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The irradiation of Babesia bovis . II. The immunogenicity of irradiated blood parasites for intact cattle and splenectomised calves
Veterinary Immunology and Immunopathology, 3 (1982) 591--601 Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- Printed in The Netherlands
591
THE IRRADIATION OF BABESIA BOVIS. I I . THE IMMUNOGENICITY OF IRRADIATED BLOOD PARASITES FOR INTACT CATTLE AND SPLENECTOMISED CALVES I.G. WRIGHTI , D.F. MAHDNEY1, G.B. MIRREI , B.V. GOODGERI and J.D. KERR2 ICSIRO, D i v i s i o n Indooroopilly, 2CSIRD, D i v i s i o n P.B.No.3, P.O.,
of Animal H e a l t h , Long Pocket L a b o a t o r i e s , P.B.No.3, P.O., Quensland, AUSTRALIA 4068. of Mathematics and S t a t i s t i c s , Long Pocket L a b o r a t o r i e s , I n d o o r o o p i l l y , Queensland, AUSTRALIA 4068.
(Accepted 2 June 1982) ABSTRACT Wright, I . G . , Mahoney, D.F., M i r r e , G.B., Goodger, B.V. and K e r r , J.D. 1982. The i r r a d i a t i o n of Babesia b o v i s . I I . The immunogenicity of i r r a d i a t e d blood p a r a s i t e s f o r i n t a c t c a t t l e and splenectomised calves. Vet. Immunol. Immunopathol., 3: 591-601. I n t r a e r y t h r o c y t i c forms of B. bovis were exposed to 350 Grays (Gy) y i r r a d i a t i o n and were then i n j e c t e d i n t r a v e n o u s l y i n t o i n t a c t two and t h r e e year old Hereford s t e e r s . One of 15 steers died on i n i t i a l i n f e c t i o n and subsequently s i x steers were given a v i r u l e n t heterologous challenge t h r e e weeks a f t e r recovery; a l l s i x animals were h i g h l y immune. The remaining e i g h t animals were kept under quarantine c o n d i t i o n s f o r i0 months and were then challenged w i t h a d i f f e r e n t v i r u l e n t heterologous s t r a i n of B. b o v i s . Seven of e i g h t were h i g h l y immune, but one animal d i e d . Subsequently a f u r t h e r 12 steers were i n j e c t e d i n t r a v e n o u s l y w i t h i x 108 i r r a d i a t e d organisms. A l l showed only m i l d t r a n s ient clinical signs. A f t e r 12 months quarantine in a t i c k - f r e e area these animals were then challenged w i t h a v i r u l e n t heterologous s t r a i n and a l l 12 were shown to be h i g h l y immune. I r r a d i a t i o n reduced the i n f e c t i v e dose from i x I0 U t o 2.5 x 103 p a r a s i t e s . These p a r a s i t e s m u l t i p l i e d at the same r a t e , and achieved the same maximum p a r a s i t a e m i a as the parent n o n - i r r a d i a t e d s t r a i n , but the disease produced by them was not severe. A dose of 2.5 x i0 s noni r r a d i a t e d p a a s i t e s was l e t h a l to a l l of the four animals which received i t . I t was concluded t h a t i r r a d i a t i o n had produced a predominantly a v i r u l e n t parasite population. INTRODUCTION In a previous paper Mahoney et a l .
(1973)
reported t h a t B. bovis p a r a s i t e s
i r r a d i a t e d w i t h doses between 200 and 500 Gy produced only a mild disease t h a t left
the
recipient
multiplication
animals
strongly
immune to
reinfection.
rate of these organisms was reduced.
In a d d i t i o n
the
These data i n d i c a t e d t h a t
p a r a s i t e s were a t t e n u a t e d by exposure to r a d i a t i o n doses of a p p r o x i m a t e l y 300350 Gy but were s t i l l
capable of immunising s u s c e p t i b l e animals.
the experiments r e p o r t e d here were designed to gain a d d i t i o n a l their
Consequently i n f o r m a t i o n on
immunogenicity and to determine the l o n g e v i t y of immunity in the h o s t ,
0165-2427/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing C o m p a n y
592
MATERIALS AND METHODS Experimental Animals Calves and a d u l t c a t t l e
of mixed breeds (Bos taurus) and sexes obtained from
a B a b e s i a - f r e e area were used in these experiments. the
P r i o r to commencement of
experiments the calves were splenectomised and a l l
ically
negative
to
B.
bovis
using
the
passive
animals were s e r o l o g -
haemagglutination
test
(PHA)
(Goodger and Mahoney, 1974). The p a r a s i t e s The
Lismore
(L)
(Mahoney et a l . , iated
strain
of
B.
boris,
described
1973) was used f o r i r r a d i a t i o n .
parasites
were challengd with v i r u l e n t
("S" and "C" s t r a i n s ) . from a n a t u r a l l y
previous
experiments
heterologous s t r a i n s
of B, bovis
The "C" s t r a i n was r e c e n t l y i s o l a t e d at t h i s
transmitted lethal
served as s t a b i l a t e s
in
Animals immunised w i t h i r r a d -
infection.
All
and stored in l i q u i d n i t r o g e n vapour,
storage they were passaged in a donor c a l f
laboratory
p a r a s i t e s t r a i n s were preA f t e r recovery from
from which the i n f e c t i v e
dose was
then o b t a i n e d . P r e p a r a t i o n and i r r a d i a t i o n Blood w i t h
at
least
splenectomised calves anticoagulant
of i n f e c t e d blood
5 x 107 p a r a s i t e s / m l
into
blood was withdrawn from donor
I% EDTA (di-sodium s a l t )
and dispensed in
in p h y s i o l o g i c a l
1-oz screw-cap b o t t l e s
s a l i n e as
in amounts of
10 ml at
4°Co The blood was i r r a d i a t e d Establishment, Sutherland, during
irradiation,
and i n o c u l a t i o n
NSW.
It
was held at 4°C during t r a n s i t
and was exposed at
Gy/min from a 60Co source. control
at the A u s t r a l i a n Atomic Energy Commission Research rates
that
varied
The periods between c o l l e c t i n g
by a i r and
between 6 and 10 the i n f e c t e d blood
i n t o the t e s t animals did not exceed 10-12 h.
Non-irradiated
samples of i n f e c t e d blood were also included in each package.
I n f e c t i o n of Animals Steers were g e n e r a l l y i n f e c t e d 1 x 108 i r r a d i a t e d was
taken d a i l y
(Mahoney parasites/
and
or n o n - i r r a d i a t e d
f o r packed c e l l
Saal,
mm3 b l o o d .
method described
intravenously
1961)
which
w i t h a standard dose of
p a r a s i t e s in whole b l o o d .
Jugular blood
volume (PCV) e s t i m a t i o n and t h i c k were
examined
to
estimate
blood f i l m s
the
number
of
P a r a s i t e doubling time was estimated according to the
by Mahoney et
i x 107 p a r a s i t e s i / v .
(i/v)
al,
(1973).
Splenectomised
calves
received
593
Experimental design Experiment 1.
F o u r groups of splenectomised, three month old calves each
composed of four animals wee infected with 1 x 108 B. bovis "L" strain parasites exposed to either 200, 300, 400 or 500 Gy, and the f i f t h group received i x 108 non-irradiated parasites of the parent strain.
Experiment 2 :
li)
Primary i n f e c t i o n .
Two year old non-splenectomised Here-
ford steers were used e x c l u s i v e l y in t h i s experiment. (a)
Group 1.
strain)
Six animals were inoculated i / v
which had been i r r a d i a t e d with 350 Gy.
enged three
weeks a f t e r
recovery with
i
with
1 x 108 parasites
(L
These animals were then c h a l l -
x 108 n o n - i r r a d i a t e d
parasites
(S
strain). (b) Group 2. Six animals were inoculated i / v with 1 x 108 non-irradiated parasites (L s t r a i n ) .
This was the parent strain of that used for production
of irradiated parasites. (c) Group 3.
Four animals were inoculated i / v with 2.5 x 103 non-irradiated
parasites (L s t r a i n ) .
