of rabbits with FP (total dosage 9 mg/kg) produced rapid and sustained reductions in total
lymphocyte counts. There was also an early weight gain (3-5 days) and subsequent weight loss (5-12
days) following treatment. Treatments with CY (total dosage 90 mg/kg) did not significantly reduce
lymphocyte counts. However, a slight lymphocytosis which occurred in the inhibition controls was not
observed in the CY-treated animals. Antibody titers to sheep erythrocytes (SRBC) were highest in
FP-treated rabbits and animals with low-level infections (4,000 L3); inhibition controls (100,000 L3)
and CY-treated animals had comparable antibody titers. Antibody titers to O. cuniculi antigen (OCA)
and sheep erythrocytes (SRBC) on day 27 p.i. showed similar relationships between treatment groups,
however, anti-OCA titers were considerably lower.
Gross pathological lesions after 27 days in rabbits with high-level O. cuniculi infections included the
presence of numerous petechial hemorrhages and folding and thickening of the gastric mucosa; only
the cardiac and fundic areas of the stomach were affected. Histopathological lesions included massive
epithelial hyperplasia, lymphocytic hyperplasia and nodule formation, lymphocyte and eosinophil
infiltration into the lamina propria and cryptitis in areas near embedded larvae. Other effects on rabbits
were weight loss and anorexia during the first 6-12 days p.i. Immunosuppressants did not appreciably
alter the formation of lesions.
The data indicate that host immunity plays an important role in inhibited development of O. cuniculi in
rabbits: (1) active immunization by repeated low-level (3,000 L3) and single high-level (100,000 L3)
infections resulted in inhibition of larvae at the 4th stage, (2)corticosteroid treatments increased the
number of worms that developed to the adult stage, (3) inhibition was larval dose dependent, and (4)
worms were damaged during inhibition in source rabbits such that some of the L4 were unable to
resume development when transferred to uninfected rabbits. Also many inhibited male L4 failed to
develop into adults, and this was further accentuated in actively immunized recipients. THE ROLE OF IMMUNITY IN INHIBITED DEVELOPMENT
OF OBELISCOIDES CUNICULI ( GRAYBIL L ), A STOMACH
NEMATODE OF RABBITS
by
JOSEPH CARL FOX
.
y
A thesis submitted in p a r tia l fu lfillm e n t
of the requirements fo r the degree
of
;
DOCTOR OF PHILOSOPHY .
in
V eterinary Science
Approved:
Chairman, Examining Commi/t^ee
/
HS)
x
Head, Major Department
Graduate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
June, 1975
ACKNOWLEDGMENTS
The author expresses appreciation to Dr. David E. Worley fo r
providing an opportunity to p a rtic ip a te in the doctoral program, at
Montana State U n iversity and fo r guidance and encouragement during the
course o f th is study.
Appreciation is also extended to Mr. R. H.
Jacobson, fo r many hours o f help and discussion in developing ideas
re la te d to th is research.
Special appreciation is extended to Dr.
L. L. Myers, fo r his encouragement, advice and friendship and to the
other members o f the graduate committee:
J. E. G a tlin , D .V .M ., Dr.
F. S. Newman, Dr. N. D. Reed and Dr. G. A. Taylor, fo r t h e ir valuable
time and council.
Thanks are also extended to J. In h e ld e r, D .V.M ., fo r help with
h is to lo g ic a l examinations; Mr. Donald F r it t s , fo r photomicrographs;
Mrs. Jonne Shearman, and Mrs. Mary R e illy , fo r technical assistance;
Mrs. Katherine S t i t t , fo r reading the manuscript; and Mrs. Carolyn
Ann Lyon, Mrs. Betty Freeland, and Mrs. Betty Larson, fo r typing the
manuscript.
Love and appreciation go to the author's lo vely w ife Lael and
th e ir children S c o tt, J e ffre y and Shalene5 fo r constant encouragement,
boundless love and un selfish s a c rific e during many years o f education.
This research was supported in p art by Regional Research Funds,
p ro jec t W -I02.
iv
TABLE OF CONTENTS
Page
VITA .................................................. ; ; ..............................................................
ACKNOWLEDGMENTS
......................................................
ii
iii
LIST OF TABLES.....................................
LIST OF FIGURES
• I
...................................... . . .............................................. ,
ABSTRACT...................................................................................................
A.
B.
C.
D.
E.
F.
I
,
Environmental e f f e c t s ..................................... . . . . ,
D i e t ...........................................................................' . . . . L .
Size o f in fe c tiv e la rv a l d o s e ......................... . , ■
Presence o f adult worms . . ......................................... ,
Host age r e s i s t a n c e ..................... - ..................... • • ;
Acquired immunity . . . . . . . .
......................... ,
In h ib itio n o f Obeliscoides c u n i c u l i .......................................... .
MATERIALS AND METHODS..........................................
,
R a b b its .................
t
Helminth cultures .........................................
. .
Inoculations with in fe c tiv e larvae ........................ . . . . I
Fecal e x a m in a tio n s ........................
Necropsies .............................................. .... . ..................................;
Helminth t r a n s f e r s ...................................................
{
Helminth measurements . .....................................
;
A n th e lm in tic s .................................................................................... ,
Immunosuppressant d r u g s ............................. • ........................... ..
'
Histopathology . .......................................................
H e m a to lo g y ...................................... .................................................... ,
Antigens i ............................. . . . . . . . . . . . . . . . .
A n t i s e r a .............................................. ....
^
;
Serological te s ts
......................... .... .
..................... ■
A.
B.
C.
v ii
ix
INTRODUCTION ........................................................................................................
Factors contrib uting to in h ib ite d
development o f nematodes ..............................................................
vi
Immunodiffusion t e s t ..........................................................
In d ire c t hemagglutination te s t . . . . . . . . .
I
Hemolysin t e s t ................................................................... I
3
. 3
5
6
7
S
10
14
17
17
T7
18
18
19
20
21
21
22
22
22
22
23
24
24
25
25
V
Page
RESULTS.............................
27
Repeated in fe c tio n s with Obeliscoides cuniculi . .................
H igh-level in fe c tio n s with Obeliscoides cuniculi ................
Elim ination o f immature Obeliscpides cuniculi with
a n th e lm in tic s ..........................
Transfer o f in h ib ite d Obeliscoides cuniculi to
normal rabbits
. . . . . .
......................................................
Experiment A
Experiment B
27
30
34
36
...............................................................................
....................
36
39
E ffects o f p re -in fe c tio n s and passive immunizations in
rabbits on development o f Obeliscoides cunicu li . . . .
Development o f Obeliscoides cunicu li in rabbits treated
with the c o rtic o s te ro id 9 - f luoroprednisol one . . . . .
Experiment A
Experiment B
43
47
..................................
47
51
Development o f Obeliscoides cunicu li in rabbits
tre a te d with cyclophosphamide or 9 - f Iuoroprednisol one .
DISCUSSION
...................................................................................
LITERATURE CITED
..............................................
. . . . . . . . . . .
A P P E N D IX .................; ................................................................................... ...
58
.
75
88
100
vi
LIST OF TABLES
Table
1.
2.
3.
4.
5.
6.
7.
8.
9.
Page
Development o f Obeliscoides cunicu li in previously
in fected (p re -in fe c te d ) rabbits follow ing a challenge
with 3,000 in fe c tiv e l a r v a e .................................................................
28
Development o f Obeliscoides cuniculi in rabbits with
hig h -level in f e c t io n s ................................. ......................................... .
32
E fficacy o f levamisole HCl (LHC) against immature
Obeliscoides cunicu li in rabbits ..................... . .........................
35
Development o f in h ib ite d and normal 4th stage larvae (L .)
o f Obeliscoides cunicu li a f t e r tra n s fe r to normal rabbits . . .
38
Development o f in h ib ite d (IL -4 ) and normal (NL-4) 4th
stage larvae o f Obeliscoides cuniculi a fte r tra n s fe r to
previously in fected (p re -in fe c te d ) or unexposed (normal
rabbits . . . . ............................................................................................
41
Development o f Obeliscoides cunicu li in p re -in fe c te d or
passively immunized rabbits ......................... .........................................
45
Development o f Obeliscoides c u n ic u li.in rabbits w ith
hig h -level in fec tio n s and treated with the co rtic o s te ro id
9 - f l uoropredni sol one (FR) . . . ..................... ... . .........................
49
Development of Obeliscoides cuniculi in rabbits with
h ig h -le v e l in fec tio n s and trea te d with the c o rtic o s te ro id
9 - f l uoroprednisol one (FR) .......................................................................
54
Development o f Obeliscoides cunicu li in rabbits tre a te d
with 9-fluoroprednisol one (FR) or cyclophosphamide (CY) . .
64
.
vi i
LIST OF FIGURES
Figure
I .
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
'
'
1
Page
Mean weight changes associated w ith repeated lowlevel (3,000 L3 ) in fec tio n s with Obeliscoides
cunic u Ii in rabbits . ...........................................................................
29
Mean weight changes associated with high-level
Obeliscoides cunicu li in fec tio n s in rabbits .............................
33
Mean egg production by Obeliscoides cuniculi
follow ing tra n s fe r o f in h ib ite d and normal 4th
stage larvae ( L ,) to previously in fected or normal
rabbits . . . ............................................................................................
42
Mean egg production by Obeliscoides cuniculi in
previously in fected or passively immunized rabbits
46
. . . .
Mean weight changes in rabbits given high-level
in fec tio n s w ith Obeliscoides cuniculi
and trea te d with 9 - f I uoropredni sol one ( F R ) .....................
50
Mean egg production by Obeliscoides cuniculi in
rabbits treated w ith 9-fluoroprednisol one (FR) . . . . . .
55
Mean white blood c e ll (WBC) counts in rabbits given
h ig h -le v e l Obeliscoides cuniculi in fec tio n s
and trea te d with 9-fluoroprednisol one ( F R ) .............................
56
Mean weight changes in rabbits given high-level
Obeliscoides cuniculi in fec tio n s and treated with
9 - f l uoropredni sol one ( F R ) ..................................... ............................
57
Mean egg production by Obeliscoides cuniculi in rabbits
trea te d with 9 - f l uoropredni sol onie (FR) or cyclophosphamide (CY) ...........................................................................................
65
Mean peripheral blood lymphocyte
counts in rabbits in fected w ith Obeliscoides cuniculi
and trea te d with 9-fluoroprednisol one (FR) or cyclophosphamide (CY) ..........................................................................
66
Mean white blood
blood o f rabbits
and trea te d w ith
cyclophosphamide
67
c e ll (WBC) counts in peripheral
in fected with Obeliscoides cuniculi
9 - f l uoroprednisol one (FR) or
(CY) ...........................................................................
v iii
Figure
12.
Mean weight changes in rabbits in fected with Obeliscoides
cuniculi and trea te d with 9-fluoroprednisolone (FP) or
cyclophosphamide ( C Y ) ...........................................................................
Page
68
13.
Mean antibody t it e r s to sheep red blood c e lls (SRBC) in
rabbits in fected w ith Obeliscoides cuniculi and treated
with 9 - f l uoroprednosolone (FR) or cyclophosphamide (CY) . . • 69
14.
A comparison o f antibody t it e r s against Obeliscoides
cuniculi antigen (OCA) and sheep red blood c e lls (SRBC)
in immunosuppressed rabbits ..............................................................
70
15.
G astric mucosa from an uninfected (normal) ra b b it . . . . .
71
16.
E p ith e lia l and lymphocytic hyperplasia in the g a s tric
musoca of a ra b b it a f t e r a 27-day in fe c tio n with 100,000
Obeliscoides cuniculi .........................
71
Area o f lymphocytic hyperplasia in the g a s tric mucosa
where lymphocytes have in f ilt r a t e d the muscuTaris mucosa
and in to the submucosa o f a ra b b it infected with 100,000
Obeli scoides cunicu li ......................... .................... . . . . . . .
72
Lymphocytic hyperplasia, c r y p t it is , embedded nematode
la rv a e , and lymphocytic in f i l t r a t i o n in the g a s tric mucosa
o f a ra b b it in fected with 100,000 Obeliscoides cuniculi . .
72
Embedded nematode la rv a e , c r y p tit i s , and eosinophils in
the g a s tric mucosa o f a ra b b it in fected with
100,000 Obeliscoides cuniculi . . . .
.........................................
73
E p ith e lia l hyperplasia o f the g a s tric mucosa in a ra b b it
during an in fe c tio n with 100,000 Obeliscdides cunicu li
. .
73
Normal adults and in h ib ite d 4th stage larvae o f Obeliscoides
cuniculi from rabbits 27 days a f t e r inoculation w ith 4,000
Lg and 100,000 Lg, resp ectively . . . . . . . . . .
. . . .
74
17.
18.
19.
20.
21.
Ix
ABSTRACT
A study was designed to examine the ro le o f immunity in the
in h ib ite d development o f Obeliscoides c u n ic u li. Rabbity, infected
per os^ with 1-3 doses of th ird stage larvae (Lcj) 9 in h ib ite d larvae in
subsequent challenge in fec tio n s a t the 4th stage o f development.
Massive in fe c tio n s in rabbits given 200,000-834,700 Lcj resu lted in
almost complete worm in h ib itio n .
In rabbits infected^w ith 100,000 Lcj, 50
75% o f the worms were in h ib ite d a t the 4th stage. Rabbits, p re -in fe c te d
w ith 75,000 Lg, treated 10 days la t e r with levamasole HCl and sub­
sequently challenged with 5,000 Lcj harbored in fectio ns comprised of
18.3% 4th stage larvae ( L * ) . A l r 5th stage worms (L5 ) recovered from
these in fe c tio n s were retarded in growth, whereas U re c o v e re d from
controls were normal. Egg production by worms in a c tiv e ly immunized
rabbits was complete suppressed, and in passively immunized rabbits
patency did not occur u n til 6 days a f t e r serum tran sfers were term inated.
When in h ib ite d L4 were tran s fe rred in to previously unexposed
ra b b its , many developed to adults. (28-87%). However, s ig n ific a n t
numbers fa ile d to develop beyond the 4th stage. Also, the sex ra tio
o f adult worms which developed from in h ib ite d larvae follow ing tra n s fe r
s h ifte d toward la rg e r numbers o f fem ales, and in a c tiv e ly immunized
re cip ie n ts the differences were fu rth e r accentuated. Egg production,
by adult worms which developed from both in h ib ite d and normal L4 a fte r
tra n s fe r, was suppressed in a c tiv e ly immunized re c ip ie n ts ; fewer eggs
were produced by worms o f the in h ib ite d type.
Two immunosuppressant drugs, 9 - f I uoroprednisol one (FR; c o rtic o ­
s te ro id ) and cyclophosphamide (CY-, a lk y la tin g a g e n t), were given to
rabbits in fected with 100,000 L3 to determine i f la rv a l development
would resume. When rabbits were treated with FR on days 0-28 there
was an increase in the numbers o f adult worms. However, development
did not resume a f t e r treatments on days 0 -6 . S ig n ific a n t numbers of
worms developed to adults when FR treatments were given days 9-15 or
20-26; also normal egg production occurred in these worms. Nontrea te d in h ib itio n control animals (100,000 LcJ passed only low number
o f eggs ( < 100) even though 25-42% of t h e ir worms were Lg . Treatment
o f rabbits w ith CY in h ib ite d fu rth e r development of larvae as only 13%
of the worms progressed to the 5th stage, and none were found th at
contained eggs.
Treatment o f rabbits with FR (to ta l dosage 9 mg/kg) produced
rapid and sustained reductions in to ta l lymphocyte counts. There
was also an e a rly weight gain (3-5 days) and subsequent weight loss
(5-12 days) follow ing treatm ent. Treatments with CY (to ta l dosage
90 mg/kg) did not s ig n ific a n tly reduce lymphocyte counts. However,
a s lig h t lymphocytosis which occurred in the in h ib itio n controls was
not observed in the CY-treated animals. Antibody t it e r s to sheep
erythrocytes (SRBC) were highest in FP-treated rabbits and animals
with lo w -level in fe c tio n s (4,000 L3) ; in h ib itio n controls (100,000 L3)
X
and CY-trea te d animals had comparable antibody t it e r s . Antibody
t it e r s to CU curiiculi antigen (OCA) and sheep erythrocytes (SRBC)
on day 27 p *.i. showed s im ila r relatio n sh ip s between treatm ent groups,
however, a n t i-OCA t it e r s were considerably lower.