T h i s dose was calculated to be equivalent to the surviv-
ing parasites after i r r a d i a t i o n of i x 108 v i r u l e n t organisms with 350 Gy. (d) Group 4.
Six control animals were inoculated i / v with 1 x 108 virulent
"S" strain parasites. (e) Group 5.
Four animals were infected subcutaneously (s/c) with i x 107
B. bovis "L" strain irradiated parasites which had been stored in l i q u i d n i t r o gen vapour for 10 months and had then been passaged through a splenectomised susceptible calf from which the inoculum was obtained. (ii)
Secondary challenge.
Hereford steers purchased at or i n i t i a l l y vaccin-
ated with irradiated organisms at the same time as those used in the primary infection experiment,
and subsequently maintained under t i c k - f r e e quarantine
conditions were used in this experiment. (a) Group 6.
Ten steers were inoculated i / v with i x 108 irradiated para-
sites (L strain) at the same time as Group 1.
One of these animals died from
the infection and a second injured i t s e l f and was destroyed.
The eight remain-
ing animals were then sent to a t i c k - f r e e quarantine area for 10 months. Upon return to the laboratory the sera of these animals were tested for B. bovis antibodies using the PHA test and all were found to be positive.
A 75 ml
sample of whole blood was withdrawn from each animal, mixed with 7.5 ml of i% sodium c i t r a t e and the pooled sample was then injected i / v into a susceptible splenectomised c a l f .
The c a t t l e were then challenged i / v with i x 108 virulent
heterologous B. bovis (C strain) parasites obtained from the donor c a l f . (b) Group 7. parasites.
Five control animals were infected i / v with 1 x 108 "C" strain
594
Experiment 3: ised
Hereford
with
1 x 108 B.
li)
Primary i n f e c t i o n .
steers
were used
bovis
"L"
(Group
strain
Secondary i n f e c t i o n . (Group i i )
ii).
These animals were i n f e c t e d
parasites
animals were then sent to a t i c k - f r e e (ii)
Twelve t w o - y e a r old non-splenectomirradiated
with
350 Gy.
i/v These
q u a r a n t i n e area f o r 12 months.
A f t e r the 12 month q u a r a n t i n e p e r i o d the 12 vacc-
inated
steers
and f o u r
susceptible
control
t h r e e - y e a r old Hereford
steers
(Group 12) were i n o c u l a t e d i / v w i t h i x 108 v i r u l e n t
B. bovis "C" s t r a i n
parasites. Control
experiments.
(a)
Group 8.
month old c a l v e s were i n f e c t e d
i/v
been s t o r e d
for
irradiated strain
in
liquid
nitrogen
parasites
with
splenectomised t h r e e -
i x 107 i r r a d i a t e d
p a r a s i t e s which had
10 months, to determine whether storage of
had any e f f e c t
as compared to i r r a d i a t e d
Four s u s c e p t i b l e
on the
stability
and i n f e c t i v i t y
of
the
organisms d e r i v e d from donor animals as used
in Control Group i 0 . (b) Group 9. strain
genicity (c) bovis
Four splenectomised calves were i n f e c t e d
non-irradiated
parasites,
of the s t r a i n Group
i0.
parasites
as a c o n t r o l
with
i x 107 " L '
to compare i n f e c t i v i t y
and patho-
compared to the i r r a d i a t e d
Four splenectomised obtained
calves
strains
in Groups 8 and 10.
were i n f e c t e d
from a donor splenectomised c a l f
the pooled blood sample from the Group 7 c a t t l e . determine whether the i r r a d i a t e d after
i/v
This
with
i
x
107 B.
which had received
group was a c o n t r o l
p a r a s i t e s had m a i n t a i n e d t h e i r
avirulent
to
state
i0 months in donor a n i m a l s .
RESULTS Experiment 1 The p a r a s i t e d o u b l i n g times were 8.23±1.25, erent.
No p a r a s i t e s
parasites time i0, 16.
in
in the 200, 300 and 400 Gy groups r e s p e c t i v e l y
10.10±0.92 and 9.50±0.20 hrs which were not s t a t i s t i c a l l y were d e t e c t e d
and these
animals
the c o n t r o l
and animals
in
The i n c r e a s e
in
survived
the the
group was 7.90 hrs. the
200,
The c o n t r o l
time was s i g n i f i c a n t
500 Gy i r r a d i a t e d
The p a r a s i t e animals a l l
300 and 400 Gy groups died
in s u r v i v a l
these data an i r r a d i a t i o n
group r e c e i v i n g experiment.
diffdoubling
died by day
between days
(P
12 and
As a r e s u l t
of
dose of 35 krad was chosen f o r experiment 2.
Experiment 2 Primary
challenge.
regressed on time
for
The
log
each animal
time to reach one p a r a s i t e / t h i c k ised in Table I .
parasitaemia
of
Groups
and the estimates
film
field
I,
2,
and
6 were
of d o u b l i n g time and the
were made.
These data are summar-
595
No s i g n i f i c a n t in
Groups
sites).
i
differences
(irradiated
in m u l t i p l i c a t i o n
parasites),
Highly s i g n i f i c a n t
differences
group 2 animals compared w i t h Groups i All
2
rate were observed f o r animals
(control),
and
6
(irradiated
and 6 animals on days 5, 6, 7 and 8.
Group 2 animals died by day nine w h i l e one animal died in Group 6.
animals
all
had haemoglobinuria,
para-
(p
haemoglobinaemia, f e v e r ,
and a t a x i a and were not examined by post-mortem.
jaundice,
These
anorexia
These data are summarised in
Table 2. The s i x Group i animals which had received i r r a d i a t e d challenged w i t h 1 x 108 v i r u l e n t ery from the i n i t i a l
challenge.
were also
with
inoculated
recov-
Six two-year old s u s c e p t i b l e Hereford steers
the same challenge dose (Group 4).
obtained from the Group 1 animals
it
dose received was 2.5 x 103 p a r a s i t e s . relatively
p a r a s i t e s were then
heterologous B. bovis three weeks a f t e r
was estimated t h a t
From the data
the actual
parasite
Consequently, to determine whether the
m i l d disease observed in the i r r a d i a t e d group was dose r e l a t e d , four
two-year old s u s c e p t i b l e Hereford steers were i n o c u l a t e d i n t r a v e n o u s l y w i t h 2.5 x 103 of the o r i g i n a l
"L" s t r a i n
(Group 3).
All
four cattle
in t h i s group died
on days 10-12, TABLE 1 Expriment 2 - estimates of the m u l t i p l i c a t i o n sites/mm 3 blood f o r c o n t r o l Group
rate and time to reach I000 para-
(group 2) and i r r a d i a t e d groups (Groups i and 6).
M u l t i p l i c a t i o n rate/24 hr
Control (group 2) Irradiated (groups i and 6) 95% LSD Group I Group 6 95% LSD
Mean
S.E.
2.06 2.07 0.56 1.69 2.46 1.13
0.03 0.08 0.08 0.15
Time (days) to reach 1000 parasites/mm 3 blood Mean
S.E.
6.0 13.2 1.1 13.8 12.9 2.3
6,16 0.58 0.83 0.92
There is no s i g n i f i c a n t difference in m u l t i p l i c a t i o n rate; the overall mean is 2.067 which is equivalent to an 11.6 hr doubling time. The mean difference in time to reach 1000 parasites/mm3 blood is 7.2 days. Group i - 6 c a t t l e subsequently challenged three weeks a f t e r recovery. Group 6 - 9 c a t t l e , 8 of which survived and were subsequently challenged 10 months l a t e r . Although only one of the Group 3 control animals died, the PCV f a l l was s i g n i f i c a n t l y d i f f e r e n t (p
No s i g n i f i c a n t f a l l
in the PCV was
Parasite m u l t i p l i c a t i o n rates in a l l three groups were
s i m i l a r but the time to reach maximumparasitaemias was dose dependent.
Immune
596
TABLE 2 Packed Cell Volumes f o r c o n t r o l
(Group 2) and i r r a d i a t e d Groups ( i and 6).
Group I - 6 c a t t l e
r e c e i v i n g i r r a d i a t e d blood challenged three weeks l a t e r .
Group 6 - 8 c a t t l e
r e c e i v i n g i r r a d i a t e d blood challenged I0 months l a t e r .
Day
0 2 3 4 5 6 7 8 9 10 ii 12
Control
Group 2
I r r a d i a t e d Groups i and 6
Group 1
Group 6
Mean
S.E.
Mean
S.E.
Mean
S.E.
Mean
S.E.
44.3 45.3 46.5 43.8 38.5 33.5 30.1 24.0
1.17 0.80 1.38 0.95 0.75 1.38 1.51 2.30
47.4 46.7 45.8 46.2 45.3 44.6 44.9 42.1
1.09 0.81 0.69 0.86 0.84 1.02 0.92 1.12
47.3 45.8 45.3 45.9 45.2 43.7 40.8 39.8 38.6 34.3 31.0 30.6
1.74 1.01 1.38 1.32 1.47 1.80 1.67 1.85 1.80 2.17 1.62 2.03
47.4 46.7 46.1 46.2 45.4 45.1 42.9 41.3 39.7 35.7 31.4 28.9
1.47 1.17 0.97 1.18 1.07 1.31 1.43 1.31 1.11 1.41 1.52 1.74
Group I animals had maximum parasitaemias on day 7, the c o n t r o l 8 and c o n t r o l
Group 3 on day I0.
Group 4 on day
These data are summarised in Table 3.
All
animals in Group 5 which were i n o c u l a t e d s/c w i t h I x 107 i r r a d i a t e d p a r a s i t e s became i n f e c t e d and underwent m i l d r e a c t i o n s . Secondary c h a l l e n g e . with
irradiated
tick-free
Eight
two-year old steers which had been vaccinated
p a r a s i t e s s i m u l t a n e o u s l y w i t h group 1 were kept in q u a r a n t i n e ,
c o n d i t i o n s f o r 10 months (Group 6).
Together with the f i v e animals
TABLE 3 Experiment 2 - comparison of
reactions
between i n t a c t
cattle
inoculated with
i r r a d i a t e d and n o n - i r r a d i a t e d B. bovis s t r a i n s . Grp Inoculum Inoculum Maximum* Max. dose/ type p a r a s i t - % PCV strain aemia change 1 2 3 4 5 6 7
lxlOBL ixlOSL IxlOTL IxlOSL
350 Gy Live 350 Gy 350 Gy
* Maximum p a r a s i t a e m i a
7.24xi03 -34.46 1.02xlO" -59.8 1.50x105 -48.62 1.74xi03 -33.54
=
No. of Challenge Maximum* Max. No.of Survivdose/ p a r a s i t - % PCV Surviv ors strain aemia change ors 6/6 0/6 0/4 8/9
lx108S
18.7
2.5xlO3L lxlOBS
5xlO 4 -58.24 4.17xi03 -45.5
0/4 5/6
ixlOBC IxlOBC
1.75x103 -17.94 4.01xi04 -47.87
7/8 2/5
number of parasites/mm 3 blood.
-6.03
6/6
597
in control Group 7 these two groups were inoculated intravenously with a virulent heterologous strain of B. bovis (C).
Both the mean maximum PCV f a l l and
mean maximum parasitaemia of Group 7 was s i g n i f i c a n t l y d i f f e r e n t from that of the immune Group 6 (p
Three animals in Group 7
died on days 9 or i0 and one animal in the immune Group 6 died from classical symptoms of babesiosis on day 12. globinaemia,
These signs included haemoglobinuria, haemo-
tetanic spasms, ataxia, fever, anorexia and jaundice.
mortem was deemed not necessary.
A post
These data are summarised in Table 3.
Experiment 33 Primary
challenge.
The maximum p a r a s i t a e m i a of
blood was observed 13 DPI.
At t h i s time the % f a l l
which was not s t a t i s t i c a l l y
significant.
in the PCV was -5.94±3.09%
significant
(p
The twelve animals in t h i s
The rate of p a r a s i t e m u l t i p l i c a t i o n
observed in Groups i ,
2 and 6 in experiment 2.
culated,
reduced
the
viable
effect
was s i m i l a r
to t h a t
There was also a delay in onset
of the i r r a d i a t i o n
parasites
in the PCV
By 25 DPI the
group were only m i l d l y
affected clinically.
of p a r a s i t a e m i a due to the l e t h a l
parasites/mm 3
However the maximum f a l l
of -19.07±2.71% 16 DPI was s t a t i s t i c a l l y PCV had returned to normal.
17.33±6.92
in
the
inoculum,
which, i t from
was c a l -
i x 108
to
2.5 x 103. Secondary c h a l l e n g e . in
Significant
differences
in the maximum % PCV f a l l
the maximum p a r a s i t a e m i a between the c o n t r o l
observed.
The maximum p a r a s i t a e m i a was observed in
both groups 6 DPI, being
6500±2843 parasites/mm 3 blood and 89.67±28.47 parasites/mm 3 blood f o r and vaccinated groups r e s p e c t i v e l y
(p
was -27.94±1.27% and -12.29±2o54% f o r ively
(p<0.0025).
The maximum % f a l l
control in
-13.09±3.77% 8 DPI, w h i l e the maximum % f a l l -55.92±4.45%
I0
DPI
(p
and
and vaccinated animals were
The % f a l l
control
in PCV on t h i s
day
and vaccinated groups r e s p e c t -
PCV in
the
vaccinated
in PCV of the c o n t r o l
The vaccinated
animals were not
group was group was clinically
a f f e c t e d during the course of the challenge whereas, although none of the control
animals d i e d , a l l were severely c l i n i c a l l y
a f f e c t e d and t h r e e had cerebral
symptoms, severe muscle wastage, t e t a n i c spasms, laboured r e s p i r a t i o n ,
ataxia,
anorhexia and haemoglobinuria, these signs being considered to be i n d i c a t i v e of terminal
babesiosis.
These animals recovered a f t e r about 6 weeks.
normal in vaccinated animals 15 DPI.
The PCV was
These data are d e t a i l e d in Table 4.
Control experiment All calves in Groups 8, 9 and 10 died and the maximumparasitaemia was sign i f i c a n t l y greater (p
There was no significant difference between PCV
598
TABLE 4 Experiment 3 - comparison of r e a c t i o n s between animals i n o c u l a t e d w i t h i r r a d i ated and n o n - i r r a d i a t e d
B. bovis s t r a i n s
Group 11 Inoculum d o s e / s t r a i n Inoculum type Maximum* p a r a s i t a e m i a Maximum % PCV change Number of s u r v i v o r s Challenge d o s e / s t r a i n Maximum* p a r a s i t a e m i a Maximum PCV % change Number of s u r v i v o r s
Group 12
i x I08L 350 Gy 17.33 -19.07 12/12 I x I08C 89.67 -13.09 12/12
i x I08C 6.5 x 103 -55.92 4/4
* Maximum p a r a s i t a e m i a = number of parasites/mm 3 b l o o d .
fall
or maximum p a r a s i t a e m i a in the c a l f
that
received
indicating
irradiated
that
organisms
Groups 8, 9 and 10 but the two groups
had an extended s u r v i v a l
time
of
4-6 days
(a) the organisms were a t t e n u a t e d and (b) the p a r a s i t e s did not
revert
to v i r u l e n c e
liquid
n i t r o g e n vapour.
after
a p e r i o d of
i0 months in e i t h e r
donor animals or in
These data are summarised in Table 5.
TABLE 5 Control
experiment
comparison
inoculated with irradiated Group
of
reactions
and n o n - i r r a d i a t e d
between
splenectomised
calves
B. b o v i s .
Inoculum dose/strain
Inoculum type
Maximum* Parasitaemia
Maximum % PCV change
Day of Death
Number of Survivors
1x107L 1x107L ixlO?L
350 Gy
1.5x10 s 1.06x10 s 1.46xi0 s
-48.62 -47.58 -55.06
13-14 8-9 13-14
0/4 0/4 0/4
8 9 10
(350 Gy) a f t e r i0 mths in c a r r i e r c a t t l e
* Maximum p a r a s i t a e m i a = number of parasites/mm 3 b l o o d .