Gross pathological lesions a f t e r 27 days in rabbits with highlevel CL c iin icu li in fe c tio n s included the presence o f numerous petechial
hemorrhages and folding and thickening o f the g a s tric mucosa; only the
cardiac and fu n d ic areas o f the stomach were a ffe c te d . Histbpathological
lesions included massive e p ith e lia l hyperplasia, lymphocytic hyperplasia
and nodule form ation, lymphocyte and eosinophil i n f ilt r a t io n into the
lamina propria and c r y p titis in areas near embedded la rv a e . Other
e ffe c ts on rabbits were weight loss and anorexia during the f i r s t 6-12
days p . i .
Immunosuppressants did not appreciably a lt e r the formation
o f lesions.
The data in d icate th a t host immunity plays an important ro le in
in h ib ite d development o f 0. cuniculi in ra b b its : ( I ) ac tiv e immunization
by repeated lo w -level (3,000 U ) and single high-level (100,000 U )
in fec tio n s resulted in in h ib itio n o f larvae a t the 4th stage, ^ ^ c o r ­
tic o s te ro id treatments increased the number o f worms th a t developed to
the adult stage, (3) in h ib itio n was la r v a l dose dependent, and (4) worms
were damaged during in h ib itio n in source rabbits such th a t some of the
I , were unable to resume development when tran sferred to uninfected
ra b b its . Also many in h ib ite d male L, fa ile d to develop in to a d u lts ,
and th is ,was fu rth e r accentuated in ^ a c tiv e ly immunized re c ip ie n ts .
INTRODUCTION
Some p a ra s ite s , a f t e r being ingested by a su ita b le host, e ith e r
f a i l to mature or remain fo r an extended period, o f time in an e a rly
stage o f development before reaching m a tu rity .
The reasons these
parasites do not develop normally are not f u l ly understood.
In most
cases the mechanisms are complex and involve e ith e r e x trin s ic or
in tr in s ic fa c to rs .
Dormant la rv a l parasites are commonly encountered w ith in the realm
o f parasitism .
Michel (1968) reported th a t impaired la rv a l development
has been reported fo r over 30 helminth sepecies.
The common gastro­
in te s tin a l nematodes of. domestic c a ttle and sheep (T h ' chostrongyloidea
and Strbngyloidea) co n sisten tly e x h ib it a high degree o f arrested de­
velopment, e s p e c ia lly in older animals or during the cold seasons of
the year.
During the e a rly p a ra s itic phase o f th e ir l i f e c y cle, many
o f these nematodes enter a h is to tro p ic stage of development (Madsen,
.1962).
At th is stage the larvae m igrate in to the mucosa and undergo 2
molts (ecdyses).
Larval development is usually impaired d u rin g .th is
process.
Dormant larvae are important in the epizootiology o f several
nematode species.
Spedding and Brown (1956) reported th a t a spring
ris e in worm egg output commonly occurred in .confined sheep.
Increased
egg output was a ttrib u te d to.worms which were dormant throughout the
w in te r and resumed development in the spring.
F ie ld , Brambell and
Campbell (I9 6 0 ) reported increased egg production by Haemonchus contortus
2
in domestic sheep.
Connan (1968a) and Gibbs (1968) stressed the
importance o f in h ib ite d larvae in the spring ris e phenomenon, and Crofton
(1954) and Brunsdon (1966) pointed out th a t the spring increase in worm
egg output was a primary source o f in fe c tio n fo r young animals.
Dormant larvae are g enerally thought to resume development a fte r
a prolonged quiescence.
Mechanisms which may be responsbile fo r re ­
newed development o f dormant larvae are: waning o f host immunity
(Soulsby, 1957), changes in host d ie t ( Connah, 1969) or seasonal
hormonal changes in the host ( Dunsmore, 1965; Connan9 1968b; B liz and
Gibbs, 1972b).
McKenna (1974a) reported th a t
contortus was expelled
from sheep w ith in 10 weeks a f t e r in h ib itio n occurred, and thus could
not contribute to spring r is e .
B lit z and Gibbs (1971a) produced patent
in fec tio n s with 1H. contortus by tra n s fe r o f in h ib ite d L4 into the
abomasa o f p a r a s ite -fr e e , pregnant ewes; however, development did not
occur u n til the time o f p a rtu rito n .
Also, Roberts and Keith (1963)
transplanted in h ib ite d Oesophagostomum radiatum in to uninfected calves,
and patent in fec tio n s developed.
In c o n tra s t, H erlich (1974) tran s­
planted 4th stage larvae o f O stertagia o s tertag i th a t were in h ib ite d
in sheep in to p a ra s ite -fre e calves and found th a t patent in fectio n s did
not develop.
However, when.larvae were grown to the 4th stage (not
in h ib ite d ) in calves and subsequently tran sferred to sheep, patent in ­
fections developed w ith in 6 days.
These data suggest th a t the 3rd and
4th ecdyses may be important in the in h ib ito ry process and th a t th is
3
process may be ir r e v e rs ib le under c e rta in circumstances.
In h ib ite d helminths are the e tio lo g ic a l agents in c e rta in disease
syndromes.
These were discussed in d e ta il by M artin , Thomas and Urquhart,
(1 9 57 ), Anderson, Armour, J a r r e t t , Jennings, R itchie and Urquhart (1965;
and Armour (1970;.
Factors Contributing to In h ib ite d Development
of Nematodes
Michel (1968) reviewed some o f the mechanisms o f retarded la rv al
development.
A.
B.
C.
D.
. E.
F.
These could be c la s s ifie d as:
Environmental e ffe c ts
D iet
Size o f in fe c tiv e la rv a l dose
Presence o f adult worms
Host age resistance
Acquired immunity
Other reviews (Taylor and M ichel, 1953; Madsen, 1962; M icpel, 1969;
Armour, 1970; J a r r e tt and Urquhart, 1971, and O g ilvie and Jones, 1973)
contain s im ila r evaluations o f the processes involved in in h ib ite d la rv a l
development.
A.
Environmental e ffe c ts
Large numbers o f dormant parasites are often observed in c a ttle
and sheep in the autumn or e a rly w in te r.
Anderson e t a l . (1965)
showed th a t calves grazing on contaminated pastures during la te autumn
in fec tio n s which were comprised p r im arily o f 4th stage la rv a e , whereas few
were found in animals.
4
which grazed the same pastures fo r s im ila r periods in the spring or
summer.
They concluded th a t la rv a l dormancy was p re c ip ita te d by autumn
environmental facto rs which induced physiological changes in e ith e r the
•host or the in fe c tiv e la rv ae .
S im ila r observations have been reported
fo r 0. o s te rta g i in calves (Armour, Jennings and Urquhart, 1969a; Reid
and Alrmour5 1972), Cooperia oncophora in calves (H e rlic h , 1965; M ichel,
Lancaster and Hong, 1970), Haemonchus contortus in sheep (Connan, 1971;
Brunsdon, 1973), _C. c u rtic e i in sheep ( Sommervil i e , 1960), Chabertia
ovina in sheep (Connan, 1974), Q stertagia spp. in sheep (M u lle r, 1968;
Reid and Armour, 1972, and Haemonchus contortus and (L circumcincta
in sheep (McKenna, 1973).
These reports in d icate th a t seasonal in ­
h ib itio n o f larvae is common with many nematode parasites of. c a ttle
and sheep.
Physiological changes in in fe c tiv e larvae as a re s u lt o f environ­
mental or season! e ffe c ts are not understood.
B litz and Gibbs (1972b)
observed th a t Hv contortus became dormant in sheep when the in fe c tiv e
larvae were cultured to the 3rd stage in the laboratory and subsequently
exposed to pasture conditions fo r several weeks during la t e autumn.
Certain s tra in s of Ov o s te rta g i were shown to be more prone to dormancy
than others.
Armour, Jenning and Urquhart (1967; 1969) demonstrated th a t
laboratory cultures of Ov o s tertag i which were seeded onto pastures
during the f a l l , did not become dormant in calves, whereas larvae
acquired from the n a tu ra lly contaminated pastures d id .
Physiological
5
differences w ith in the laboratory and f ie ld strain s o f larvae were not
defined.
Storage o f in fe c tiv e larvae a t 4C fo r 3-8 weeks also greatly,
enhanced the tendency fo r 0. o s te rta g ia to become quiescent (Bruce and
Armour, 1974; Armour and Bruce, 1974; M ichel, Lancaster and Hong, 1975).
A s im ila r e ffe c t was observed w ith
contortus a f t e r the L3 were
stored at SC fo r 60 days (McKenna, 1974a,b ).
Rod-1ik e , c r y s ta llin e
structures have been observed w ith in the in te s tin a l c e lls o f in h ib ite d
H.. cbntorus ( B lit z and Gibbs, 1971b).
The sign ificance o f these
crys ta ls was not known, but they were not found in worms which had
resumed development.
The presence o f such c rystals in in h ib ite d H_.
cortortus was v e r ifie d by McKenna (1974a,b ) .
B.
D iet
An anim al's n u tritio n a l status is important in it s a b il it y to r e s is t
parasitism (Ackert and Beach, 1933; Ross and Gordon, 1933; Hunter, 1953;
Kates and Wilson, 1955; Geiman, 1958; Brunddon, 1962a; Gibson, 1963;
Bawden, 1969).
Several studies have shown th a t resistance to helminth
in fec tio n s is closely linked to an anim al's protein intake (B aird ,. Vegors
S ell and Stew art, 1956; W ells, 1962, 1963).
Sheep on high protein
rations exhibited g re a ter resistance to Oesophagostomum radiatum and
produced less in te s tin a l mucin Dobson and Bawden (1 9 74 ), Frick and
Ackert (1948) and Dobson (1967) demonstrated th a t mucin was important
in resistance to in te s tin a l helminths.
Vegors e t a l . (1956) fu rth e r
showed th a t animals on low -protein d ie ts harbored la rg e r numbers o f both
6
immature and mature worms than those on a high-protein ra tio n .
A
s im ila r observation in sheep infected w ith 0. columbianum was described
by Dobson and Bawden (1974).
These observations indicated th a t the
host's d ie t probably does not have a d ire c t e ffe c t on worm development.
This conclusion was also expressed by Connan (1969) concerning the
e ffe c ts o f d ie t on the re ta rd a tio n o f O stertagia spp. in lambs.
C.
Size o f in fe c tiv e la rv a l dose
Retarded la rv a l development associated with massive worm in fectio n s
has been a ttrib u te d to competition between individual parasites (Taylor
and M ichel, 1953), however, immunological responses may also be involved.
Michel (1952a,b) observed th a t the course o f an in fe c tio n with
Trichostronqylus reto rtaeform is in rabbits varied according to the size
o f the in fe c tiv e la rv a l dose.
In some cases, massive in fec tio n s re ­
sulted in worm expulsion, while a t other tim es, th e .la rv a e were in h ib ite d
a t the la te Lg stage.
The in h ib ite d t .
retortaeform is were hot rejected
immediately but were slowly elim inated as they matured.
Martin e t a l .
(1957) reported th a t development of Graphidium strigosum in rabbits was
impaired in heavy in fe c tio n s .
In fec tio n s in it ia t e d with 5,000 Lg
developed norm ally, whereas most of the worms were retarded in in fectio ns
with Cooperia pectinata and £ . oncophora in calves indicated th a t these
species were in h ib ite d in g reater -htmbers as the la rv a l dose increased.
Moreover, Mapes, Coop and Angus (1973) demonstrated th a t the number o f
in h ib ite d N. battus in lambs was d ir e c tly proportional to the number of
7
in fe c tiv e larvae given.
The number o f Ostertagia spp. which become
retarded in sheep is also proportional to la rv a l dose.
Dunsmore (1960)
observed th a t in in fe c tio n s in it ia t e d w ith I ,000 L35 1% o f the worms
remained a t the 4th la rv a l stage as compared to almost complete in h ib i­
tio n o f larvae in in fe c tio n s with 100,000 L3 .
Also, the l a t t e r in fe c ­
tions were characterized by long prepatent periods and small adult worms.
D.
Presence of adult worms
Larval development may be c o n tro lled by the presence o f adult worm
populations.
Gibson (1953) found th a t worm eggs ( Trichonema spp.)
appeared in the feces o f horses follow ing treatment w ith phenothiazine,
even though they were not re-exposed to in fe c tiv e la rv ae .
Dormant
larvae were thought.to resume development to replace the adult worms
which were removed with anthelm in tic.
Dunsmore (1963) observed a s im ila r
s itu a tio n in sheep th a t were in fected w ith Ostertagia spp.
Following
treatm ent with phenothiazine, the numbers o f dormant larvae decreased
by a proportion approximately equal to the numbers o f new adults.
Michel (1969, 1970, 1971) reviewed and stressed the importance of
adult worms in c o n tro llin g the development o f O^ O stertagia in calves.
However, such a control mechanism was discounted by Anderson e t a l .
(1965) and la t e r by M ichel, Lancaster and Hong (1973).
E.
Host age resistance
I t has been recognized fo r many years th a t mature animals are
usually more re s is ta n t to p a ra s itic in fec tio n s than are the young.
f
8
'
H erlich (1960) studied age resistance against nematode in fec tio n s in
c a ttle .
P a ra s ite -fre e calves (5-8% months old) and steers (18 months
old) were pastured in contaminated paddocks.
Necropsy re su lts on
these animals showed th a t the calves had worm burdens comparable to
those o f the s te e rs , even though the steers consumed more forage.
More im po rtantly, the steers were less susceptible than the calves
to the d e b ilita tin g e ffe c ts o f parasitism .
These re su lts were thought
to r e fle c t a c e rta in degree of resistance in the older animals.
Gibson (1959) observed th a t development of Nematodirus spp. was
often retarded in mature sheep and th a t the capacity to in h ib it worm
development was acquired by lambs a t approximately 2 months of age.
In a d d itio n , Gibson and E verett (1963) demonstrated a reduced fecundity
in adult Nr battus harbored by older sheep.
Brunsdon (1962b) stated
th a t resistance to Nematodirus spp. in sheep was characterized by I )
lim ite d establishment of la rv a e , 2) increased length o f prepatent
periods, and 3) decreased egg production by adult worms.
Age re s is ­
tance to Nr battus in fec tio n s in sheep was fu rth e r documented by Mapes
and Coop (1973) who demonstrated th a t the percentages o f worms th at
were in h ib ite d a f t e r 5 inoculations with 60,000 Lg were sm aller in
3 month-old lambs (1 2 .4 - 22.2%) than in 8 month-old animals (47.8 72.3%).
G a llie (1973) showed th a t development of Nr battus was
s im ila r ly retarded in old versus young ra b b its .
the age
These reports in d icate
o f the host can have a d ire c t e ffe c t on p arasite development.
9
Studies on HL cdntortus in fec tio n s in sheep (Manton9 Peacock,
Poyter9 Silverman and T e rry 9 1962) showed th a t lambs (2 -4 months old)
were
not re s is ta n t to a challenge with 15,000 L3 .
Furthermore9
Urquhart9 J a r r e t t 9 Jennings9 McIntyre and Mulligan (1966) observed th a t
lambs (5-week old) were not immunized against 11. contortus using e ith e r ■=
sing le or repeated inoculations with irra d ia te d la rv ae .
This was
v e r ifie d by Lopez and Urquhart (1968) who demonstrated th a t sheep under
6 months of age did not develop immunity to
contortus a f t e r vaccina­
tio n with irra d ia te d la rv a e , whereas older animals re a d ily developed
strong immunity using the same vaccine.
Recently, Knight and Rodgers
(1974) observed th a t sheep o f d iffe r e n t ages exhibited various degrees
o f resistance to. primary in fec tio n s w ith H^. contortus; animals older
than 12 months c o n s isten tly harbored fewer worms than younger sheep.
Michel (1963) was able to in h ib it the development of CL ostertagi
in calves (96-272 days old) using repeated oral in oculatio ns.
In
con trast, Anderson e t a ). (1967) were .unsuccessful in in h ib itin g 0.
o s tertag i in 63-day old calves by a s im ila r method.
Other workers
(Armour, Jennings and Urquhart9 1969b; Smith, 1974) have stated th a t
in h ib itio n o f (L o s tertag i in calves has nothing to do with host
resistance.
S im ila r opinions were expressed concerning in h ib ite d
O stertagia spp. and -N. f i l i c o l l i s
in sheep (Reid and Armour, 1972)
and H_. contortus in sheep (Brunsdon, 1973).
10
F.
Acquired immunity
Several e x ce lle n t review a r tic le s are a v a ila b le which discuss the
ro le o f acquired immunity in parasite development ( Urquhart, J a rre tt
and M ulligan, 1962; M ichel, 1968; J a r r e tt and Urquhart, 1971; O g ilvie
and Jones, 1973).
Because dormant parasites tend to accumulate in
animals during autumn and w in te r, regardless of the age o f the host,
and because in h ib itio n often occurs in i n i t i a l in fe c tio n s , many people
believe th a t la rv a l in h ib itio n cannot be re la te d to host immunity.