DISCUSSION These generally
experiments
showed t h a t
produced a m i l d ,
B.
transient
bovis
parasites
disease
in
irradiated
with
non-immune c a t t l e .
p a t e n t p e r i o d was p r o l o n g e d , but the p a r a s i t e d o u b l i n g time was s i m i l a r observed
with
non-irradiated
parasites
of
the
same i s o l a t e .
i n c u b a t i o n p e r i o d was due t o the r e d u c t i o n of the i n f e c t i v e i
x 108 to
2.5
x 103 as a r e s u l t
of
irradiation.
350 Gy The p r e to t h a t
The prolonged
p a r a s i t e dose from
Infection
of
susceptible
animals with 2.5 x 103 non-irradiated parasites of the parent i s o l a t e k i l l e d
599
all
animals and both the prepatent period and the p a r a s i t e d o u b l i n g time were
similar
to t h a t
observed in the group r e c e i v i n g I x 108 i r r a d i a t e d
parasites,
but the maximum p a r a s i t a e m i a and PCV d e c l i n e were s i g n i f i c a n t l y
higher.
indicated
but
that
irradiation
produced a predominantly
normal p a r a s i t e p o p u l a t i o n . lent
form
The f a i l u r e
10 months a f t e r
suggests t h a t avirulent
irradiation
strains
for
a single may well
avirulent
otherwise
of p a r a s i t e s to r e v e r t to a more v i r u -
infection
of
susceptible
be a new and e f f e c t i v e
vaccination.
This
In a d d i t i o n ,
cattle
further
means of producing
parasites
stored
in
liquid
n i t r o g e n vapour f o r 10 months maintained t h e i r a v i r u l e n t s t a t e . Other evidence t h a t
irradiated
p a t h o p h y s i o l o g i c a l studies
p a r a s i t e s were a v i r u l e n t
(Wright et a l . ,
was obtained from
1980) which showed t h a t there was no
involvement of the c o a g u l a t i o n - k i n i n system in animals i n f e c t e d w i t h the i r r a d iated strains, ated s t r a i n s irradiated
in c o n t r a s t to the animals i n f e c t e d w i t h the parent n o n - i r r a d i -
which showed marked involvement of these systems. and n o n - i r r a d i a t e d
maximum p a r a s i t a e m i a l e v e l s effect
parasites indicating
had s i m i l a r that
is not r e l a t e d as much to red c e l l
However, both
multiplication
virulence
rates
damage by emerging p a r a s i t e s ,
damage by o t h e r means such as release of p r o t e o l y t i c
and
and hence p a t h o l o g i c a l as to
enzymes (Wright et a l . ,
1981). The data obtained obtained
with
(Purnell
et a l . ,
in
these experiments are in
irradiated
B.
major
(Brocklesby
et
broad agreement w i t h those al.,
1972),
B.
divergens
1977) and B. bovis (Halachera and Kasarizova, 1977).
these workers found t h a t a dose of 400 ot 500 Gy was l e t h a l t h a t these dead suspensions were non-immunogenic.
All
of
to p a r a s i t e s and
Mahoney et a l .
(1973) how-
ever found t h a t some B. bovis p a r a s i t e s did survive a dose of 500 Gy and a f t e r a mild disease the animal recovered spontaneously. that
an i r r a d i a t i o n
inoculation
into
dose of
animals,
strong immune response. irradiated
parasites
immune response
a mild
transient
disease which
is
that
cause,
followed
on
by a
In the c u r r e n t experiment the animals immunised w i t h
displayed
after
There is general concensus
around 350 Gy produces p a r a s i t e s
the
a strong
intravenous
immunity.
The f a i l u r e
administration
of
to
induce an
antigenic
material
e q u i v a l e n t to I x 1010 dead B. bovis organisms has been demonstrated (Mahoney et a l . , al.
1973; Mahoney and Wright,
(1978),
Purnell
1976).
Thus the suggestions of Purnell
and Lewis (1981) and Lewis et a l .
et
(1979) t h a t i r r a d i a t i o n
of B. major and B. divergens induced immunity p a r t l y by the simultaneous i n j e c t i o n of a small number of v i a b l e p a r a s i t e s and p a r t l y by a l a r g e number of dead parasites small
are
difficult
to
substantiate.
number of v i a b l e a v i r u l e n t
These workers probably
p a r a s i t e s by i r r a d i a t i o n
immunogenic by v i r t u e of t h e i r a b i l i t y
selected
a
which were s t r o n g l y
to m u l t i p l y in the host.
600
The i n f e c t i o n
of a l l
f o u r animals in the group 5 w i t h
1 x 107 i r r a d i a t e d
organisms subcutaneously is an encouraging r e s u l t even though the experimental group was s m a l l . (s/c)
of
calves
Callow
blood vaccine
was
i
infectivity
x
107 .
(1971)
determined t h a t
attenuated Thus
by m u l t i p l e
parasites
the optimum i n f e c t i v e
passage through
attenuated
r e q u i r e d f o r development as a l i v i n g
by
may induce
against highly virulent group, the f i r s t
a strong
is
of
the
immunity of
at
of B. bovis attenuated
least
12 months d u r a t i o n
heterologous c h a l l e n g e , the death of two animals in one The i n i t i a l
IgM antibody
predominant
protective
Mahoney
al.
et
have
during immunisation and the second a f t e r the challenge i n f e c -
t i o n cannot be o v e r l o o k e d . infection
splenectomised
irradiation
vaccine.
Although these experiments have shown t h a t a s t r a i n by i r r a d i a t i o n
dose
(1979)
type,
antibody have
immune response of bovines to B. bovis
whilst
class
is
within IgG
6 months of
(Mahoney,
infection
1972),
shown t h a t passive p r o t e c t i o n
the
Futhermore,
may be conferred
a g a i n s t homologous but not a g a i n s t heterologous B. bovis p a r a s i t e s .
Thus the
failure
only
to
attributed
induce to
experiment w i t h the
later
protective
a poor
immunity
in
these
immune response by them.
12 animals,
it
was shown t h a t
two
animals
However,
in
both the i n i t i a l
heterologous challenge produced only mild r e a c t i o n s
Further studies on the e f f e c t
of i r r a d i a t i o n
can
repeating infection in a l l
be this and
animals.
on B. bovis are warranted.
REFERENCES Brocklesby, D.W., P u r n e l l , R.E. and s e l l w o o d , S.A., 1972. The e f f e c t of i r r a d i a t i o n on i n t r a e r y t h r o c y t i c stages of Babesia major. Br. Vet. J . , 1 1 8 : i i i v. Callow, L . L . , 1971. The c o n t r o l of babesiosis w i t h a h i g h l y i n f e c t i v e , a t t e n u ated vaccine. Proc. 19th World Vet. Congr., 1:357-360. Goodger, B.V. and Mahoney, D.F., 1974. Evaluation of the passive haemagglutina t i o n t e s t f o r the diagnosis of Babesia a r g e n t i n a i n f e c t i o n in c a t t l e . Aust. Vet. J . , 50:246-249. Halacheva, M. and K a r a r i z o v a , L . , 1977. E f f e c t of i o n i z i n g r a d i a t i o n on the v i r u l e n c e and the immunogenic p r o p e r t i e s of Babesia o v i s . Vet. Med. Nauki, 14:32-38. Lewis, D., P u r n e l l , R.E. and Brocklesby, D.W., 1979. Babesia divergens: the immunisation of splenectomised calves using i r r a d i a t e d p i r o p l a s m s . Res. Vet. S c i . , 25:220-222. Mahoney D.F., 1972. Immune response to hemoprotozoa. I I . Babesia spp. In: Immunity to Animal P a r a s i t e s . E.J.L. Soulsby ( E d i t o r ) , Academic Press, New York, pp.-30-1---]~T~. Mahoney, D.F., K e r r , J . D . , Goodger, B.V. and Wright, I . G . , 1979. The immune response of c a t t l e to Babesia bovis (syn. B. a r g e n t i n a ) . Studies on the nature and s p e c i f i c i t y of p r o t e c t i o n . I n t . J. P a r a s i t o l . 9:297-306. Mahoney, D.F. and Saal, J . R . , 1961. B o v i ~ a - b - e - s i o s i s : t h i c k blood f i l m s f o r the d e t e c t i o n of p a r a s i t a e m i a . Aust. vet. J. 37:44-47. Mahoney, D.F, and Wright, loG., 1976. Babesia a r g e n t i n a : immunization of c a t t l e w i t h a k i l l e d antigen a g a i n s t i n f e c t i o n w i t h a heterologous s t r a i n . Vet. P a r a s i t o l . 2:273-282.
601 Mahoney, D.F., Wright, I.G., and Ketterer, P.J., 1973. Babesia argentina: the i n f e c t i v i t y and immunogenicity of i r r a d i a t e d blood p ~ s for splenectomized calves. I n t . J. P a r a s i t o l , 3:209-217. Purnell, R.E., Brocklesby, D.W., Stark, A.J. and Young, E.R., 1977. Babesia divergens: reactions of splenectomized calves to the inoc u la t io n of t ~ r a t e d or i r r a d i a t e d piroplasms. Parasitology, 75:xxxi. Purnell, R.E., Brocklesby, D.W. and Stark, A.J., 1978. Protection of c a t t l e against Babesia major by the inoculation of i r r a d i a t e d piroplasms. Res. Vet. Sci. 25:388-390. Purnell, R.E. and Lewis, D., 1981, Babesia divergens: combinationof dead and l i v e parasites in an i r r a d i a t e d vaccine. Res. Vet. Sci. 30:18-21. Wright, I.G., Goodger, B.V, and Mahoney, D.F., 1980, The i r r a d i a t i o n of Babesia bovis. 2. The difference in pathogenicity between i r r a d i a t e d and non-irradiated populations. Z. Parasitenkde, 63:47-57. Wright,I.G., Goodger, B.V. and Mahoney, D,F., 1 9 8 1 . Virulent and a v i r u l e n t strains of Babesia bovis: the r e l a t i o n s h i p between parasite protease content and p a t h o ~ o ~ a l effect of the s t r a i n . J. Protozool, 28:118-120.
591
THE IRRADIATION OF BABESIA BOVIS. I I . THE IMMUNOGENICITY OF IRRADIATED BLOOD PARASITES FOR INTACT CATTLE AND SPLENECTOMISED CALVES I.G. WRIGHTI , D.F. MAHDNEY1, G.B. MIRREI , B.V. GOODGERI and J.D. KERR2 ICSIRO, D i v i s i o n Indooroopilly, 2CSIRD, D i v i s i o n P.B.No.3, P.O.,
of Animal H e a l t h , Long Pocket L a b o a t o r i e s , P.B.No.3, P.O., Quensland, AUSTRALIA 4068. of Mathematics and S t a t i s t i c s , Long Pocket L a b o r a t o r i e s , I n d o o r o o p i l l y , Queensland, AUSTRALIA 4068.
(Accepted 2 June 1982) ABSTRACT Wright, I . G . , Mahoney, D.F., M i r r e , G.B., Goodger, B.V. and K e r r , J.D. 1982. The i r r a d i a t i o n of Babesia b o v i s . I I . The immunogenicity of i r r a d i a t e d blood p a r a s i t e s f o r i n t a c t c a t t l e and splenectomised calves. Vet. Immunol. Immunopathol., 3: 591-601. I n t r a e r y t h r o c y t i c forms of B. bovis were exposed to 350 Grays (Gy) y i r r a d i a t i o n and were then i n j e c t e d i n t r a v e n o u s l y i n t o i n t a c t two and t h r e e year old Hereford s t e e r s . One of 15 steers died on i n i t i a l i n f e c t i o n and subsequently s i x steers were given a v i r u l e n t heterologous challenge t h r e e weeks a f t e r recovery; a l l s i x animals were h i g h l y immune. The remaining e i g h t animals were kept under quarantine c o n d i t i o n s f o r i0 months and were then challenged w i t h a d i f f e r e n t v i r u l e n t heterologous s t r a i n of B. b o v i s . Seven of e i g h t were h i g h l y immune, but one animal d i e d . Subsequently a f u r t h e r 12 steers were i n j e c t e d i n t r a v e n o u s l y w i t h i x 108 i r r a d i a t e d organisms. A l l showed only m i l d t r a n s ient clinical signs. A f t e r 12 months quarantine in a t i c k - f r e e area these animals were then challenged w i t h a v i r u l e n t heterologous s t r a i n and a l l 12 were shown to be h i g h l y immune. I r r a d i a t i o n reduced the i n f e c t i v e dose from i x I0 U t o 2.5 x 103 p a r a s i t e s . These p a r a s i t e s m u l t i p l i e d at the same r a t e , and achieved the same maximum p a r a s i t a e m i a as the parent n o n - i r r a d i a t e d s t r a i n , but the disease produced by them was not severe. A dose of 2.5 x i0 s noni r r a d i a t e d p a a s i t e s was l e t h a l to a l l of the four animals which received i t . I t was concluded t h a t i r r a d i a t i o n had produced a predominantly a v i r u l e n t parasite population. INTRODUCTION In a previous paper Mahoney et a l .
(1973)
reported t h a t B. bovis p a r a s i t e s
i r r a d i a t e d w i t h doses between 200 and 500 Gy produced only a mild disease t h a t left
the
recipient
multiplication
animals
strongly
immune to
reinfection.
rate of these organisms was reduced.
In a d d i t i o n
the
These data i n d i c a t e d t h a t
p a r a s i t e s were a t t e n u a t e d by exposure to r a d i a t i o n doses of a p p r o x i m a t e l y 300350 Gy but were s t i l l
capable of immunising s u s c e p t i b l e animals.
the experiments r e p o r t e d here were designed to gain a d d i t i o n a l their
Consequently i n f o r m a t i o n on
immunogenicity and to determine the l o n g e v i t y of immunity in the h o s t ,
0165-2427/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing C o m p a n y
592
MATERIALS AND METHODS Experimental Animals Calves and a d u l t c a t t l e
of mixed breeds (Bos taurus) and sexes obtained from
a B a b e s i a - f r e e area were used in these experiments. the
P r i o r to commencement of
experiments the calves were splenectomised and a l l
ically
negative
to
B.
bovis
using
the
passive
animals were s e r o l o g -
haemagglutination
test
(PHA)
(Goodger and Mahoney, 1974). The p a r a s i t e s The
Lismore
(L)
(Mahoney et a l . , iated
strain
of
B.
boris,
described
1973) was used f o r i r r a d i a t i o n .
parasites
were challengd with v i r u l e n t
("S" and "C" s t r a i n s ) . from a n a t u r a l l y
previous
experiments
heterologous s t r a i n s
of B, bovis
The "C" s t r a i n was r e c e n t l y i s o l a t e d at t h i s
transmitted lethal
served as s t a b i l a t e s
in
Animals immunised w i t h i r r a d -
infection.
All
and stored in l i q u i d n i t r o g e n vapour,
storage they were passaged in a donor c a l f
laboratory
p a r a s i t e s t r a i n s were preA f t e r recovery from
from which the i n f e c t i v e
dose was
then o b t a i n e d . P r e p a r a t i o n and i r r a d i a t i o n Blood w i t h
at
least
splenectomised calves anticoagulant
of i n f e c t e d blood
5 x 107 p a r a s i t e s / m l
into
blood was withdrawn from donor
I% EDTA (di-sodium s a l t )
and dispensed in
in p h y s i o l o g i c a l
1-oz screw-cap b o t t l e s
s a l i n e as
in amounts of
10 ml at
4°Co The blood was i r r a d i a t e d Establishment, Sutherland, during
irradiation,
and i n o c u l a t i o n
NSW.