Such views were expressed in regard to in h ib itio n o f
contortus in
sheep by B lit z and Gibbs (1971a) and Brunsdon (1 9 73 ), CL ostertaq i in
calves by Anderson e t a l . , (1967) and Cooperia oncophora in calves by
M ichel, Lancaster and Hong (1970).
Smith (1974) fu rth e r suggested
th a t host resistance had no e ffe c t on the development of 0. o s te r ta q i,
C. oncophora or N_. helvetianus in calves experiencing m u ltip le in fectio ns
. there is substantial evidence th a t retarded development of
nematodes in the host is enhanced by acquired immunity.
spp. e l i c i t strong immune responses in sheep.
Nematodirus
Donald, Dineen, Turner
and Wagland (1964) demonstrated th a t JL spathiger in fe c tio n s were
regulated about threshold le vels in sheep according to the immune status
o f the host.
Immune responses were characterized by I ) elim ination of
Lg, 2) retarded development o f L^, 3) reduced fecundity o f adults or
4) expulsion of ad u lts.
repeated in fe c tio n s .
In h ib itio n o f
was most pronounced a fte r
11
Retarded development o f Oesophagostomum spp. occurs rath er
c o n s isten tly,
Roberts, Elek and Keith (1962) showed th a t the immune
response against 0. radiatum in calves occurred w hile the larvae were
in the h is to tro p ic stage.
The immune response to 0. radiatum was shown
to be directed against 4th stage larvae by Keith and Bremner (1973),
who demonstrated th a t the
were highly e ffe c tiv e fo r immunization
o f calves.
Keith (1967) observed abnormal spicules in male Cooperia pectinate
obtained from immune calves.
In a d d itio n , the prepatent periods of
the £ . pectinata in fec tio n s were twice as long in immune animals.
Sommerville (19,60) demonstrated th a t immunity to £ . c u r t ic e i, a
s im ila r p a ra s ite in sheep, was directed against the L^.
The ro le of immunity in in h ib itio n o f Ostertagia spp. has
stim ulated controversy among workers.
Michel (1963, 1970) described
the dynamics o f 0. o s te rta g i in fec tio n s in calves which were inoculated
d a ily with 1,500 Lg.
He reported th a t I ) worms were constantly being
e lim in a ted , 2) in h ib itio n o f larvae seemed to be c o rrelated w ith the
size of the adult worm population, 3). a d u lt worms were.stunted in growth,
4) ovulation was suppressed in adult female worms or 5) the host became
re s is ta n t to the establishment of new la rv ae .
Active immunization by
repeated in fe c tio n s , th e re fo re , increased the numbers o f in h ib ite d
la rv a e .
L a te r, M ichel, Lancaster and Hong (1973) v e r ifie d th a t acquired
immunity was important in the re ta rd a tio n o f CL o s tertag i in calves.
12
Haemonchus placei is more susceptible to reta rd a tio n in calves
than is H. contortus in sheep.
Roberts (1957) showed th a t calves
developed a strong resistance to H. placei during an i n i t i a l in fe c tio n .
R esistant calves were found to harbor many more in h ib ite d L4 follow ing
a subsequent challenge in fe c tio n than unexposed calves.
In con trast,
Dineen and Wagland (1956a) observed th a t pre-exposed sheep showed no
appreciable increase in the proportions o f in h ib ite d L4 a f t e r I
challenge with 3,000 II. contortus; although a fte r a second challenge
a small increase was evident.
I t is possible th a t the pre-exposing
doses o f larvae used in the l a t t e r experiment (498-2,713 L^) were not
s u ffic ie n t to e l i c i t a strong resistance to challenge.
Repeated inoculations with in fe c tiv e larvae have been shown to
immunize sheep e ffe c tiv e ly against H^. contortus.
Dineen, Donald,
Wagland and O ffner (1965) observed th a t 3,000 IH. contortus, administered
a t the ra te of 100 Lg/day fo r 30 days, yield ed in fec tio n s comprised of
350-387 in h ib ite d L4 , whereas 3,000 Lg in a single in oculation resulted
in only 17-63 retarded la rv ae .
In addition when large numbers of
in fe c tiv e larvae are superimposed upon e x is tin g populations of ji.
contortus, sheep became immunologicalIy exhausted or "to le ra n t" (Dineen
and Wagland, 1966b).
This is an important concept to consider when
animals are repeatedly infected or when natural pasture in fectio n s are
employed to evaluate the dynamics o f worm populations.
Wagland and
Dineen (1967) fu rth e r observed th a t animals became to le ra n t a fte r only
13 .
6 inoculations with 3,000 L3 , however, resistance returned 4-8 weeks
la t e r .
As previously discussed, t h e .h is to tro p ic la rv a l stages of many
nematodes are highly immunogenic, and such is also the case with
contortus in sheep (C h r is tie , Brambell and Charleston, 1965; Bi takarami re ,
1966; and Wagland and Dineen, 1967).
Immunosuppression has been used to study the ro le o f immunity in
the in h ib ite d development of helminths.
Dunsmore (1961) combined X-
ir r a d ia tio n and cortisone treatments in sheep in fected w ith Ostertagia
spp. and found th a t fewer larvae were in h ib ite d in trea te d animals
(0.7-31.0% ) than in untreated controls (28.2-71.4% ).
Michel and
S in c la ir (1969) tested the e ffe c ts o f the c o rtico sterio d s B-methazone
and prednisolone on in h ib itio n o f 0. o s te rta g i in calves but did not
fin d a reduction in the number o f in h ib ite d la rv ae .
They did show, .
however, th a t worm egg production was g re a tly enhanced follow ing
treatm ent.
S im ila rly , P ritc h a rd , Donald and Hennessy (1974) fa ile d to
induce development of in h ib ite d 0. ostertag i using the co rtic o s te ro id
dexamethazone trim e th y la c e ta te .
Soulsby (1966) observed increased
worm egg counts in sheep follow ing treatm ent with chloram bucil, an
a lk y la tin g agent, and suggested th a t the increased worm egg production
occurred a f t e r in h ib ite d larvae had matured (also Soulsby and Owen,
1965).
Other studies regarding the e ffe c ts , o f immunosuppression on worm
populations were reviewed by Gibbs (1968).
Such studies should be
in te rp re te d c a re fu lly , however, as i t has been demonstrated th a t the
14
time at' which a drug is administered ( r e la tiv e to the time of antigenic
s tim u la tio n ), dosage r a te , and the type o f immunosuppressant used are
extremely important in the degree and type of suppression produced by
a drug (Gabrielson and Good, 1967: Lagrange, Mackaness and M ille r , 1974
Kerckhaert, 1974; and Kerckhaert, van den Berg, and H ofhius, 1974).
Work, heretofore reported, indicates th a t many facto rs may be
involved in the in h ib itio n phenomenon.
In the past some discrepancies
have existed among workers using the same or s im ila r ho st-parasite
systems.
I t seems reasonable th a t nematode species should vary in
th e ir s u s c e p tib ility to or p r o c liv ity fo r retarded development.
V a ria tio n may also occur because immune responses in animals vary
according to the species and/or the degree o f immunbcompetence which
they p o ss e s s .- These factors may account fo r much o f the confusion
regarding the ro le of immunity in the in h ib ito ry process.
In h ib ite d Development o f Qbeliscoides cuniculi
The bionomics and development o f Oj, cuniculi were described by
A lic a ta (1932) from in fec tio n s in guinea-pigs.
The 3rd ecdysis was
found to occur between 3 and 5 days p o s t-la rv a l-in o c u la tio n .
Fourth
stage females measured 2.3< mm by day 5 and the genital structures were
beginning to take form.
Fourth stage males measured 4 .3 -4 .8 mm and
possessed a prominant t a i l spike.
adults were present.
By day 12 many 5th stage larvae and
S o iled , Hayes and Soulsby (1968) described the
15 •
development o f 0. cuniculi in ra b b its .
They reported th a t 3rd ecdysis
occurred a t approximately 72 hr p o s ta la rv a l-in o c u la tio n .
By day 10
some larvae were in the 5th la rv a l stage, but 93% were s t i l l
stage.
in the 4th
The asynchronous development o f the larvae could not be explained,
but i t was postulated th a t many of the L4 were retarded.
Samuel (1970)
observed retarded 0. cuniculi w ith in the g a s tric mucosa of rabbits up
to 225 days p o s t-la rv a l-in o c u la tio n .
Retarded development of 0. cuniculi may re s u lt from several fa c to rs .
Russel I , Baker and Raizes (1966) showed th a t the number o f in h ib ite d
L4 depended on the size of the la rv a l dose.
In rabbits infected with
2,500 and 25,000 Lg/kg of body w eight, 6.0% and 70.9% o f the worms,
re s p e c tiv e ly , were in h ib ite d .
Also, prepatent periods were lengthened,
and worm egg production was reduced in worms in the hig h -level
in fe c tio n s .
. Obeliscoides cunicu li also becomes. dormant in rabbits i f the
in fe c tiv e larvae are stored under cold conditions fo r a prolonged period
(Fernando, Stockdale and Ashton, 1971; Stockdale, Fernando and Lee, 1970).
When Lg were stored a t 15 C fo r 28 days and then cooled to 5 C and l e f t
fo r an addition al 5 days, 42% o f the worms became retarded a t the 4th
stage (hutchinson, Lee and Fernando, 1972).
The physiological changes
which occurred in these larvae were not defined, nor was any reason
given fo r a ll the larvae not becoming dormant.
Studies have not been done to determine whether development of CL
cuniculi is a lte re d by an immunological process.
This study was
16
designed to determine whether host immunity is a fa c to r in the in h ib itio n
of development of 0_; cuniculi in domestic ra b b its .
Such parameters as
single repeated in fe c tio n s , hig h -level in fe c tio n s , passive immunizations
and immunosuppression o f the host were evaluated as to t h e ir e ffe c ts on
worm development.
The re su lts o f the present study show th a t the
immunological status and the degree o f antigenic stim ulation in rabbits
d ir e c tly influenced the development o f (h cuniculi such th a t large
numbers o f worms became in h ib ite d a t the 4th la rv a l stage.
I t is
th erefo re concluded th a t host immunity plays a s ig n ific a n t ro le in
imparing the development o f 0. cunicu li in ra b b its .
MATERIALS AND METHODS
Rabbits
Domestic rabbits ( OryctoTagus cuniculus L .) were obtained from 3
lo cal ra b b it producers.
a v a ila b ilit y .
Several breeds were used depending on th e ir
C a lifo rn ia n , New Zealand White and Palomino breeds
were used in contro lled te s ts , while Champagne d 'Argent and some cross­
bred rabbits were used as source animals.
A n tib io tic s were occasionally
administered to rabbits with diarrhea or re s p ira to ry in fe c tio n s .
Test
animals were usually allowed to acclim ate fo r a t le a s t 2 weeks and were
fed medicated Peavey p e lle te d ra tio n ( Peavey C o., Minneapolis, Minnesota)
ad lib itu m .
Helminth Cultures
The Obeliscoides cunicu li s tra in used in th is study was o r ig in a lIy
is o la te d from a c o tto n ta il ra b b it ( Sylvilagus floridanus mearnsi)
collected in Ohio in 1959 and was maintained in labo ratory rabbits th a t
were ro u tin e ly in fected with 3 ,000-5,000 th ird stage larvae (L g ).
s tra in was usually passaged every 3-5 months.
The
In fe c tiv e larvae were
cultured from the feces o f in fected source ra b b its .
To prevent
desiccation, feces were co llected in trays containing damp wood
shavings covered with paper towels.
Fecal p e lle ts were f i r s t softened
by soaking in tap water fo r 2-3 h r, a f t e r which peat moss was added
(approx. 2 /3 peat moss and 1/3 feces) and mixed with an e le c tr ic mixer.
- 18
This m aterial was then placed in 10-inch p la s tic p ie tte s (P o lly -fle x
Products, Chicago, 111.) and incubated a t ambient temperature (approx.
25 C) fo r 7-10 days.
Larvae were recovered from the cultures by the
standard Baermann technique ( Baermann, 1917).
Baermannization was
done through I la y e r o f c e llu lo s e tissue ( Kimwipe, Kim berly-Clark)
supported by a 60-mesh screen in an 8 - inch polyethylene fu n n e l.
The
larvae were washed several times and e ith e r used immediately in tests
or stored a t 4 C u n til s u ffic ie n t larvae were a v a ila b le fo r s p e c ific
experiments.
Some larvae were stored a t -20 C to be used in the
preparation o f worm antigen.
Inoculations with In fe c tiv e Larvae
The inocula were prepared from an aqueous suspension o f Lg by
counting the number o f ac tiv e larvae in an a liq u o t.
Volumes o f the
suspension containing the appropriate number of larvae were then
inoculated in to rabbits by gavage using a size 6 Bard catheter (Bard
woven venous cannula, C. R. Bard I n c . , Murray H i l l , N. J .) which was
covered with a t i g h t - f i t t i n g piece of Tygon tubing (Ui S. Stoneware,
Akron, Ohio) as a stomach tube.
Fecal Examinations
Fecal egg counts were determined using e ith e r a modified McMaster
technique or a Lane flo ta tio n procedure.
In the McMaster technique.
19
5 gm o f ra b b it feces and 150 ml water were blended fo r 2 min.
T h irty ml
o f the mixture were co llected in a graduated te s t tube and sedimented
in a centrifu g e a t I ,500 g fo r 3 min.
The supernate was poured o f f ,
and the sediment was resuspended in saturated NaCl solution to a volume
o f 15 ml.
A fte r m ixing, a portion o f the suspension was examined in
a McMaster chamber.
Both grids o f the chamber were scanned a t 60 X
m agnification on a compound microscope.
The worm eggs were counted and
the to ta ls were m u ltip lie d by a correction fa c to r (50) to obtain the
number o f eggs per gram o f feces (EPG).
For Lane flo ta tio n s , 5 gm o f feces were macerated in. 150 ml of
water as described above.
Duplicate 15 ml aliquots were collected in
15 ml c a lib ra te d te s t tubes and sedimented a t 1,500 g fo r 3 min.
The
supernate was removed and replaced with saturated NaCl solu tio n .
The
sedimented m aterial was resuspended and addition NaCl solution was
added u n til a meniscus formed a t the top of the tube.
A cover s lip
was placed on the. top o f each te s t tube a fte r which they were
centrifuged a t 700 g fo r 2 min.
A cover s lip was removed from each
sample, placed on a microscope s lid e and. examined under lowm agnification using a compound microscope.
A ll eggs were counted, and
a correction fa c to r (2) was used to obtain the EPG count.
Necropsies
A ll rabbits were euthanatized w ith Beuthanasia Special (H. C.
Burns Pharmaceuticals, Oakland, C a l i f . ) administered via an ear vein.
20 '
Each ra b b it was,immediately opened and the stomach removed.
The
stomach contents were rinsed in to a container and form alin was added
(5-10% fin a l concentration) to f i x the worms.
In some cases a I cm2
piece o f stomach tissue was removed from the dorsal aspect of the
fundus in the area d ir e c tly across from the cardiac sphincter.
These
tissue specimens were stored in 10% phosphate buffered form alin fo r
subsequent h is to lo g ic a l examinations.
The remaining stomach tissue was digested fo r 8 hr a t 37 C with
a g ita tio n in 200 ml o f a solution containing I gm o f pepsin (1 :10 ,00 0 ,
D ifc o )/3 0 0 ml of 0.1 normal HCl.
Both the stomach contents and the
digested m aterial were rinsed on a 200-mesh sieve, and the remaining
m aterial was stored in 10% formal in fo r l a te r worm counts.
The numbers of 4th stage larvae and 5th stage worms were counted
in aliquots o f both the stomach contents and the tissue digests.
The .
size of the aliq uots depended on the number o f worms in the sample but
usually ranged from 1/20 to 1/2 the to ta l m a te ria l.
Occasionally i t
was necessary to examine a ll the m aterial to obtain an accurate worm
count.
Male
were distinguished by the presence o f a t a i l spike
instead o f a bursa and females
by t h e ir size and the presence of
rudimentary reproductive organs (F ig . 2 1 ).
Helminth Transfers
Larval 0. cuniculi fo r use in tra n s fe r experiments were collected
from in fected source rabbits by placing stomach tissue in to an "8"
21
diameter glass funnel f i l l e d with warm (37-40 C) physiological salin e
solution (PSS).
When s u ffic ie n t
had migrated from the tissue
(1 /2 -1 hr) they were counted and administered per Osl to re c ip ie n t
ra b b its .
Helminth Measurements
Worms were mounted on microscope slid es and images o f the worms
were projected onto a white background using a Bausch and Lomb
M icroprojector.