It
was held at 4°C during t r a n s i t
and was exposed at
Gy/min from a 60Co source. control
at the A u s t r a l i a n Atomic Energy Commission Research rates
that
varied
The periods between c o l l e c t i n g
by a i r and
between 6 and 10 the i n f e c t e d blood
i n t o the t e s t animals did not exceed 10-12 h.
Non-irradiated
samples of i n f e c t e d blood were also included in each package.
I n f e c t i o n of Animals Steers were g e n e r a l l y i n f e c t e d 1 x 108 i r r a d i a t e d was
taken d a i l y
(Mahoney parasites/
and
or n o n - i r r a d i a t e d
f o r packed c e l l
Saal,
mm3 b l o o d .
method described
intravenously
1961)
which
w i t h a standard dose of
p a r a s i t e s in whole b l o o d .
Jugular blood
volume (PCV) e s t i m a t i o n and t h i c k were
examined
to
estimate
blood f i l m s
the
number
of
P a r a s i t e doubling time was estimated according to the
by Mahoney et
i x 107 p a r a s i t e s i / v .
(i/v)
al,
(1973).
Splenectomised
calves
received
593
Experimental design Experiment 1.
F o u r groups of splenectomised, three month old calves each
composed of four animals wee infected with 1 x 108 B. bovis "L" strain parasites exposed to either 200, 300, 400 or 500 Gy, and the f i f t h group received i x 108 non-irradiated parasites of the parent strain.
Experiment 2 :
li)
Primary i n f e c t i o n .
Two year old non-splenectomised Here-
ford steers were used e x c l u s i v e l y in t h i s experiment. (a)
Group 1.
strain)
Six animals were inoculated i / v
which had been i r r a d i a t e d with 350 Gy.
enged three
weeks a f t e r
recovery with
i
with
1 x 108 parasites
(L
These animals were then c h a l l -
x 108 n o n - i r r a d i a t e d
parasites
(S
strain). (b) Group 2. Six animals were inoculated i / v with 1 x 108 non-irradiated parasites (L s t r a i n ) .
This was the parent strain of that used for production
of irradiated parasites. (c) Group 3.
Four animals were inoculated i / v with 2.5 x 103 non-irradiated
parasites (L s t r a i n ) .
T h i s dose was calculated to be equivalent to the surviv-
ing parasites after i r r a d i a t i o n of i x 108 v i r u l e n t organisms with 350 Gy. (d) Group 4.
Six control animals were inoculated i / v with 1 x 108 virulent
"S" strain parasites. (e) Group 5.
Four animals were infected subcutaneously (s/c) with i x 107
B. bovis "L" strain irradiated parasites which had been stored in l i q u i d n i t r o gen vapour for 10 months and had then been passaged through a splenectomised susceptible calf from which the inoculum was obtained. (ii)
Secondary challenge.
Hereford steers purchased at or i n i t i a l l y vaccin-
ated with irradiated organisms at the same time as those used in the primary infection experiment,
and subsequently maintained under t i c k - f r e e quarantine
conditions were used in this experiment. (a) Group 6.
Ten steers were inoculated i / v with i x 108 irradiated para-
sites (L strain) at the same time as Group 1.
One of these animals died from
the infection and a second injured i t s e l f and was destroyed.
The eight remain-
ing animals were then sent to a t i c k - f r e e quarantine area for 10 months. Upon return to the laboratory the sera of these animals were tested for B. bovis antibodies using the PHA test and all were found to be positive.
A 75 ml
sample of whole blood was withdrawn from each animal, mixed with 7.5 ml of i% sodium c i t r a t e and the pooled sample was then injected i / v into a susceptible splenectomised c a l f .
The c a t t l e were then challenged i / v with i x 108 virulent
heterologous B. bovis (C strain) parasites obtained from the donor c a l f . (b) Group 7. parasites.
Five control animals were infected i / v with 1 x 108 "C" strain
594
Experiment 3: ised
Hereford
with
1 x 108 B.
li)
Primary i n f e c t i o n .
steers
were used
bovis
"L"
(Group
strain
Secondary i n f e c t i o n . (Group i i )
ii).
These animals were i n f e c t e d
parasites
animals were then sent to a t i c k - f r e e (ii)
Twelve t w o - y e a r old non-splenectomirradiated
with
350 Gy.
i/v These
q u a r a n t i n e area f o r 12 months.
A f t e r the 12 month q u a r a n t i n e p e r i o d the 12 vacc-
inated
steers
and f o u r
susceptible
control
t h r e e - y e a r old Hereford
steers
(Group 12) were i n o c u l a t e d i / v w i t h i x 108 v i r u l e n t
B. bovis "C" s t r a i n
parasites. Control
experiments.
(a)
Group 8.
month old c a l v e s were i n f e c t e d
i/v
been s t o r e d
for
irradiated strain
in
liquid
nitrogen
parasites
with
splenectomised t h r e e -
i x 107 i r r a d i a t e d
p a r a s i t e s which had
10 months, to determine whether storage of
had any e f f e c t
as compared to i r r a d i a t e d
Four s u s c e p t i b l e
on the
stability
and i n f e c t i v i t y
of
the
organisms d e r i v e d from donor animals as used
in Control Group i 0 . (b) Group 9. strain
genicity (c) bovis
Four splenectomised calves were i n f e c t e d
non-irradiated
parasites,
of the s t r a i n Group
i0.
parasites
as a c o n t r o l
with
i x 107 " L '
to compare i n f e c t i v i t y
and patho-
compared to the i r r a d i a t e d
Four splenectomised obtained
calves
strains
in Groups 8 and 10.
were i n f e c t e d
from a donor splenectomised c a l f
the pooled blood sample from the Group 7 c a t t l e . determine whether the i r r a d i a t e d after
i/v
This
with
i
x
107 B.
which had received
group was a c o n t r o l
p a r a s i t e s had m a i n t a i n e d t h e i r
avirulent
to
state
i0 months in donor a n i m a l s .
RESULTS Experiment 1 The p a r a s i t e d o u b l i n g times were 8.23±1.25, erent.
No p a r a s i t e s
parasites time i0, 16.
in
in the 200, 300 and 400 Gy groups r e s p e c t i v e l y
10.10±0.92 and 9.50±0.20 hrs which were not s t a t i s t i c a l l y were d e t e c t e d
and these
animals
the c o n t r o l
and animals
in
The i n c r e a s e
in
survived
the the
group was 7.90 hrs. the
200,
The c o n t r o l
time was s i g n i f i c a n t
500 Gy i r r a d i a t e d
The p a r a s i t e animals a l l
300 and 400 Gy groups died
in s u r v i v a l
these data an i r r a d i a t i o n
group r e c e i v i n g experiment.
diffdoubling
died by day
between days
(P
12 and
As a r e s u l t
of
dose of 35 krad was chosen f o r experiment 2.
Experiment 2 Primary
challenge.
regressed on time
for
The
log
each animal
time to reach one p a r a s i t e / t h i c k ised in Table I .
parasitaemia
of
Groups
and the estimates
film
field
I,
2,
and
6 were
of d o u b l i n g time and the
were made.
These data are summar-
595
No s i g n i f i c a n t in
Groups
sites).
i
differences
(irradiated
in m u l t i p l i c a t i o n
parasites),
Highly s i g n i f i c a n t
differences
group 2 animals compared w i t h Groups i All
2
rate were observed f o r animals
(control),
and
6
(irradiated
and 6 animals on days 5, 6, 7 and 8.
Group 2 animals died by day nine w h i l e one animal died in Group 6.
animals
all
had haemoglobinuria,
para-
(p
haemoglobinaemia, f e v e r ,
and a t a x i a and were not examined by post-mortem.
jaundice,
These
anorexia
These data are summarised in
Table 2. The s i x Group i animals which had received i r r a d i a t e d challenged w i t h 1 x 108 v i r u l e n t ery from the i n i t i a l
challenge.
were also
with
inoculated
recov-
Six two-year old s u s c e p t i b l e Hereford steers
the same challenge dose (Group 4).
obtained from the Group 1 animals
it
dose received was 2.5 x 103 p a r a s i t e s . relatively
p a r a s i t e s were then
heterologous B. bovis three weeks a f t e r
was estimated t h a t
From the data
the actual
parasite
Consequently, to determine whether the
m i l d disease observed in the i r r a d i a t e d group was dose r e l a t e d , four
two-year old s u s c e p t i b l e Hereford steers were i n o c u l a t e d i n t r a v e n o u s l y w i t h 2.5 x 103 of the o r i g i n a l
"L" s t r a i n
(Group 3).
All
four cattle
in t h i s group died
on days 10-12, TABLE 1 Expriment 2 - estimates of the m u l t i p l i c a t i o n sites/mm 3 blood f o r c o n t r o l Group
rate and time to reach I000 para-
(group 2) and i r r a d i a t e d groups (Groups i and 6).
M u l t i p l i c a t i o n rate/24 hr
Control (group 2) Irradiated (groups i and 6) 95% LSD Group I Group 6 95% LSD
Mean
S.E.
2.06 2.07 0.56 1.69 2.46 1.13
0.03 0.08 0.08 0.15
Time (days) to reach 1000 parasites/mm 3 blood Mean
S.E.
6.0 13.2 1.1 13.8 12.9 2.3
6,16 0.58 0.83 0.92
There is no s i g n i f i c a n t difference in m u l t i p l i c a t i o n rate; the overall mean is 2.067 which is equivalent to an 11.6 hr doubling time. The mean difference in time to reach 1000 parasites/mm3 blood is 7.2 days. Group i - 6 c a t t l e subsequently challenged three weeks a f t e r recovery. Group 6 - 9 c a t t l e , 8 of which survived and were subsequently challenged 10 months l a t e r . Although only one of the Group 3 control animals died, the PCV f a l l was s i g n i f i c a n t l y d i f f e r e n t (p
No s i g n i f i c a n t f a l l
in the PCV was
Parasite m u l t i p l i c a t i o n rates in a l l three groups were
s i m i l a r but the time to reach maximumparasitaemias was dose dependent.
Immune
596
TABLE 2 Packed Cell Volumes f o r c o n t r o l
(Group 2) and i r r a d i a t e d Groups ( i and 6).
Group I - 6 c a t t l e
r e c e i v i n g i r r a d i a t e d blood challenged three weeks l a t e r .
Group 6 - 8 c a t t l e
r e c e i v i n g i r r a d i a t e d blood challenged I0 months l a t e r .
Day
0 2 3 4 5 6 7 8 9 10 ii 12
Control
Group 2
I r r a d i a t e d Groups i and 6
Group 1
Group 6
Mean
S.E.
Mean
S.E.
Mean
S.E.
Mean
S.E.
44.3 45.3 46.5 43.8 38.5 33.5 30.1 24.0
1.17 0.80 1.38 0.95 0.75 1.38 1.51 2.30
47.4 46.7 45.8 46.2 45.3 44.6 44.9 42.1
1.09 0.81 0.69 0.86 0.84 1.02 0.92 1.12
47.3 45.8 45.3 45.9 45.2 43.7 40.8 39.8 38.6 34.3 31.0 30.6
1.74 1.01 1.38 1.32 1.47 1.80 1.67 1.85 1.80 2.17 1.62 2.03
47.4 46.7 46.1 46.2 45.4 45.1 42.9 41.3 39.7 35.7 31.4 28.9
1.47 1.17 0.97 1.18 1.07 1.31 1.43 1.31 1.11 1.41 1.52 1.74
Group I animals had maximum parasitaemias on day 7, the c o n t r o l 8 and c o n t r o l
Group 3 on day I0.
Group 4 on day
These data are summarised in Table 3.
All
animals in Group 5 which were i n o c u l a t e d s/c w i t h I x 107 i r r a d i a t e d p a r a s i t e s became i n f e c t e d and underwent m i l d r e a c t i o n s . Secondary c h a l l e n g e . with
irradiated
tick-free
Eight
two-year old steers which had been vaccinated
p a r a s i t e s s i m u l t a n e o u s l y w i t h group 1 were kept in q u a r a n t i n e ,
c o n d i t i o n s f o r 10 months (Group 6).
Together with the f i v e animals
TABLE 3 Experiment 2 - comparison of
reactions
between i n t a c t
cattle
inoculated with
i r r a d i a t e d and n o n - i r r a d i a t e d B. bovis s t r a i n s . Grp Inoculum Inoculum Maximum* Max. dose/ type p a r a s i t - % PCV strain aemia change 1 2 3 4 5 6 7
lxlOBL ixlOSL IxlOTL IxlOSL
350 Gy Live 350 Gy 350 Gy
* Maximum p a r a s i t a e m i a
7.24xi03 -34.46 1.02xlO" -59.8 1.50x105 -48.62 1.74xi03 -33.54
=
No. of Challenge Maximum* Max. No.of Survivdose/ p a r a s i t - % PCV Surviv ors strain aemia change ors 6/6 0/6 0/4 8/9
lx108S
18.7
2.5xlO3L lxlOBS
5xlO 4 -58.24 4.17xi03 -45.5
0/4 5/6
ixlOBC IxlOBC
1.75x103 -17.94 4.01xi04 -47.87
7/8 2/5
number of parasites/mm 3 blood.
-6.03
6/6
597
in control Group 7 these two groups were inoculated intravenously with a virulent heterologous strain of B. bovis (C).
Both the mean maximum PCV f a l l and
mean maximum parasitaemia of Group 7 was s i g n i f i c a n t l y d i f f e r e n t from that of the immune Group 6 (p
Three animals in Group 7
died on days 9 or i0 and one animal in the immune Group 6 died from classical symptoms of babesiosis on day 12. globinaemia,
These signs included haemoglobinuria, haemo-
tetanic spasms, ataxia, fever, anorexia and jaundice.
mortem was deemed not necessary.
A post
These data are summarised in Table 3.
Experiment 33 Primary
challenge.
The maximum p a r a s i t a e m i a of
blood was observed 13 DPI.
At t h i s time the % f a l l
which was not s t a t i s t i c a l l y
significant.
in the PCV was -5.94±3.09%
significant
(p
The twelve animals in t h i s
The rate of p a r a s i t e m u l t i p l i c a t i o n
observed in Groups i ,
2 and 6 in experiment 2.
culated,
reduced
the
viable
effect
was s i m i l a r
to t h a t
There was also a delay in onset
of the i r r a d i a t i o n
parasites
in the PCV
By 25 DPI the
group were only m i l d l y
affected clinically.
of p a r a s i t a e m i a due to the l e t h a l
parasites/mm 3
However the maximum f a l l
of -19.07±2.71% 16 DPI was s t a t i s t i c a l l y PCV had returned to normal.
17.33±6.92
in
the
inoculum,
which, i t from
was c a l -
i x 108
to
2.5 x 103. Secondary c h a l l e n g e . in
Significant
differences
in the maximum % PCV f a l l
the maximum p a r a s i t a e m i a between the c o n t r o l
observed.
The maximum p a r a s i t a e m i a was observed in
both groups 6 DPI, being
6500±2843 parasites/mm 3 blood and 89.67±28.47 parasites/mm 3 blood f o r and vaccinated groups r e s p e c t i v e l y
(p
was -27.94±1.27% and -12.29±2o54% f o r ively
(p<0.0025).
The maximum % f a l l
control in
-13.09±3.77% 8 DPI, w h i l e the maximum % f a l l -55.92±4.45%
I0
DPI
(p
and
and vaccinated animals were
The % f a l l
control
in PCV on t h i s
day
and vaccinated groups r e s p e c t -
PCV in
the
vaccinated
in PCV of the c o n t r o l
The vaccinated
animals were not
group was group was clinically
a f f e c t e d during the course of the challenge whereas, although none of the control
animals d i e d , a l l were severely c l i n i c a l l y
a f f e c t e d and t h r e e had cerebral
symptoms, severe muscle wastage, t e t a n i c spasms, laboured r e s p i r a t i o n ,
ataxia,
anorhexia and haemoglobinuria, these signs being considered to be i n d i c a t i v e of terminal
babesiosis.