Measurements were made by bending a piece o f sm all-
diameter cath eter tubing along the conformation o f the projected image.
The actual length was calculated using a conversion fa c to r obtained from
the projected image o f a I mm stage micrometer.
Anthelm intics
Two anth elm in tics, thiabendazole (TBZ) and levamisole HCl (LHC),
were tested fo r t h e ir e ffic a c y against the immature larvae of 0.
cuniculi in ra b b its .
The OmnizoTe form ulation of TBZ (Merck, Sharp,
and Dohme, Rahway, N. J .) was administered a t a dosage ra te of 500
mg/kg.. The LHC (American Cyanamid, Princeton, N. J i ) was dissolved in
water and administered to rabbits at the ra te of 70 mg/kg.
were administered via gavage.
Both drugs .
22
Immunosuppressant Drugs
Two drugs were used fo r immunosuppression o f ra b b its .
These
included the c o rtic o s te ro id 9 - f Iuoropredniso lene ( Predef 2X; Upjohn C o.,
Kalamazoo,. M ich .) and the a lk y la tin g agent cyclophosphamide ( Cytoxin;
Mead-Johnson, E v a n s v ille , In d .) .
Both drugs were administered in t r a ­
muscularly a t the dosage described in protocols o f the respective
experiments.
Histopathology
Tissues were fix e d in 10% phosphate buffered formal in , p a ra ffin
embedded, th in sectioned, and stained with a hematoxylin-eosin (H & E ).
The tissue sections were examined using a compound microscope fo r the
presence of lesions or embedded p a ra sites .
Hematology
Packed red blood c e ll volumes, to ta l leukocyte counts and white
blood c e ll d if fe r e n t ia ls were measured using standard hematological
techniques.
The number of lymphocytes/mm
3
of blood was calculated from
concurrent to ta l WBC counts and WBC d if fe r e n t ia l counts.
Antigens
Obeliscoides cuniculi antigen (OCA) was prepared from a pool of
th ird stage larvae using a lip id -e x tr a c tio n tecnhique.
Following 4
23
washings in tap water the larvae were rinsed 4 times in cold absolute
ethyl alcohol and extracted in the same solution fo r I 1/2 hr a t 4 C.
The l a t t e r step was then repeated using d ieth yl e th er.
The larvae were
removed from the e th e r, placed on a watch glass and allowed to desiccate
a t 4 C fo r 12 hr.
The dry preparation was weighed (168 mg), placed in
a mortar with glass fragments, and ground in to a powder.
The powdered
preparation was rehydrated with 10 ml or PBS (ph 7 .4 ) and sonicated 3
times (2 min a t an in te n s ity o f 73.5 W/cm2 ) using a Biosonik I I I
Sonicator ( Bronwi11 S c ie n t ific , Rochester, N. Y. ) .
eluted in PBS a t 4 C fo r 22 hr.
The antigen was then
A fte r subsequent c e n trifu g a tio n the
supernate containing the soluble antigen was stored in ampoules a t -20 C
Sheep red blood c e lls (SRBC) fo r use as antigen were collected in
Al s e v e r's solution and stored a t 4 C.
A 5% suspension o f washed SRBC
in PSS was prepared ju s t p rio r to use.
Each ra b b it received .5 ml via
an ear vein.
Antisera
A ntisera against CL cunicu li were obtained from rab b its which
previously had been inoculated w ith 100,000-200,000 Lg.
collected 5-10 weeks p o s t-la rv a l-in o c u la tio n ( p. i . ) .
Serum was
Sera th a t reacted
with OCA in gel d iffu s io n te s ts were incorporated in a pooled a n ti-OCA
anti-serum which was used fo r passive immunization o f rab b its and as a
control serum in serological te s ts .
24
Sera from te s t rabbits were s im ila r ly processed and examined
separately fo r antibody t it e r s against OCA or SRBC.
Normal rab b it
serum (NRS)' was collected from non-infected rabbits fo r use in serological
tests and passive immunization experiments.
Serological Tests
Antibody t it e r s against OCA and SRBC were determined by
immunodiffusion, in d ire c t hemagglutination, and hemolysin te s ts .
A.
Immunodiffusion te s t
The immunodiffusion te s t was patterned a fte r a method described
by Campbell (1970).
Agar gel was prepared by mixing I gm o f agarose
( SeaKem, Marine C o llo id s , I n c . , S p rin g fie ld , N. J .) with 94 ml of
PBS (pH 7 .4 ) and 5 ml of borate b u ffe r.
A fte r heating the mixture
to dissolve the agarose, I ml o f 1% m erthiolate in PSS was added as
a preservative.
The agar was then stored in 10 ml aliq uots a t 4 C.
Just p rio r to use the agar was melted by heating and poured into formvar
( 1% formvar in carbon te tra c h lo rid e ) coated glass p e tri dishes to a
depth o f 3 mm.
A fte r the agar s o lid if ie d , w ells (3 mm in diameter and
1-2 mm apart) were cut in the g e l.
Antisera were added to the outer
W ells, and antigen was placed in the center w e ll.
The agar plates were
then placed in a humidity chamber fo r 12-18 h r, a fte r which.they were
examined fo r the presence o f p re c ip itin lin e s .
25
B.
In d ire c t hemagglutination (IHA) te s t
The IHA t e s t , used to determine OCA antibody t i t e r s , was patterned
a fte r a m ic ro tite r method described by Herbert (1973).
Antigen coated
horse red blood c e lls (HRBC) were prepared by mixing 2 ml o f 0.1%
chromic chloride in PSS and 5 ml o f OCA solution (0 .3 7 ml OCA d ilu te d
to 5 ml in PSS) with I ml o f a 50% suspension of washed HRBC in PSS.
Following absorption fo r I hr a t 25 C the erythrocytes were washed 6
times in PSS.
The IHA te s ts were done in V-w ell M ic r o tite r Plates (Cooke
Laboratory Products, A lexandria, V a .).
S e ria l 2 -fo ld d ilu tio n s of
0.025 ml of h e a t-in a c tiv a te d serum were prepared in 0.025 ml of PSS
a f t e r which 0.025 ml o f a 1% suspension o f the OCA-coated HRBC was
added to each w e ll.
co n tro ls.
Each te s t also, included HRBC, NRS, PSS and a n ti-OCA
The plates were covered w ith cellophane ta p e , agitated and
l e f t at 25 C fo r 2-3 hr.
Anti-OCA t it e r s were determined by examination
o f the plates fo r a g g lu tin atio n o f c e lls .
C.
Hemolysin (HL) te s t
The HL te s t was used to determine antibody t it e r s to SRBC., S erial
2-fo ld d ilu tio n s o f h e a t-in a c tiv a te d serum were made as described above
using PBS in place o f PSS.
Afterwards, 0.025 ml of a 0.5% suspension
of SRBS in PSS and 0.01 ml of guinea-pig complement (1:10 d ilu tio n in
PSS) were added to each w e ll.
The plates were then covered with cellophane
tape, agitated and incubated a t 37 C fo r I hr.
Antibody t it e r s were .
26
determined by examination o f the plates fo r hemolysis o f the erythrocytes
These te s ts also included PBS, NRS and complement controls.
RESULTS
Repeated In fectio n s with Obeliscoides cuniculi
The o b je ctiv e o f th is experiment was to determine whether repeated
exposures to lo w -level in fe c tio n s with 0 . cuniculi would enable rabbits
to r e s is t development o f subsequent challenge in fe c tio n s .
For th is
purpose 5 groups o f rabbits (3 .4 kg mean w t), each consisting of I
C a lifo rn ia n , I New Zealand White, and 2 animals o f unknown breed, were
p re -in fected by in oculatin g them with 3,000 L3 a t 2-week in te rv a ls
according to the schedule in Table I .
Following p re -in fe c tio n s , a ll
20 rabbits were trea te d twice (days 42 and 49) with TBZ a t a dosage
ra te of 500 mg/kg, and subsequently challenged (day 68 ). w ith 3,000 L3 .
Packed red blood c e ll volumes (h e m a to c rits ), white blood c e ll (WBC)
d iffe r e n t ia l counts, and weight changes were determined a t weekly or
biweekly in te rv a ls .
On day 94, a ll animals were necropsied, and feces
were co llected and examined fo r helminth eggs using the Lane flo ta tio n
method.
One ra b b it in group 5 died before the end o f the experiment.
Necropsy data and mean eggs per gram (EPG) a t day 26 post-challenge
(day 94) fo r the remaining animals are lis te d in Table I .
Group I
animals (c o n tro ls) harbored the la rg e s t number o f worms (x" 587) of
which 12.8% were fourth stage larvae (L ^ ).
In co ntrast, the animals
in the 4 p re -in fe c te d groups ( I I - V ) contained fewer worms (x 245-411)
o f which 19.3%-56.5% were in h ib ite d a t the 4th la rv a l stage.
Table I ,
Group
Development of Qbelisco id es cuniculi in previously infected ( pre­
infected ) RABBITS FOLLOWING A CHALLENGE^ WITH 3,000 INFECTED LARVAE.
Number
Day
of
of
MEAN WORM COUNT
rabbits
pre- infection
(SlE1F U c
I
4
Control
587 (± 49,6)
II
4
0
258 (± 59,9)
III
4
0, 14, 28
IV
4
V
3
a Rabbits challenged day
46.4
498
341 (±161.3)
19,3
525
14,. 28
411 (±173.8)
52,3
403
28
245 (± 45.7)
56,5
353
68.
3,000 L3
per dose .
post- challenge
( day
94
of experiment ) ,
.
EPG
count0
908
cStandard error of the mean,
26
Mean
12,8
b Rabb its pre- infected with
dEgg counts day
Per cent
k
Control#—
P re-i nfected
Pre-infected
Pre-infected
Pre-infected
(Day 0) o —
(Days 0, 14, 2 8 )o - -(Days 14, 2 8 )* —
(Day 28) • —
rv>
10
Challenge
Anthelmintic
14
Figure I .
21
28
35
42
49
56 63 70
77
Day Post-larval-inoculation
84
91
98
Mean weight changes associated with repeated low-level (3,000 L3 ) in fectio ns
with Obeliscoides cuniculi in rab b its.
30
P re -in fe c tio n s , th e re fo re , produced conditions in these rabbits
under which both establishment and development o f worms in challenge
in fec tio n s were impaired.
Although an F -te s t showed th a t the v a ria tio n
between treatm ent means was not s ig n ific a n t, the data suggest th at
in h ib itio n o f la rv a l development was enhanced in the previously in fected
animals.
Mean EPG counts 26 days a f t e r challenge were almost twice as
high in control ra b b its .
This fu rth e r indicates th a t the worms in
p re -in fe c te d animals did not develop norm ally.
I t is. also possible th a t
immune responses in it ia t e d by previous in fectio n s in rabbits may have
reduced the fecundity o f worms in the challenge in fe c tio n s .
Pathological changes in rabbits with repeated lo w -level in fectio ns
were only minor.
Mean weight changes (F ig . I ) indicated th a t the
p re -in fe c te d rabbits gained weight a t a ra te comparable to the controls.
Hematological values fo r rabbits with sing le or repeated.low -level
in fec tio n s are provided in the Appendix.
Changes were not s ig n ific a n t
in e ith e r the packed red c e ll volumes or WBC d iffe r e n tia l counts.
H igh-level In fectio n s with Obeliscoides cuniculi
The purpose of th is experiment was to determine the e ffe c t of
massive in fec tio n s with (L cuniculi in ra b b its .
Eight New Zealand White
(12-14 weeks old) were allo cated in to 4 groups consisting o f 2 animals
each.
Group I rabbits served as uninfected co n tro ls, w hile those in
groups I I ,
I I I and IV were inoculated o r a lly with 200,000, 400,000 and
834,700 Lg re sp ec tiv e ly (Table 2 ).
Sera were collected a t biweekly
31
in te rv a ls and tested fo r antibody against CL cuniculi antigen (OCA)
using a gel d iffu s io n te s t.
Fecal samples were collected d a ily (day
14-80) and examined by the Lane flo ta tio n procedure.
fecal examinations were negative fo r helminth ova.
Pretreatm ent
Animals in groups
I I and I I I were necropsied on day 80, while those in groups I and IV
were used in other te s ts .
One ra b b it (IV -a ) showed periodic symptoms of diarrhea and
anorexia throughout the te s t.
Anorexia was observed in a ll the
infected animals from approximately day 3 to day 9 p . i .
ra b b it in each o f the in fected groups became patent.
Only I
Rabbit IV-a
became patent day 47, whereas rabbits I l - a and H l - a began to pass
worm eggs on days 61 and 63, re s p e c tiv e ly .
In a ll cases the worm egg
counts were low and egg output ceased a f t e r 3-10 days.
The prolonged
prepatent periods (47-63 days) in d ic a te th a t the worms did not develop
normally.
The mean weight changes in rabbits with hig h -level 0_. cuniculi .
in fec tio n s (F ig . 2 ) showed a weight loss up to day 10 , but th e re a fte r
weight increased a t a ra te comparable to the uninfected controls.
At day 59, the in fected animals had not gained as much weight as the
c o n tro ls ; groups I I I ,
I I and IV weighed an average of 200, 420 and
630 gm le s s , re sp ec tiv e ly .
At necropsy, group I I rabbits harbored a mean o f 38,850 worms
and group I I I , 22,286.
These were predominantly 4th stage larvae
(L q) , w ith only a few 5th stage worms (Lg) present.
These data
'
Table 2,
Development of Qreliscoides cuniculi in . rabbits with high - level
INFECTIONS,
■
Number Larvae
ADMINISTERED
<CL
ZD
O
CX
up
DAY
PATENT
Mean worm count®
(S ,E ,M ,)C
None
—
NDd
II
2 0 0 /0 0 0
63
38/850 (± 3,250)
III
400/000
61
22/286 (±21,413
834/700
47
ND
I
IV
aTwo aminals per group.
b Rabbits
in groups
IT and I I I were necropsied on day 80 p . i .
cStandard error of the mean,
dNot done,
* □
Weight Change (gm)
• Controls
□ 200,000 L3
o 400, OOOU
★ 834,700 U
.★ — ★ - *
Day Post-larval-inoculation
Figure 2.
Mean weight changes associated with high-level Obeliscoides cuniculi infections
in rabbits
34
in d ic a te th a t massive in fec tio n s with 0 . cuniculi resulted in almost
to ta l reta rd a tio n o f the larvae a t the 4th stage o f development.
Gross pathological lesions found in the g a s tric mucosa included
numerous petechial hemorrhages and a thickened, rough, w hitish
epithelium .
Only the fu n d ic and cardiac regions of the stomach
were a ffec te d .
Al I rabbits in groups I I and I I I developed s u ffic ie n t antibody
t it e r s by day 80 p . i . to give a p o s itiv e gel d iffu s io n te s t.
Serum
from ra b b it IV-b produced the most prominent p re c ip itin lin e s , whereas
ra b b it I V -a did not develop a measurable antibody response.
This may
explain the e a rly patency (day 47) observed in the la t t e r anim al.
Elim ination o f Immature Obeliscoides cuniculi with Anthelmintics
. In order to use hig h -level in fec tio n s o f Cf cuniculi as a means
o f a c tiv e ly immunizing ra b b its , a method of exp ellin g large numbers
o f worms had to be devised.
An i n i t i a l attempt to expel in h ib ite d
0^ cuniculi from rabbits in group IV o f the previous experiment fa ile d
These animals were given 834,700 Lg and a f t e r approximately 5 months
p . i . were trea te d w ith TBZ a t a dosage ra te o f 500 mg/kg.
there were s t i l l
30,000-50 ,000
present.
At necropsy
A study was then designed
to te s t the e ffic a c y o f LHC against immature Cf cuniculi la rv ae .
For th is t e s t , 8 C a lifo rn ia n rabbits (10-12
weeks old) were
allo cated to 4 groups each consisting o f 2 anim als, which were
inoculated per os with 100,000 Lg.
Beginning day 8 p . i . , rabbits in
Tab Le 3.
Group8
I
II
III
IV
Efficacy of levamisole
CUNICULI IN RABBITS,A
Treatment0
(70 MG/KG LHC)
HGl (LHC)
against immature
Day .
NECROPSIED
(p ,"I,)
Obeliscoides
Mean number
k
Per CENT
REDUCTION
Control
16
10,614
I DOSE
10
3
. 99,9
2 DOSES
14
15
99,8
3 DOSES
16
8
99,9
aRabbits were inoculated with
™---
100/000 INFECTIVE LARVAE 8 DAYS BEFORE TREATMENT,
8Two ANIMALS PER GROUP,
cAnthelmintic was administered b y gavage at
2 - day intervals .
36
g ro u p s II, I I I and IV were given e ith e r I , 2 or 3 doses o f LHC a t a
ra te o f 70 mg/kg, re sp ec tiv e ly .
A 2-day in te rv a l was allowed between
doses when more than I treatm ent was given.
as untreated controls.
Group I rabbits served
A ll treated rabbits were necropsied 2 days
a fte r they received t h e ir la s t dose o f a n th e lm in tic , and the controls
were necropsied on day 16.
At necropsy the control group harbored a mean o f 10,614
compared to 3-15 L4 in the treated groups (Table 3 ).
as
Therefore, LHC
was >99.8% e ffe c tiv e in removing the immature worms in a ll 3 groups.
Toxic e ffe c ts were not noticeable in the treated animals, even a fte r
repeated treatm ents.
Transfer o f In h ib ite d Obeliscoides cuniculi to Normal Rabbits
Experiment A:
Two experiments were designed to determine whether in h ib ite d L4
could resume development when tran s fe rred to previously uninfected
(normal) ra b b its .
In experiment A, 5 rabbits were inoculated with 4,000
Lg and served as sources o f normal L4 .
Normal 4th stage larvae were
removed from the rabbits on day 5 p . i .
In h ib ite d L4 were obtained from
a ra b b it which had been in fected w ith 5,000 Lg and subsequently
challenged (5 months la t e r ) with 200,000 Lg; the in h ib ite d L4 were
harvested day 14 p . i .
Four C a lifo rn ia n rabbits (14-16 weeks old) were
inoculated per os w ith 500 in h ib ite d L4 , w hile 4 others received 500
normal L4 .
Fecal samples were examined by the Lane flo ta tio n procedure
37
on day 18 p . i .
tra n s fe r.
A ll rabbits were necropsied on day 33 p o s t-la rv a l-
One hundred adult male and 100 adult female worms from each
group were measured, and the resu lts analyzed.using the Student's
I - te s t.
The remaining data were analyzed using an F -te s t.
Table 4 contains the necropsy re su lts (day 33 p o s t-la rv a l-tra n s fe r)
fo r experiment A as well as EPG counts fo r feces co lle cte d on day 18
p o s t-la r v a l-tr a n s fe r .
Approximately 50% o f both the in h ib ite d and
normal 4th stage larvae th a t were tran sferred to the re c ip ie n t rabbits
were recovered a t necropsy.
Of these 87.6% of the in h ib ite d and 98.2%
o f the normal larvae developed to adulthood.
in h ib ite d
However, the 12.4% o f the
tran sfers which remained in the 4th developmental stage
represented a s t a t i s t ic a l ly s ig n ific a n t number (P C 005).
These data
suggest th a t some o f the in h ib ite d larvae were damaged during in h ib itio n
and were not capable of fu rth e r development.
An analysis o f the a d u lt worm sex ra tio s (F -te s t) showed a .
s ig n ific a n t s h if t in the in h ib ite d L4 re cip ie n ts (P < .0 2 5 ) to higher
numbers o f female worms.
Evidently male worms were more adversely
a ffected during in h ib ito ry process than females;
Fecal examinations
showed th a t worms which developed from normal L4 produced s lig h tly
higher EPG counts.
Measurements o f 100 adult males and 100 adult females
from each group showed th a t the worms were not s ig n ific a n tly d iffe re n t
in to ta l length.
In h ib ite d males measured 9 .7 0 (+ 0 .5 ; S .E .M .), normal
males 9 . 58(+0.6) mm, in h ib ite d females I 3 .4 7 ( + l.2 ) mm, and normal
females 13 .8 6 (+ 0 .9 ) mm.
Table 4,
Development o f in h ib it e d and normal 4th stage larvae (L ^ ) a
o f Qbeliscoides cuniculi after transfer to normal r a b b it s ,
Mean worm c o u n t
La r v a e
TRANSFERRED . P er CENT r e c o v e r e d
I n h ib it e d 6
Worm s e x r a t io
P er CENT ADULTS
( m a le: fem ale)
Mean
EPG c o u n t
d a y 18
45,3
87,6
1:1.61
600
55,8
98,2
1:1.01
900
(500 L4)
No r m a l 0
(P < .0 0 5 ) d
(500 L4)
a F our
r a b b it s
per
group.
bObtained from
L ^ - day infection with 200,000 L^,
cObtained from
5 - day infection with 4,000 L3.
d F-TE S T
FOR.ANALYSIS OF VARIANCE,
( P C ,025)
39
Experiment B:
Experiment B d iffe re d from A in th a t re c ip ie n t animals were
included which were p re -in fe c te d with 0 . cuniculi p rio r to tra n s fe r of
the in h ib ite d and normal L4 .
Seventeen rabbits (14-16 weeks old) were
allo cated to 3 experimental groups.
Groups I and I I contained subgroups
o f 4 p re -in fe c te d (A) and 3 normal (B) rabbits (Table 5 ); each subgroup
was comprised of 2 New Zealand Whites and e ith e r I or 2 Palomino ra b b its .
Group I I I was made up o f 3 New Zealand Whites which served as prein fe c tio n con tro ls.
Rabbits in Group I I I and subgroups I-A and II- A
were p re -in fe c te d using oral inoculations w ith 75,000 L3 .
A ll rabbits
(groups I - I I I ) were tre a te d 7 days a f t e r p re -in fe c tio n w ith I dose of
LHC a t a rate o f 70 mg/kg.
Ten days l a t e r , the 4th stage larvae were
tran s fe rred to a ll re c ip ie n t ra b b its .
The L4 fo r tra n s fe r were obtained,
as in experiment A except th a t the in h ib ite d L4 were obtained.from a
ra b b it only 8 days a f t e r inoculation with 200,000 L3.
The L3 used to
produce both the in h ib ite d and normal L4 came from .the same la rv a l pool.
Group I rabbits were inoculated with 400 in h ib ite d L4 each, and group
I I animals received 325 normal L4 each.
receive a tra n s fe r o f la rv ae .
tra n s fe r.
Group I I I rabbits did not
Necropsies were done 26 days a f t e r la rv a l
Fecal samples, co lle cte d days .14-26 p o s t-1 a rv a l-tra n s fe r,
were examined by a modified McMaster technique.
an F -te s t fo r analysis o f variance.
Data were compared using
40
The combined data o f subgroups A & B (Table 5) showed s ig n ific a n t
differences in the numbers o f in h ib it e d .(28.5%) versus normal (64.6%) L4
which became established in re c ip ie n t rabbits follow ing tr a n s fe r *
A s ig n ific a n tly sm aller percentage o f the in h ib ite d larvae developed
to a d u lts , o f which females were more numerous than males.
These data
in d ic a te the larvae involved in a h ig h -le v e l in fe c tio n fo r only 8 days
were s u b s ta n tia lly a lte re d in t h e ir a b il it y to develop, prop erly, and i t
appears th a t the males were more a ffected than females.
Worm populations were fu rth e r a lte re d in p re -in fected re c ip ie n ts .
Approximately 5% more worms were present in the pre -in fe c te d rabbits
o f both tra n s fe r groups.
This increase cannot be a ttrib u te d to residual
worms from p re -in fe c tio n s , as only 17 worms were found in the prei nvection controls.
Larger numbers o f in h ib ite d larvae developed to
the adult stage in p re -in fe c te d re c ip ie n ts (34.3%) than in normal .
re cip ie n ts (18.2% ).
In c o n trast, a ll o f the normal L4 developed to
m aturity in normal re c ip ie n ts .
The m ale-to-fem ale r a tio o f adult worms
which developed from in h ib ite d L4 in p re -in fe c te d re cip ie n ts was 1 :8 .3
as compared to 1 :3 .3 in the normal re c ip ie n t ra b b its .
A s h if t in sex
ra tio s was not observed in animals which received normal L4 .
Therefore,
ac tiv e immunization o f rabbits a lte re d worm development with respect to
I ) the percentages o f worms which were recovered a t necropsy, 2) the
percentage o f worms which remained in the L4 stage and 3) the number o f
male worms which developed to m atu rity.
Although some o f the retarded
Table
5 , Development
of
in h ib it e d
( I -Llt) a
and normal
(N-L^)b 4th
stage
LARVAE OF Qb e LISCOIDES CUNICULI AFTER TRANSFER TO PREVIOUSLY
INFECTED (PR E~IN FEC TED )C OR UNEXPOSED (NORMAL) RABBITS,
Per cent
Per cent
adults
—
Pre- infected
(4 rabbits )
30,6
65,5
34,3
91,2
1 :8 ,3
1 :1 ,2
Normal
(3 rabbits )
25,7
60.5
18,2
100,0
1 :3 ,3
1 :1 ,2
Combined data
(7 rabbits )
28,5
98.4
1 :7 ,4
*
*
St a t is t ic a l l y
SIGNIFICANT
64,6
(P <,001)
I
I
JCr
N-L4
I
1 -4
I
I—
-Cr
recovered
Z
I
I—
-Cr
Re c ip ie n t
r abbits .
. Adult worm sex
RATIO (MALE:FEMALE)
*
28,1
.
(P <,001)
#
1 :1 ,2
(P C O D
A I- L it OBTAINED FROM 8-DAY INFECTIONS WITH 200>000 Lg; 400 LARVAE TRANSFERRED,
5N-Lit OBTAINED FROM 5-DAY INFECTIONS WITH 4,000 LgJ 325 LARVAE TRANSFERRED.
cRabbits pre- infected with
"
75,000 Lg and dewormed with levamisol HCl,
Inhibited L4to Rre-infected rabbits*— Inhibited L4to Normal rabbits#—
Normal L4to Pre-infected rabbits*—
Norms! L4 to Normal rabbits#—
/
J
Day Post-larva I-transfer
Figure 3.
Mean egg production by Obeliscoides cuniculi follow ing tra n s fe r of inhibited
and normal 4th stage larvae (L 4 ) to previously infected or normal rabbits.
43
larvae were impaired in t h e ir a b il it y to resume development follow ing
tra n s fe r to normal ra b b its , many L4 did develop to m aturity in dicating
th a t short term in h ib itio n was not completely irr e v e r s ib le .
Differences also were found in worm egg production follow ing
tra n s fe r o f in h ib ite d and normal
(F ig . 3 ) .
Most re c ip ie n t animals
developed patent in fec tio n s before day 14 p o s t-la r v a l-tr a n s fe r .
Patency
normally occurs about day 18 when in fec tio n s are in it ia t e d w ith 3rd
stage la rv ae .
In general, EPG counts were lower in rabbits which
received the in h ib ite d L^.
Also egg counts were lowest in the pre­
in fected re c ip ie n ts w ith in both groups.
These data suggest th a t adult
worms which developed from the in h ib ite d
were not as p r o lif ic as
those from the normal L^y and th a t a c tiv e ly immunized animals fu rth e r
suppressed egg production by adult worms.
E ffects o f P re -in fe c tio n s and Passive Immunizations
in Rabbits on Development of Obeliscoides cuniculi
The purpose of th is experiment was to ascertain whether active
immunization via exposure to p re -in fe c tio n s or passive-transfers of
immune serum in rabbits would a lt e r the development o f 0-. cunicul i .
F ifte e n New Zealand White rabbits (12-16 weeks old) were allo cated to
5 groups o f 3 animals.
Rabbits in groups I and I I were p re -in fe c te d
with 75,000 Lg per os.
Ten days la t e r , rab b its in a ll groups were
tre a te d w ith LHC a t a ra te of 70 mg/kg.
F ifte e n days a f t e r anthelm intic
treatm ent, rabbits in groups I , I I I , IV and V were challenged with
44
5,000 L3 , whereas animals in group I I were not challenged and served
as pre -in fe c te d controls.
At challenge, animals in group I I I were
in je c te d (in tr a p e r ito n e a lly ) with 12 ml o f pooled ra b b it a n t i-OCA serum.
S im ila r in je c tio n s were continued a t 2-day. in te rv a ls fo r a to ta l of 9
in je c tio n s (108 m l).
Group IV rabbits received a s im ila r regimen of
NRS and served as normal serum c o n tro ls, whereas animals in group V did
not receive treatments a f t e r challenge and served as patency controls.
Fecal samples were collected days 14-24 and were examined by the modified
McMaster method.
A ll animals were necropsied 26 days post-challenge.
Although the p fe -in fected animals (group I ) harbored the largest
number of worms, there was no evidence th a t th is was due to residual
in fe c tio n p e rs is tin g other treatm ent (Table 6 ) .
Immune serum recipients
(group I I I )
Because worm counts in
did not have increased worm counts.
both o f the immunized groups were highly v a ria b le i t is possible th at
in divid ual rabbits did not respond to the in fectio n s to the same degree.
P re-in fected animals (group ,I ) harbored the highest percentages of
in h ib ite d L^.
In a d d itio n , the 5th stage worms in the p re -in fe c te d
animals were e ith e r stunted in growth or i n f e r t i l e .
P re -in fe c tio n s in
rabbits th erefo re resulted in abnormal development o f the young adult
worms (Lg) and enhanced in h ib itio n of the 4th stage la rv a e , whereas
a n ti-OCA serum did n e ith e r.
The p re -in fe c tio n controls (not challenged) did not pass helminth
eggs during the te s t.
Patent in fec tio n s developed in the NRS controls
and the patency controls by days 17 and 18, re sp ec tiv e ly .
In both cases
. Table 6 ,
D e v e l o p m e n t o f Qb e l i s c o id e s
AND PASSIVELY
c u n ic u l i
in
pre-
I nfecteda
IMMUNIZED RABBITS,
Mean worm c o u n t c
T reatm ent
Gr o u p b
Co n d it io n
(S .E .M ,)d
P er c e n t
701 (±248.2)
18,3
of
adult
I
Pr e - i n f e c t e d
II
a n t h e l m in t ic
III
I mmune seru m
505 (±318,2)
0,4
Normal
No r m a l seru m
459 (± 27.2)
0 ,0
Norm al
Pa t e n c y c o n t r o l
594 (±112,0)
0 ,1
Norm al
IV
V
a Ra b b i t s
p r e - in f e c t e d
CHALLENGE WITH
bT h r e e
r a b b it
c Ra b b it s
d St a n d a r d
6 (± 2, 8)
control
w it h
75,000
5,000 L3,
and
dew ormed
.
groups,
n e c r o p s ie d
error
of
26
the
days
post- c h allen g e,
m e a n ...
worms
S t u n t e d or i n f e r t i l e
—
w it h
l e v a m is o l e
HCl
p r io r
to
800
Pre-infected e —
Immune Serum © —
Normal Serum © —
Control • —
Last Serum Injection
22
23
24
Day Post-larval-challenge
Figure 4.
Mean egg production by Obeliscoides cuniculi in previously infected
passively immunized ra b b its .
47
worm egg production increased normally (F ig . 4 ).
The p re -infected
animals (group I ) passed only a few worm eggs a fte r a s lig h tly prolonged
patency.
This fu rth e r supports the observation th a t many o f these worms
were e ith e r stunted or i n f e r t i l e .
Worm egg production was suppressed in
the passively, immunized rabbits u n til day 23 a fte r which EPG counts
increased normally.
Immune serum e v id e n tly obviated worm egg production
u n til the amount o f passively tran s fe rred antibody f e l l below c r it ic a l
le v e ls .
c u n ic u li:
Therefore, rab b its possibly produce 2 types o f response to £ .
one which may or may not be antibody dependent and results
in la rv a l in h ib itio n , and another which is antibody dependent and causes
suppression o f worm egg production.
. The Development o f ObeliscoideS fclihiculi in Rabbits
Treated w ith the C o rticostero id 9 - f Iuoroprednisol one
. Experiment A:
Two experiments were designed to examine the e ffe c ts of 9 -flu o ro ­
predni sol ene (FP) treatments in rabbits on the development o f concurrent
in fec tio n s w ith 0. cuniculi .
In experiment A, 8 rabbits (12-14 weeks
old) were separated in to 2 groups.
Whites and 2 C a lifo rn ia n s .
with 100,000 Lg.
Each group consisted o f 2 New Zealand
On day 0 , a ll rabbits were inoculated per os
Animals in the treatm ent group were given in je c tio n s
(ih tra-m uscular) with FP a t dosage rated o f 2 mg/kg/day (days 0-6) and
0 .5 mg/kg a t 2-day in te rv a ls th e re a fte r (days 8 -2 8 ).
Fecal egg counts
were determined days 17-40 using the Lane flo ta tio n technique.
Total
48
WBC counts were determined on days 19 and 42.
A ll rabbits were,
necropsied on day 42.
Two o f the FP-treated rabbits died during the i n i t i a l phase o f the
test,an d I control animal was s a c rific e d fo r a worm burden comparison.
The necropsy data fo r the remaining rab b its are lis te d in Table 7.
The
FP-treated rabbits harbored s lig h tly la rg e r number o f worms than co n tro ls.
In the control animals, 49.5% o f the 0. cuniculi present a t necropsy had
developed to the L5 stage, but only 3.5% were la te L5 ( f u l l y mature).
Comparable data fo r the FP-treated group were 57.5% and 20.9%, respect­
iv e ly .
Therefore, s u b s ta n tia lly more worms attain ed m aturity in the
FP-treated animals.
However, the egg count data revealed th a t worm egg
production Was impaired in animals o f both groups.
None o f the controls
became p a te n t, and the FP-treated animals passed only small numbers o f
eggs (8-72 mean EPG) fo r a short period (days 3 0 -36 ).
The reason egg
production was suppressed even though large numbers of adult worms were
present is not understood.
Mean WBC counts in the FP-treated rabbits were considerably Tower
than in the controls; counts on days 19 and 42 were 5,138 and 5,075
3
c e ll s/mm o f blood, re s p e c tiv e ly , in FP-treated animals and 9,979. and
3
8,100 c e ll s/mm in co n tro ls. In a d d itio n , FP produced e a rly weight
losses (F ig . 5) in the trea te d rabbits (days 4-12) a f t e r which they
began to gain weight slowly.
By day 29 the FP-treated rabbits weighed
an average of 892 gm less than the controls.
T able 7,
Development of Qbeliscoides cuniculi in rabbits with high - levela
INFECTIONS AND TREATED WITH THE CORTICOSTEROID 9-FLUOROPREDNISOLONE
(FR),
Number
Group
.
of
(D a y s
.
Pe r _ c e n t
P er c e n t
CS.E,fUc
l 5d
. 2
7,382 (±2013)
57,5
20.9
3
6,911 (±2110)
49,5
3,5
RABBITS
FP-TREATED
Mean worm c o u n t 6
.
LATE
Lg
0-28)
Co n t r o l s
a I nfections
in it ia t e d with an oral inoculation of
bNecropsies performed
.
100,000 L^,
42 days post- larval- inoculation ,
cStandard error of the mean,
dEarly and la t e .5 th stage worms
( late - fully mature) .
800
Figure 5.
-
• Inhibition Control
o FR-treated
Mean weight changes in rabbits given h igh-level in fectio ns with Obeliscoides
cunic u I i and treated with 9-fluoroprednisolone (F P ).
—
Necropsy examinations on the FP-treated rabbits th a t died on days
5 and 9 revealed numerous petechial hemorrhages w ith in the mucosa of
the fu n d ic and cardiac regions of the stomachs, and 10,848 and 14,1.70
4th stage larvae were recovered from these 2 animals.
The control ra b b it
s a c rific e d on day 9 had s im ila r petechial hemorrhages and harbored
8,364 Ly,.
4
■
i
Experiment B:
Because FP treatm ent in rabbits resu lted in increased numbers of
mature O^ c u n ic u li, a second te s t was designed to determine the e ffe c ts
o f FP on worm development when administered to rabbits a t 2 d iffe r e n t
time in te rv a ls .
In experiment B, 15 rabbits (12-14 weeks old) were
allo cated to 3 groups (Table 8 ) .
Whites and 2 C a lifo rn ia n s .
Each group consisted o f 3 New Zealand
An addition al group consisting o f 2
C alifo rn ian s (6 months o ld ) were used as patency co n tro ls.
On day 0,
animals in groups I , I I and I I I were inoculated per os with 100,000 L3
and those in group IV , with 3,000 L3 .
The rabbits in group I (FP-O)
were given FP in je c tio n s ( i.m .) on days 0-6 a t the dosage rates of
2 mg/kg/day fo r 2 days and I mg/kg/day fo r an additional 5 days:
A
s im ila r regimen o f FP was administered to animals in group I I (FP-20)
on days 20-26.
Rabbits in groups I I I
(in h ib itio n controls) and IV
(Patency controls) did not receive FP in je c tio n s .
Weight changes and
WBC counts were determined re g u la rly throughout the te s t fo r animals in
groups I , I I and I I I .
Fecal samples were collected on days 14-38 and
52
examined using a modified McMaster technique.
Serum samples were
co llected a t 6-day in te rv a ls and examined fo r antibodies against OCA
using the gel d iffu s io n te s t.
The animals in groups I , I I and I I I were
necropsied on day 42.
Necropsies were not performed on 2 FP-O rabbits th a t died ( I on
day 6 and one on day 7 ) , and the rabbits in group IV were not s a c rific e d
a t the end o f the te s t.
found in Table 8 .
Necropsy data fo r the remaining animals are
Rabbits in both FP-treated groups harbored an average
o f approximately 3,000 more worms than the in h ib itio n controls.
The
percentages o f worms which did not develop beyond the 4th stage were
68.0% (FP-O ), 28.5% (FP-20) and 58.1% (in h ib itio n c o n tro ls ).
Thus, FP
treatments administered days 20-26 produced a s ig n ific a n t reduction in
in h ib ite d la rv a e , whereas treatments on days 0-6 did not.
In a d d itio n ,
only 12.2% o f the FP-O worms and 29.6% o f the in h ib itio n control worms
progressed to the la te 5th stage as compared to 50.1% o f those from the
FP^20 group.
These data in d ic a te th a t the FP treatments given days
20-26 allowed in h ib ite d L4 to resume development.
I t was not c le a r why
FP treatments administered days 0-6 resulted in an enhancement of worm
in h ib itio n .
However, i t is known th a t immunosuppressants have varied
e ffe c ts on immune responses in animals depending on the time the drug
is administered r e la tiv e to an antigenic stim ulus. .
Rabbits in the patency control group began to pass helminth eggs
on day 19, followed by a normal ris e in mean EPG counts to a peak o f
3,300 (F ig . 6 ) .
In c o n tra s t, the other groups exhibited prolonged
t
53
prepatent periods.
The FP-O and in h ib itio n control groups started
passing eggs on days 35 and 37, re s p e c tiv e ly , and the mean EPG counts,
remained below 100 in both cases.
Yet#eggs f i r s t appeared in the feces
of rabbits in the FP-20 group only 6 days a f t e r FP treatments were begun
(day 2 6 )j and the mean EPG counts rose to a peak of 780.
The egg count
data c o rre la te well w ith the necropsy re su lts which showed th a t many
adult worms were present in the FP-20 groups.
Treatment o f rab b its w ith FP resulted in s ig n ific a n t changes in
WBC counts (F ig . 7 ).
The FP-O group showed an immediate drop, and counts
remained a t low le vels throughout the te s t..
Both the FP-20 and
in h ib itio n controls exhibited markedly depressed counts on day 6
followed by an immediate r is e to an elevated le v e l.
The WBC drop may
r e fle c t a period in which lymphocytes were migrating to the g a s tric
mucosa, w hile the ris e probably coincided with m obilizatio n o f c e lls
from lymphoid tissues.
The WBC counts in the FP-20 animals also dropped
ra p id ly a f t e r treatm ent began (day 20 ) but did not.remain as low as in
the. FP-O group.
Treatments with FP produced a s p e c ific weight change pattern (F ig .
8 ) which was characterized by an immediate and extensive increase in
weight followed by a precipitous drop.
During the period o f i n i t i a l
weight increase, the rabbits engorged themselves with food, a f t e r which
they stopped eating fo r a period o f 6-9 days.
Once the rabbits again
s tarted eating they gained weight a t a somewhat normal ra te but never
achieved the weight o f the untreated animals.
The l a t t e r observations
T able 8.
Development of Qbeliscoides cuniculi in rabbits with h i .gh- levela
INFECTIONS AND TREATED WITH THE CORTICOSTEROID 9-FLUOROPREDNISOLONE
(FP)i
Number
Group
of
Treatment
Mean worm count®
(Sl El I U c
RABBITS
.
Per cent
•
4
.
Per cent
ADULTS
3
Fp-TREATEDd
(Day 0-6)
8,,969 (±2067)
6 8 ,0
1 2 ,2
5
FP-TREATEDn x
(Day 20-26)
8,326 (±1612)
28,5
50.1
III
5
I n h ib it io n control
5,513 (± 751)
58,1
29,6
a I nfections
in it ia t e d with
bNecropsies
42 days post- larval - inoculation .
I
II
'
100,000 I 3 ,
cStandard error of the mean.
dTwo injections
I mg/ kg/ day ,
( i . m .) at a rate of 2 mg/ kg/ day followed by 5 injections at
Inhibition control
FR-treated (Day 0-6)
FR-treated (Day 20-26)
Patency control
Eggs Per Gram of Feces
•
10,000 o
□
★
Day Post-larval-inoculation
Figure 6 .
Mean egg production by Obeliscoides cuniculi in rabbits treated with
9-fluoroprednisolone (FP)
• Inhibition Controls
<D FR-treated (Day 0-6)
□ FR-treated (Days 20-26)
l o ix )
14.0 -
□
e
pooia jo ,umi/oaM
□ / X
<D
Day Post-larval inoculation
Figure
7.
Mean w h i t e bl ood c e l l
Obeliscoides cuniculi
T fp T
(WBC) count s i n r a b b i t s g i v e n h i g h - l e v e l
i n f e c t i o n s and t r e a t e d w i t h 9 - f l u o r o p r e d n i sol one
Weight Change (gm)
• Inhibition Controls
o FR-treated (Days 0-6)
□ FR-treated (Days 20-26)
h o —0 '
Day Post-larval-inoculation
Figure 8 .
Mean weight changes in rabbits given high-level Obeliscoides cuniculi
in fectio ns and treated with 9-fluoroprednisolone (FR).
58
are probably in d ic a tiv e o f the hormonal e ffe c ts of the c o rtic o rs te ro id
on glucose metabolism.
Serum antibodies against OCA were detected in only 2 rabbits during
the 42-day observation period.
One ra b b it in the FP-20 group was p o s itiv e
on days 11 and 17 but antibody was not detected a fte r FP treatments began.
Also, I ra b b it in the in h ib itio n control group was p o s itiv e on day 17.
The gel d iffu s io n te s t probably was not sen sitive enough to detect low
concentrations of antibody.
Development o f Obeliscoides cunicu li in Rabbits Treated With
Cyclophosphamide or 9 - f Iuoroprednisol one
This te s t was designed to study the development o f CL cuniculi in
rabbits given immunosuppressive drugs with d iffe r e n t b io lo g ic a l a c t iv it ie s .
Three Palomino and 2 New Zealand White rabbits (approximately 6 months o ld )
were allo cated to each of 4 experimental groups (Table 9 ).
On day 0, a ll
rabbits in groups I , I I and I I I were inoculated with 100,000 Lg, and
rabbits in group IV with 4,000 Lg.
in te rv a ls .
Total WBC counts were done a t 2-day
Serum (3-5 ml blood) was collected and WBC d iffe r e n tia l
counts were determined every 6 days.
On day 9, a ll rabbits were in jected
w ith 5 ml o f a 5% suspension o f SRBC as a te s t antigen.
Rabbits in group
I were given a regimen o f FP (2 days a t 2 mg/kg and 5 days a t I mg/kg)
on days 9-15.
A regimen o f CY (2 days a t 20 mg/kg and 5 days a t 10 mg/kg)
and was given days 9-15 to animals in group I t .
Group I I I rabbits served
as in h ib itio n controls and Group IV as patency controls.
A ll rabbits
were weighed d a ily during the time o f drug treatment and on a lte rn a te
days th e re a fte r.
Fecal egg counts were measured on days 14-26 using the
modified McMaster technique.
A ll rabbits were necropsied on day 27.
Serum antibody t it e r s against SRBC were measured via a hemolysin te s t,
and antibody against OCA was detected by in d ire c t hemagglutination.
Stomach tissues were examined h is to lo g ic a lly fo r lesio ns.
One ra b b it in each group died e a rly in the te s t.
fo r the remaining 16 animals, are lis te d in Table 9.
Necropsy data
The FP-treated
rabbits harbored a mean o f 14,562 worms, of which 61.7% were in h ib ite d
L^.
One FP-treated ra b b it did not have a sustained reduction in numbers
o f peripheral blood lymphocytes a f t e r treatment as did the other animals
in the group, nor was it s worm burden comparable (16,340 worms; 83.2%
in h ib ite d ).
Comparable data fo r groups I I ,
I I I and IV were 10,785,.
85.2%% 8 ,9 7 8 , 75.6%*and 416, 47.8% re sp ec tiv e ly .
A Duncan's m ultip le
range analysis showed th a t the percentage of in h ib ite d larvae in FPtreated rabbits and patency control animals were s t a t i s t ic a l ly s im ila r.
Likewise, the percentages o f retarded worms in the CY-treated group were,
s im ila r to those o f the in h ib itio n co n tro ls.
These data in d icate th a t FP
treatments given days 9-15 allowed a large number o f L4 larvae to resume
development.
Conversely, CY treatments days 9-15 prevented development
o f L4 to mature ad u lts.
in h ib ito ry process.
numbers of
The data suggest th a t CY may have enhanced the
This conclusion is substantiated by the small
stages found in the CY-treated animals.
Therefore, FP
and CY appeared to have opposite e ffe c ts on the a b il it y o f rabbits to
retard development of CL c u n ic u li.
Furthermore, the e ffe c ts o f CY
60
administered days 9-15 were s im ila r to the e ffe c ts o f FR given days
0-6 in the previous te s t.
Egg production by Ov cuniculi in immunosuppressed rabbits is shown
in Fig. 9.
Egg production was not observed in the CY-treated rabbits
(F ig . 9 ).
Also very few helminth eggs were passed by the in h ib itio n
c o n tro ls.
Patency controls developed patent in fec tio n s on day 18 and
EPG counts increased norm ally.
Egg output in the FP-treated group also
showed a normal rapid increase, but patency was delayed u n til day 22
(7 days a f t e r FP treatments began).
These data in d ic a te th a t FP
treatments in rabbits allowed development o f retarded larvae to resume,
whereas an opposite e ffe c t occurred in the CY-treated animals.
Changes, in the numbers o f ra b b it peripheral blood lymphocytes
(PBL) are shown in Fig. 10.
Treatment with FP produced the greatest
reduction in the numbers o f PBL.
The reduction occurred w ith in 6 days
a fte r treatm ent began and persisted to the end of the te s t.
Treatment
with CY did not s ig n ific a n tly depress PBL counts, however, i t did prevent
the lymphocytosis th a t was observed in the in h ib itio n controls at
approximately day 16.
These resu lts show th a t FP had a s ig n ific a n tly
greater e ffe c t on lymphocyte populations in the peripheral blood than did
CY.
These data also.suggest th a t some lymphoid c e ll type may mediate
the suppression o f worm development.
Figure 11 shows th a t FP was more e ffe c tiv e in suppressing to ta l
leukocyte counts than was CY.
A drop in WBC counts occurred in the FP .
group 2 days a f t e r treatm ent began.
Reductions in to ta l leukocyte
61
counts were not as great as the reduction in numbers o f lymphocytes.
This suggests th a t other cel I -types were m obilized as the c irc u la tin g
lymphocytes numbers were depleted.
The WBC d iffe r e n tia ls indicated
th a t the greatest increase in leukocyte numbers occurred in the
neutrophil populations; eosino philia was not evident in the peripheral
blood.
Both drugs produced weight losses in rabbits (F ig . 12).
Treatment
w ith FR caused an i n i t i a l weight increase followed by a rapid drop.
pattern was also described in the previous experiment.
Treatments with
CY caused a rapid weight loss without an i n i t i a l increase.
e ffe c ts o f CY on rab b its are d iffe r e n t than those o f FR.
This
Thus,the
Animals in
the 2 control groups gained a t a f a i r l y constant ra te throughout the
te s t.
Even though CY did not reduce the numbers c irc u la tin g lymphocytes,
the depressed weight gains in the re c ip ie n t rabbits in d ic a te th a t the
drug produced an adverse physiological e ffe c t.
T h e.resu lts o f the hemolysin te s ts to detect antibodies against
SRBC are shown in Fig. 13.
Six days a f t e r SRBC were administered (day
15), a ll rabbits which were in fected with 100,000 Lg had s im ila r mean
a n ti-SRBC antibody t i t e r s .
However, those rabbits which were inoculated
with only 4,000 Lg had considerably higher antibody t it e r s against SRBC.
Thus, the massive worm in fe c tio n s may have suppressed the antibody
response to SRBC.
These findings again suggest th a t FR and CY had
d iffe r e n t e ffe c ts on the immune systems in these ra b b its . .
62
A comparison o f the antibody t it e r s against OCA and SRBC on day
27 is shown in Fig. 14. ’ In both cases the CY-treated animals had the
lowest t it e r s and the FP-treated animals, the highest, w hile the
in h ib itio n controls had suppressed t it e r s to both antigens.
Even
though the differences in a n t i-OCA t it e r s were not s t a t is t ic a lly
.
s ig n ific a n t (F -te s t) and differences in the a n ti-SRBC t it e r s were
s ig n ific a n t a t only the P < 610 l e v e l , the r e la tiv e s im ila r itie s in.
t it e r s to both antigens suggest th a t CY and FP exerted opposite e ffe c ts
on both antibody le v e ls and worm development.
The gross pathological lesions in the stomachs o f these animals
were s im ila r to those previously described.
The h is to lo g ic a l lesions
(F ig s. 14-20) w ith in the fu n d ic and cardiac regions o f the g a s tric
mucosa were characterized by focal lymphoid hyperplasia in the base of
the mucosa and lymphocytic in f i l t r a t i o n in to the lamina p ro p ria.
There
was mild eosino philic in f i l t r a t i o n throughout the mucosa but es p e cia lly
in areas associated with p a ra s itic larvae or lymphoid f o l l i c l e s .
Some
lymphoid c e lls in f ilt r a t e d through the muscularis mucosa in to the
submucosa.
hyperplasia.
The mucosa was thrown in to folds as a re s u lt o f e p ith e lia l
Occasionally a mild c r y p titis was associated with the
presence o f nematode la rv a e .
Lesions in the FP and CY tre a te d animals
were s im ila r to those in the in h ib itio n controls but in general there
was less lymphocytic p r o life ra tio n in the treated ra b b its .
However,
considerable v a ria tio n occurred w ith in the immunosuppressed groups, and
63
in some animals the lesions were almost as severe as in the in h ib itio n
co n tro ls.
Lesions associated with lo w -level in fectio n s (patency
controls) were not severe and consisted o f mild lymphocytic hyperplasia
and only lim ite d e p ith e lia l hyperplasia.
J
Table 9.
Development of Qbeliscoides cunicu 'l i in rabbits trEAteda
with 9 - fluoroprednisolone (FP) or cyclophosphamide (CY),
Number
OF INFECTIVE.
LARVAE
Groupb
Mean
worm countc
(S,E.M .) d
Per cent
>4
FP-TREATED (DAYS 9-15)
1 0 0 ,0 0 0
14,562 (±1996)
61. Zy
CY-treated (Days 9-15)
1 0 0 ,0 0 0
10,785 (±1565)
8 7 ,2X
I n h ib it io n control
1 0 0 ,0 0 0
8,978 (±1254)
7 5 ,6X
Patency control
4,000
a Rabbits given a total of
416 (±
33)
-
4 7 .8y
9 mg/ kg of FR and CY-treated 90 mg/ kg CY.
b Four rabbit groups.
cNecropsies day
27 post- larval- inoculation .
dStandard error of the mean.
xyMu lt i
PLE range designations to denote differences I n treatment means.
/
® Inhibition Control
• Patency Control
© FR-treated
* CY-treated
18
19
20
21
22
23
24
25
26
Day Post-larval-inoculation
Figure 9.
Mean egg production by Obeliscoides cuniculi in rabbits treated with
9-fluoroprednisolone (FR) or cyclophosphamide (CY).
Lymphocytes/mm of Blood (X10 )
©
. •
. ©
. *
Inhibition Control
Patency Control
FR-treated
CY-treated
Treatments:
First
Day Post-larval inoculation
Figure
10.
Mean p e r i p h e r a l bl ood l y mphocy t e count s i n r a b b i t s i n f e c t e d w i t h
O b e l i s c o i d e s c u n i c u l i and t r e a t e d w i t h 9 - f l u o r o p r e d n i sol one (FP)
or c y c l oph os pha mi de ( C Y ) .
WBC/mm of Blood (XlO )
O Inhibition Control
# Patency Control
o FR-treated
* CY-treated
Treatments:
First
Day Post-larvaI inoculation
Figure
11.
Mean w h i t e bl ood c e l l (WBC) count s i n p e r i p h e r a l bl ood o f r a b b i t s
i n f e c t e d w i t h O b e l i s c o i d e s c u n i c u l i and t r e a t e d w i t h 9 - f I u o r o p r e d n i sol one ( FP) or c y s I oph os pha mi de ( C Y ) .
<d Inhibition Control
Weight Change (gm)
# Patency Control
e FR-treated
* CY-treated
-100
-200
•
P
-
-
Treatments:
Day Post-larval inoculation
Figure 12.
Mean weight changes in rabbits in fected with Obeliscoides cuniculi and
treated with 9-fluoroprednisolone (FP) or cyclophosphamide (CY).
CD
I
C
<
C
ro
$
I njected
SRBC
<d Inhibition Control
• Patency Control
o FR-treated
* CY-treated
Day Post-larval-inoculation
Figure 13.
Mean antibody t it e r s to sheep red blood c e lls (SRBC) in rabbits infected
w ith Obeliscoides cuniculi and treated with 9 - f Iuoroprednosolone (FR)
or cyclophosphamide (CY).
70
12
Antibody Titers (Log9)
11
SRBC
10
9
8
7
6
5
4
3
2
OCA
I
:
ABCD
ABCD
Treatment Groups
Figure 14.
A comparison antibody t it e r s against Obeliscoides cuniculi
antigen (OCA) and sheep red blood cel l s (SRBC) in immunosuppressed ra b b its . Sera collected day 27 p o s t-la rv a linoculation (18 days a f t e r SRBC in je c tio n s ): (A) infected
with 100,000 0 .£. and treated w ith cyclophosphamide;
(B) infected with 100,000 Ov c; (C) infected w ith 4,000
CLcv ; and (D) infected with 100,000 Ov^. and treated with
9-fluoroprednisolone.
71
Figure 16.
E p ith e lia l (E) lymphocytic (L) hyperplasia in the g astric
mucosa o f a ra b b it a f t e r a 2 7 -day in fe c tio n with 100,000
Obeliscoides c u n ic u li. H & E s ta in , x 40.
72
Figure 17.
Area o f lymphocytic hyperplasia in the g a s tric mucosa where
lymphocytes have in f ilt r a t e d the muscularis mucosa and in to
the submucosa o f a ra b b it in fected with 100,000 Obeliscoides
c u n ic u li. H & E s ta in , x 64.
Figure 18.
Lymphocytic hyperplasia ( L ) , c r y p titis (C) embedded nematode
larvae ( N ) , and lymphocytic i n f ilt r a t io n in the g astric
mucosa of a ra b b it in fected with 100,000 Obeliscoides
c u n ic u li. H & E s ta in , x 100.
73
Figure 19.
Embedded nematode larvae ( N ) , c r y p titis (C) and eosinophils
(EO) in the g a s tric mucosa of a ra b b it infected with
100,000 Obeliscoides c u n ic u li. H&E s ta in , x 375.
Figure 20.
E p ith e lia l hyperplasia (E) of the g a s tric mucosa in a ra b b it
during an in fe c tio n with 100,000 Obeliscoides c u n ic u li.
H&E s ta in , x 100.
74
Figure 21.
Normal adults (A; x 10) and in h ib ite d 4th stage larvae
(B; x 40) o f Obeliscoides cunicu li from rabbits 27 days
a f t e r in oculation w ith 4,000 Lg and 100,000 Lg, re sp ec tiv e ly .
DISCUSSION
Rabbits which were repeatedly inoculated with 3,000 L3 developed
the capacity to retard the development o f 0. c u n ic u li.
This was de­
monstrated by the presence o f large numbers o f in h ib ite d 4th stage
larvae in rabbits which were p re -in fected and dewormed p rio r to re ­
ceiving a challenge dose o f in fe c tiv e th ird stage larvae (Table I ) .
Enhanced in h ib itio n associated w ith repeated in fectio n s was also re­
ported fo r Nematodirus spathiqer in sheep (Donald e t a l . , 1964),
Haemonchus contortus in sheep ( Dineen e t a l . , 1965), Oesophagostomum
radiatum in sheep (Roberts e t a l . , 1962), Q stertagia ostertaq i in
calves (M ichel, 1963; Michel e t a l . , 1973) and jl. placei in calves
(Roberts, 1957).
la rv a l stage.
In each case th e .la rv a e were in h ib ite d a t the 4th
There is much evidence th a t the 4th stage larvae of
nematodes are highly immunogenic (Sommerville, 1960; C h ris tie e t a l . ,
1964; B itakarm ire , 1966; Wagland and Dineen, 1967; K eith, 1967; Keith
and Bremner, 1973; Michel e t a l . , 1973).
Soulsby, Sommerville and
Steward (1959) and Silverman, Poynter and Podger (1962) demonstrated
th a t immunogenicity is re la te d to excretion o f exsheathing
f lu id by worms w hile m olting.
Soiled e t a l . (1966) showed th a t ().
cuniculi larvae molt twice w hile embedded in the g a s tric mucosa; once
at 72 hr and again a t 5-10 days p o st-in o c u latio n .
This molting
process l i k e l y e l i c i t s an immune response in rabbits which in h ib its
development o f worms.
76
Development o f Obeliscoides cuniculi. was almost completely in ­
h ib ite d during i n i t i a l in fec tio n s with 200,000-834,700 in fe c tiv e larvae
(Table 2 ).
These re su lts f i t a pattern demonstrated by Russell e t a l .
(1966) in which the percentages of in h ib ite d 0 . cunicu li in rabbits in ­
creased with the size o f the in fe c tiv e la rv a l doses.
In the present
study in fec tio n s in it ia t e d with 100,000 L3 were comprised o f 50-75% in 'I
h ib ite d larvae (Tables 7, 8 and 9 ). Therefore, dose dependent in ­
h ib itio n in the (D. c u n ic u li-ra b b it system is rath er consistent.
Trichostrongylus reto rtaeform is (M ichel, 1952a) and Graphidium strigosum
(M artin e t a l . , 1957) are also in h ib ite d in rabbits in proportion to the
size o f the in fe c tiv e la rv a l dose.
The degree of in h ib itio n possibly
is re la te d to the amount of antigenic stimulus the ra b b it receives.
Dose response curves (D avis, e t a l . 1973) in d icate th a t an optimal amount
o f antig en ic stimulus is necessary to e l i c i t maximum antibody production.
I t would be d i f f i c u l t to measure the amount o f antigenic stimulus a
ra b b it receives from nematode larvae which are embedded in the mucosal
lin in g o f the g a s tro in te s tin a l t r a c t .
However, small numbers of larvae
probably do not e l i c i t a s u ffic ie n t immune response to in h ib it worm
development, unless the animal is repeatedly in fec te d .
In con trast,
massive in fe c tio n s produce a high degree of antigenic stim ulus, thus
the animal would develop a strong immune response.
Such a re la tio n s h ip
could explain why almost complete in h ib itio n occurs in h ig h -le v e l in ­
fections w ith 0 . c u n ic u li.
77
Previous reports (Gibson, 1953; Reid, Armour, Jennings, K irk p a tric k ,
and Urquhart, 1968; Armour, 1970) have indicated th a t dormant larvae are
not susceptible to c e rta in anthelm intics.
I t was found in th is study
th a t immature 0 . cunicu li were not elim inated from rabbits by 2 t r e a t ­
ments with thiabendazole (500 mg/kg).
Obeliscoides cuniculi may be
atypical in th is respect as Armour, Bairden and Reid (1975) reported
th a t thiabendazole (55-75 mg/kg) was e ffe c tiv e against dormant Ostertagia
spp. in sheep.
Levamisole HCl was tested against in h ib ite d (L cuhiculi
in the present study and was found to be highly e ffic a cio u s a t the rate
o f 70 mg/kg (Table 3 ).
McKenna (1974b) demonstrated th a t levamisole
was e ffe c tiv e against in h ib ite d Haemonchus contortus in sheep a t a ra te
o f only 8 mg/kg.
Further in v e s tig a tio n is th erefo re necessary to determine
i f lower dosage rates o f levamisole HCl would be e ffe c tiv e against Ch
c u n ic u li.
These reports in d icate th a t in h ib ite d larvae o f nematodes
are not invulnerable to anthelm intics as previously suggested (Armour,
197Q).
The time of the 4th ecdysis and the emergence of 0_. cuniculi
from the g a s tric mucosa have not been d e f in it e ly established in ra b b its .
Soiled e t a l.
(1968) thought th a t the molt to the 5th stage occurred
8-10 days p . i . , however9 41 % o f the worms were s t i l l in the 4th la rv a l
stage on day 14.
These data in d icate th a t the in fe c tiv e larvae used
in th e ir tests were in h ib ite d , thus these figures do not represent
normal molting periods.
A subsequent report (Soiled and A lle n , 1971)
78
indicated th a t most worms in s im ila r in fec tio n s (1,000 L3 ) had developed
to the 5th stage and had migrated to the surface of the mucosa by day 8 .
In the present study normal 4th stage larvae fo r use in tra n s fe r
experiments could not be obtained from source rabbits a f t e r 8 days p . i .
but were e a s ily recovered ph day 5.
Therefore, the molt to the 5th
stage and emergence from the g a s tric mucosa must occur between days
5 and 8 o f in fe c tio n .
When the larvae remain in the tissues fo r more
than 8 days they probably do so as a re s u lt o f in h ib ito ry factors
whether in tr in s ic or e x trin s ic in nature.
The question as to whether larvae resume development follow ing
re ta rd a tio n has not been answered fo r most nematode species.
In th is
study, in h ib ite d 4th stage larvae were obtained from source animals with
hig h -level in fec tio n s (200,000 L3 ) and tran sferred to e ith e r normal or
pre -in fe c te d (a c tiv e ly immunized) ra b b its .
In experiment A, 87.6%
(Table 4) o f the in h ib ite d larvae developed to m aturity follow ing
tra n s fe r to normal rabbits as compared to only 18.2% (Table 5) in
experiment B.
The differences in re su lts suggest th a t the extent to
which 4th stage larvae are affected during the in h ib ito ry process
varies from ra b b it to ra b b it.
Thus, f a ilu r e to mature was a re s u lt o f
damage incurred under in h ib itin g conditions in the source ra b b its .
Under the conditions of th is study, large numbers o f 2- cuniculi
were able to resume development a f t e r tra n s fe r to other r a b b it s ..
S im ila r observations have been reported fo r in h ib ited Oesophagostomum
79
radI aturn in calves (Roberts e t a l . , 1963) and in h ib ite d Haemonchus
contorus in ewes ( B lit z and Gibbs, 1971a).
In c o n trast, Ostertagia
o s tertag i which were in h ib ite d in sheep and tran sferred to calves did
not resume development (H e rlic h , 1974).
Following tra n s fe r to normal ra b b its , in h ib ite d male L4 did not
develop to m aturity as re a d ily as normal male L4 (Tables 4 and 5 ).
In
experiment B, the male to female ra tio n was 1:3.3 fo r in h ib ite d L4 and
1: 1. 2 fo r normal L4 tran sfers (Table 5 ).
reduced numbers o f males are:
Possible explanations fo r the
I ) fewer males were tran s fe rred to the
re c ip ie n t animals or 2 ) some of the in h ib ite d males fa ile d to resume
development a f t e r tra n s fe r.
The l a t t e r explanation is most lik e ly as
fu rth e r reductions in the numbers o f males occurred in the p re -in fected
re c ip ie n ts ( 1 : 8 . 3 ) .
The in h ib ite d L4 , tran sferred to both the pre­
in fected and the normal ra b b its , came from the same la rv a l pool and
should have produced s im ila r adult worm r a t i o s ,,
Changes in sex ra tio s
were not evident in p re -in fe c te d rabbits which received normal L4 .
Thus,
the damage to male worms occurred during the i n i t i a l in h ib ito ry process
in the source ra b b it and was potentiated by some fa c to r in the a c tiv e ly
immunized re c ip ie n t.
Why male worms fa ile d to resume development a fte r
tra n s fe r is not understood.
E ith e r they remained dormant follow ing
tra n s fe r or were elim inated from the re c ip ie n t animals v ia unknown
mechanisms.
The l a t t e r re su lts were unique in th a t no such immun­
ological Iy medicated e ffe c t has been described in the development of
larvae o f other nematode species.
80
In h ig h -level in fec tio n s (200,000-834,000 L3 ) , almost a ll worms
became in h ib ite d , and they remained w ith in the mucosa fo r periods of
3 months and longer.
Normally about 50-75% of the worms in in fectio ns
in it ia t e d with 100,000 L3 were retarded a t the 4th la rv a l stage
(Tables 7, 8 , 9 ).
Russell e t a l . (1966) showed th a t graded doses of
larvae (2,500 Lg/kg-25,000 Lg/kg) resulted in corresponding increases
in the percentages o f worms which became in h ib ite d .
Samuel (1970)
reported th a t 0. cuniculi were 'found in rabbits up to 225 days postla rv a l-in o c u la tio n in a lo w -level in fe c tio n (3,000 Lg).
Thus, the
s ize o f the in fe c tiv e la rv a l dose does not determine the length of
time larvae remain in h ib ite d , but only the number which become in h ib ite d
The la t t e r observation fu rth e r indicated th a t crowding o f worms
(Taylor and M ichel, 1952) does not account fo r a ll la rv a l in h ib itio n .
In one experiment in the present study (Table 9 ) , 47.8% o f the worms
in the patency control group (4,000 Lg) were in h ib ite d .
In th is case,
la rv a l in h ib itio n may have been re la te d to the age of the rabbits
(6 months).
Compare these data w ith those in Table 6 where patency
control animals (5,000 Lg) which were 12-16 weeks old did not contain
in h ib ite d L^.
Active immunization o f rabbits resulted in 2 changes in the
developmental pattern o f CL c u n ic u li.
augmented (Table 6 ) .
F ir s t , in h ib itio n o f L^ was
Almost a ll a d u lt worms recovered from the pre-
in f ected animals were stunted in growth, and adult females contained
81
few i f any eggs.
These data indicated th a t a host immune response
d ire c tly in h ib ite d worm development.
Second, egg production was s lig h tly
reduced in adult worms which developed from in h ib ite d and normal L4 in
a c tiv e ly immunized rabbits (F ig . 4 ) .
S im ila r observations have been
described in Nematodirus spathiger in fec tio n s in sheep (,Donald e t a l . ,
1964) and Ov o s tertag i in calves (M ic h e l, 1963; 1970).
A lso>abnormal
spicule development occurs in male Cooperia pectinata in immune calves
(K e ith , 1967).
Worm egg counts were influenced through reduction in the to ta l
number of mature worms and by suppression of egg production irf adult
worms.
Passive immunizations o f rabbits with immune serum did not
in h ib it development of worms (Table 6 ) but did suppress egg production
(F ig . 4 ).
These data in d icate th a t antibodies suppressed egg production
in mature worms.
Antibody-mediated suppression o f egg production has
not been reported fo r 0. c u n ic u li.
However, reduced egg production in
second in fec tio n s have been demonstrated many times with other nematode
species (M ichel, 1969).
Worley (1963) demonstrated th a t Ov cuniculi
produced higher worm egg counts in rabbits in fected with 1,000 Lg than
with 10,000 Lg.
The la t t e r observation could represent e ith e r an ti body-
suppressed egg production or re s tric te d development o f worms.
In summarizing the re su lts o f ac tiv e and passive immunizations,
i t is evident th a t in h ib itio n o f worm development and suppression
o f egg production in adult worms are under separate control mechanisms.
82
Egg production was suppressed with hyperimmune serum and is th erefo re
antibody dependent.
Also, follow ing tra n s fe r o f in h ib ite d and normal
L4 , egg counts were lower in a c tiv e ly immunized rabbits than in normal
animals (F ig . 3 ) , even though comparable numbers o f adult worms were
present in both types o f re cip ie n ts (Table 5 ).
Egg production was
lowest in worms which were tra n s fe rre d to the pre -in fe c te d ra b b its ,
and in both cases these
(Table 5 ).
ra b b its harbored large numbers o f adult worms
Larval in h ib itio n may or may not involve humoral antibody
because worm in h ib itio n was not augmented with immune serum.
Cyclophosphamide treatments in rabbits did not.promote worm de- ■
velopment.
Instead i t resulted in an increase in |th e percentages o f
in h ib ite d L4 , and few o f the 5th stage worms were normal (3.5%).
CY seemed to enhance the in h ib ito ry process.
Thus,
Immunosuppressive agents
have not a lte re d in h ib ito ry processes in some ho st-p arasite systems
(M ichel, 1969; B lit z and Gibbs, 1972a; P ritchard e t a l . , 1974).
How­
ever, Dunsmore (1961) was successful in causing O stertagia spp. in
sheep to develop follow ing combined c o rtic o s te ro id and X -irra d ia tio n
treatm ents.
Also, SolTod and A llen (1971) reported th a t cyclophosphamide
treatments in rabbits suppressed antibody response to jD. cuniculi
antigen and conalbumen, and produced a concomitant increase in worm
burdens and maturation o f 0 . cuniculi .
I t has been demonstrated th a t cyclophosphamide is e ffe c tiv e in
in h ib itin g antibody production to SRBC in mice a t dosage rates of
83
200-300 mg/kg.
When the drug was given to mice before s e n s itiz a tio n with
sheep erythrocytes the re s u lt was an enhanced DTH or cell-m ediated immune
response (Lagrange Mackaness and M ille r , 1974; Kerckhaert, 1974;
Kerckhaert e t a l . , 1974).
.
The dosage rate used in the present study
was considerably lower (90 mg/kg to ta l \ and antibody production was not
s ig n ific a n tly suppressed.
However, Askenase, Hayden and Gershon (1975)
demonstrated th a t CY treatments o f 20 mg/kg enhanced DTH responses to
SRBC in mice, even though i t did not abrogate antibody production.
If
CY has s im ila r e ffe c ts in ra b b its , in h ib itio n o f 0 . cuniculi may have
been controlled by cel I -mediated immune mechanisms.
Immunosuppression o f rabbits w ith FP provided strong evidence th a t
development o f ( L
cuniculi was under immunological c o n tro l.
In 3
separate experiments (Table 7 , 8 and 9 ) , FP treatm ent o f rabbits with high
level in fec tio n s (100,000 Lg) w ith 0_-. cuniculi resulted in an increased
number o f a d u lt worms.
The e ffe c ts produced by FP varied according to
the time the drug was adm inistered.
Treatments given to rabbits on days
9-15 and 20-26 p . i . resulted in s ig n ific a n tly la rg e r numbers o f adult
worms; patency occurred on days 22 (F ig . 9) and 26 (F ig . 6 ) , re s p e c tiv e ly .
Also worm egg counts increased a t a steady ra te .
Treatments given days
0-6 did not a lt e r worm development (Table 8 ) , whereas prolonged ad­
m in istra tio n o f c o rtic o s te ro id fo r 28 days (Table 7) resulted in
development o f s lig h tly la rg e r numbers o f worms.
The pathology of CL cunicu li in fe c tio n s , in it ia t e d with 100,000 Lg,
84
has been described (Russell e t a l . , 1970).
Gross lesions included
petechial hemorrhages and fo ld in g and thickening o f the g a s tric mucosa.
Russell e t a l . suggested th a t th is process was caused by mucosal edema.
In the present Study5 h is to lo g ic a l examination o f stomach tissue a t 27
days p . i . revealed th a t the fo ld in g o f the mucosa was caused by
e p ith e lia l hyperplasia ( F i g s . 16, 20) and only mild mucosal edema was
present.
Also, by day 27 p . i . , mucous-cell hyperplasia was not evident.
Russell found th a t the l a t t e r condition began e a rly (24 hr) and sub­
sided by day 22.
Other h is to lo g ic a l lesions seen in rabbits in the
present study included lymphocytic hyperplasia, lymphocytic and
eosino philic i n f i l t r a t i o n in to the lamina propria and in the v ic in it y
o f embedded parasites (Figs. 1 7 -2 0 ).
In some areas lymphocytic c e lls
were observed to i n f i l t r a t e through the muscular!s mucosa in to submucosa.
S im ila r lesions also occurred to a M im ited extent in rabbits w ith, lowlevel CL cun icu li in fec tio n s (Samuel, 1970).
The e ffe c t o f FR in rabbits points to the p o s s ib ility th a t la rv a l
in h ib itio n is re la te d to cell-m ediated immunity.
Results shown in Fig. 10
indicated th a t FR s ig n ific a n tly reduced the numbers o f c irc u la tin g
lymphocytes in ra b b its .
In v it r o transform ation of ra b b it peripheral
blood lymphocytes with T -c e ll mitogens (PHA and Con A) in d ic a te th a t
most peripheral blood lymphocytes in rabbits are T - c e lls (S e ll and
Sheppard, 1973).
I f so, FR may have s e le c tiv e ly reduced the T -c e ll
populations (the lymphocytes involved in DTH reactions) in these ra b b its .
85
Many studies have shown th a t T -c e lls are susceptible to co rtico stero id s
(Davis e t a l . , 1973; Fauci, 1975; Fauci and D ale, 1974; Yu, Clements,
Paul us, P e te r, Levy and B arn e tt, 1974).
Animals d i f f e r in t h e ir s e n s itiv ity to c o rtic o s te ro id s .
The
hamster, mouse, ra t and ra b b it are c o rtic o s te ro id -s e n s itiv e species,
whereas f e r r e t , monkey, guinea-pig and man. are re s is ta n t.
Sub­
populations o f lymphocytes in s e n s itiv e species vary in s u s c e p tib ility
to c o rtic o s te ro id s .
Claman (1972) reported th a t bone marrow derived
lymphocytes ( E- c e l l s ) are most susceptible during the precursor stage
or p rio r to being activated by antigen when in lymphoid compartments
other than the bone marrow.
Thymus drived lymphocytes (T -c e lls ) in
the thymus cortex are susceptible but not those in the medulla,
Also,
T -c e lls are s e n s itiv e during b la s t transform ation by mitogens but not .
when activated by antigen.
Therefore, antibody producing c e l l s , B-
lymphocytes or plasma cel Is?are not e ffected by c o rtib co stero id s.
Also,
antigen activated T -c e lls are not destroyed by c o rtic o s te ro id s , however,
in some cases, t h e ir e ffe c to r mechanisms may be s e n s itiv e .
The fa c t th a t activated E -c e lls are not e ffe c te d by c o rtico stero id s
would explain why antibody t it e r s against OCA and SRBC were not
pressed (Figs. 13 and 14).
sup­
Yet the mass destruction of T -c e lls could
have produced a d e f ic it in T -c e lls necessary fo r the regulation o f
the worm populations. The enhanced antibody t it e r s against sheep
erythrocytes (F ig . 13) also suggest a s e le c tiv e e ffe c t by FP on the
86
T -c e ll populations in ra b b its .
The FP treated animals Had higher
antibody le v e ls to SRBC than the in h ib itio n controls.
Taniguchi and
Tada (1974) showed th a t s e le c tiv e depletion o f T - c ll compartments in
rabbits using antithymocyte serum or adult thymectomy resulted in
elevated a n t i-DINP antibody t i t e r s .
The re su lts of th is study only in fe r an involvement o f T - c e lls in
the regulation o f development o f (L cuniculi in ra b b its .
I t has been
shown th a t T-Iymphocytes are involved in the regulation o f several other
helminth parasites (T a rg e tt, 1973; K e lly , 1973; O g ilv ie and Jones, 1973).
Recent studies using co ng en itally athymic mice showed th a t resistance
to the mouse pinworms A spicularis te tra p te ra and Syphacia obvelata
(Jacobson.and Reed, 1974a) and the r a t heligmosomid Nippostrongylus
b ra s iT ien sis (Jacobson and Reed, 1974b) was thymus-cell dependent.
These reports provide evidence th a t T - c e lls function in the regulation
o f several unrelated helminth systems.
Therefore, T - c e lls could be
involved in the regulation o f other parasites as wel l .
The conclusion from th is study is th a t host immunity plays an
important ro le in in h ib ite d development of 0. c u n ic u li.
The data
show th a t ( I ) ac tiv e immunization of rabbits by single or repeated lowlevel (3,000 Ls) or single h ig h -le v e l (75,000 l_3) in fec tio n s resulted
in .in h ib itio n o f larvae a t the 4th stage, (2) 9-fluoroprednisolone
treatment increased the numbers o f worms which, developed to the adult
stage, (3) the amount o f in h ib itio n varied with la rv a l-d o s e , and
87
(4) worms were damaged during in h ib itio n (e s p e c ia lly males) such th a t
not a ll in h ib ite d L4 were able to resume development when tran s fe rred in to
uninfected ra b b its .
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APPENDIX
Mean percent packed red blood c e ll volumes in rabbits given single and repeated infections3
with Obeliecoides cuniculi.
Day
of
test
Group
III (4)
V (2)
IV (3)
I (l)b
II (2)
14
43. 3
46.3
42.8
42.5
28
40. 0
44.0
42.5
43.3
45.5
42
41.5
46.0
42.5
41.8
43.0
49
40.8
45.8
42.5
44.3
43.3
68
42.0
44.8
43.5
43.5
43.3
75
41.8
44.8
42.0
42.3
42.7
82
40.5
42.3
40.5
41.3
41.3
94
'40.3
41.3
37.8
38.0
41.7
aInfections w ith '3,000 in fective larvae.
.^Number of inoculations.
1
41.3
Mean percent lymphocytes from white blood c e ll d iffe r e n tia l counts on rabbits given single
and reported infections9- with Obeliscoides cu h ic u li.
••
Day
of
test
I (I)
II (2)
III (4)
28
61.8
68.5
59.3
67.3
71.5
42
69.8
74.0
77.3
80.0
82.7
.49
67.3
83.0
73.0
79.8
83.7
68.
78.3
78.8
73.5
78.0
79.0
75
74.5
66.8
80.8
73.5
74.7
82 .
73.0
73.3
72.5
82.0
73.7
94.
68.3.
86.0
83.3
. 88.3
89.7
Group
9Infections with 3,000 in fective larvae.
^Number of inoculations.
'IV (3)
. V (2)
Mean percent monocytes from white blood c e ll d iffe r e n tia l counts on rabbits given single
and repeated infectionsa with Obeliscoides cuniculi.
Day
of.
test
• .I (l)t>
II (2)
Group
. Ill (4)
IV (3)
V (2)
28
3.8
6.0-
6.0
■5.0
5.5
42
1.8
3.0
1.8
2.0
0.3
49
2.3
1.5
1.0
1.5
2.0
68
1.5
2.0
1.3
1.0
1.0
75
0.5
0.8
0. 0
• 1.0
0.0
82
.
1.5
2.8
2.5
• 0.8
2.0
94
'
' 1.3
' 1.8
' 1.5
1.3
3.0
aInfections with 3,000 in fe c tiv e larvae.
umber of inoculations.
Mean percent neutrophils from white blood c e ll d iffe r e n tia l counts on rabbits given single
and repeated infections3- with Obeliscoides cu n icu li.
Day
O f
Group
I ( D ti-
II (2)
III (4)
IV (3)
V (2)
28 '
30.8
23.0
30.5
16.8
19.0
42
26.3
■19.0
16.3
13.0
7.0
49
26.5
14.3
21.5
■ 12.0
12.0
68
17.3
16.8
21.3
15.0
18.3
75
22.3
18.8
13.8
20.0
21.0
82
22.3
19.8
19.3
12.5
19.3
94
28.5
10.3
12.8
11.0
5.0
aInfections with 3,000 infective larvae.
^Number of inoculations.
■
104
Test
Mean percent eosinophils from white blood c e ll d iffe r e n tia l counts on rabbits given single
and repeated infections^ with Obeliscoides cu n ic u li.
Day
of
test
Group
I (1)0
II (2)
III (4)
IV (3)
V
(2)
28
1.3
0.5
1.3
4.3
1.0
42
0.0
2.0
1.3
1.0
3.0
49
'1.1
0.5
0.5
1.5
1.0
68
0.3
1.5
■ 0.5
1.5
0.0
75
1.3
2.0
3.0
2.0
3.0
82
1.3
. 1-0
2.8
■3.0
94
0.8
1.6
1.0
0.7
0.8 '
^Infections with 3,000 infective larvae.
Number of inoculations.
'
Mean percent basophils from white blood c e ll d iffe r e n tia l counts on rabbits given single
and repeated infections3- with Obeliscoides cu n ic u li.
Day
of
test
I (Db
II (2)
Ill (4)
IV (3)
' V (2)
28
2.5
2.0
3.0
6.8
3.3
42
2.3
2. 0
3.5
4.3
3.0
.3.0'
0.8
4.0
5.0
1.3
68
2.8
0.3
3.5
4.5
3.7
75
. 1.8
2.3
2.5
3.3
1.7
82
2.3
3.3
4.8
2.0
2.3
94
1.3
. 1.5
1.5
1.0
1.0
■ 49
"Group
^Infections with 3,000 infective larvae.
Number of inoculations.
MONTANA STATE UNIVERSITY LIBRARIES
762
001 041
D378
F832
cop. 2
DATE
Fox, Joseph C
The role of immunity
in inhibited development
of Obeliscoides cuniculi
IS S U E D
TO