These animals recovered a f t e r about 6 weeks.
normal in vaccinated animals 15 DPI.
The PCV was
These data are d e t a i l e d in Table 4.
Control experiment All calves in Groups 8, 9 and 10 died and the maximumparasitaemia was sign i f i c a n t l y greater (p
There was no significant difference between PCV
598
TABLE 4 Experiment 3 - comparison of r e a c t i o n s between animals i n o c u l a t e d w i t h i r r a d i ated and n o n - i r r a d i a t e d
B. bovis s t r a i n s
Group 11 Inoculum d o s e / s t r a i n Inoculum type Maximum* p a r a s i t a e m i a Maximum % PCV change Number of s u r v i v o r s Challenge d o s e / s t r a i n Maximum* p a r a s i t a e m i a Maximum PCV % change Number of s u r v i v o r s
Group 12
i x I08L 350 Gy 17.33 -19.07 12/12 I x I08C 89.67 -13.09 12/12
i x I08C 6.5 x 103 -55.92 4/4
* Maximum p a r a s i t a e m i a = number of parasites/mm 3 b l o o d .
fall
or maximum p a r a s i t a e m i a in the c a l f
that
received
indicating
irradiated
that
organisms
Groups 8, 9 and 10 but the two groups
had an extended s u r v i v a l
time
of
4-6 days
(a) the organisms were a t t e n u a t e d and (b) the p a r a s i t e s did not
revert
to v i r u l e n c e
liquid
n i t r o g e n vapour.
after
a p e r i o d of
i0 months in e i t h e r
donor animals or in
These data are summarised in Table 5.
TABLE 5 Control
experiment
comparison
inoculated with irradiated Group
of
reactions
and n o n - i r r a d i a t e d
between
splenectomised
calves
B. b o v i s .
Inoculum dose/strain
Inoculum type
Maximum* Parasitaemia
Maximum % PCV change
Day of Death
Number of Survivors
1x107L 1x107L ixlO?L
350 Gy
1.5x10 s 1.06x10 s 1.46xi0 s
-48.62 -47.58 -55.06
13-14 8-9 13-14
0/4 0/4 0/4
8 9 10
(350 Gy) a f t e r i0 mths in c a r r i e r c a t t l e
* Maximum p a r a s i t a e m i a = number of parasites/mm 3 b l o o d .
DISCUSSION These generally
experiments
showed t h a t
produced a m i l d ,
B.
transient
bovis
parasites
disease
in
irradiated
with
non-immune c a t t l e .
p a t e n t p e r i o d was p r o l o n g e d , but the p a r a s i t e d o u b l i n g time was s i m i l a r observed
with
non-irradiated
parasites
of
the
same i s o l a t e .
i n c u b a t i o n p e r i o d was due t o the r e d u c t i o n of the i n f e c t i v e i
x 108 to
2.5
x 103 as a r e s u l t
of
irradiation.
350 Gy The p r e to t h a t
The prolonged
p a r a s i t e dose from
Infection
of
susceptible
animals with 2.5 x 103 non-irradiated parasites of the parent i s o l a t e k i l l e d
599
all
animals and both the prepatent period and the p a r a s i t e d o u b l i n g time were
similar
to t h a t
observed in the group r e c e i v i n g I x 108 i r r a d i a t e d
parasites,
but the maximum p a r a s i t a e m i a and PCV d e c l i n e were s i g n i f i c a n t l y
higher.
indicated
but
that
irradiation
produced a predominantly
normal p a r a s i t e p o p u l a t i o n . lent
form
The f a i l u r e
10 months a f t e r
suggests t h a t avirulent
irradiation
strains
for
a single may well
avirulent
otherwise
of p a r a s i t e s to r e v e r t to a more v i r u -
infection
of
susceptible
be a new and e f f e c t i v e
vaccination.
This
In a d d i t i o n ,
cattle
further
means of producing
parasites
stored
in
liquid
n i t r o g e n vapour f o r 10 months maintained t h e i r a v i r u l e n t s t a t e . Other evidence t h a t
irradiated
p a t h o p h y s i o l o g i c a l studies
p a r a s i t e s were a v i r u l e n t
(Wright et a l . ,
was obtained from
1980) which showed t h a t there was no
involvement of the c o a g u l a t i o n - k i n i n system in animals i n f e c t e d w i t h the i r r a d iated strains, ated s t r a i n s irradiated
in c o n t r a s t to the animals i n f e c t e d w i t h the parent n o n - i r r a d i -
which showed marked involvement of these systems. and n o n - i r r a d i a t e d
maximum p a r a s i t a e m i a l e v e l s effect
parasites indicating
had s i m i l a r that
is not r e l a t e d as much to red c e l l
However, both
multiplication
virulence
rates
damage by emerging p a r a s i t e s ,
damage by o t h e r means such as release of p r o t e o l y t i c
and
and hence p a t h o l o g i c a l as to
enzymes (Wright et a l . ,
1981). The data obtained obtained
with
(Purnell
et a l . ,
in
these experiments are in
irradiated
B.
major
(Brocklesby
et
broad agreement w i t h those al.,
1972),
B.
divergens
1977) and B. bovis (Halachera and Kasarizova, 1977).
these workers found t h a t a dose of 400 ot 500 Gy was l e t h a l t h a t these dead suspensions were non-immunogenic.
All
of
to p a r a s i t e s and
Mahoney et a l .
(1973) how-
ever found t h a t some B. bovis p a r a s i t e s did survive a dose of 500 Gy and a f t e r a mild disease the animal recovered spontaneously. that
an i r r a d i a t i o n
inoculation
into
dose of
animals,
strong immune response. irradiated
parasites
immune response
a mild
transient
disease which
is
that
cause,
followed
on
by a
In the c u r r e n t experiment the animals immunised w i t h
displayed
after
There is general concensus
around 350 Gy produces p a r a s i t e s
the
a strong
intravenous
immunity.
The f a i l u r e
administration
of
to
induce an
antigenic
material
e q u i v a l e n t to I x 1010 dead B. bovis organisms has been demonstrated (Mahoney et a l . , al.
1973; Mahoney and Wright,
(1978),
Purnell
1976).
Thus the suggestions of Purnell
and Lewis (1981) and Lewis et a l .
et
(1979) t h a t i r r a d i a t i o n
of B. major and B. divergens induced immunity p a r t l y by the simultaneous i n j e c t i o n of a small number of v i a b l e p a r a s i t e s and p a r t l y by a l a r g e number of dead parasites small
are
difficult
to
substantiate.
number of v i a b l e a v i r u l e n t
These workers probably
p a r a s i t e s by i r r a d i a t i o n
immunogenic by v i r t u e of t h e i r a b i l i t y
selected
a
which were s t r o n g l y
to m u l t i p l y in the host.
600
The i n f e c t i o n
of a l l
f o u r animals in the group 5 w i t h
1 x 107 i r r a d i a t e d
organisms subcutaneously is an encouraging r e s u l t even though the experimental group was s m a l l . (s/c)
of
calves
Callow
blood vaccine
was
i
infectivity
x
107 .
(1971)
determined t h a t
attenuated Thus
by m u l t i p l e
parasites
the optimum i n f e c t i v e
passage through
attenuated
r e q u i r e d f o r development as a l i v i n g
by
may induce
against highly virulent group, the f i r s t
a strong
is
of
the
immunity of
at
of B. bovis attenuated
least
12 months d u r a t i o n
heterologous c h a l l e n g e , the death of two animals in one The i n i t i a l
IgM antibody
predominant
protective
Mahoney
al.
et
have
during immunisation and the second a f t e r the challenge i n f e c -
t i o n cannot be o v e r l o o k e d . infection
splenectomised
irradiation
vaccine.
Although these experiments have shown t h a t a s t r a i n by i r r a d i a t i o n
dose
(1979)
type,
antibody have
immune response of bovines to B. bovis
whilst
class
is
within IgG
6 months of
(Mahoney,
infection
1972),
shown t h a t passive p r o t e c t i o n
the
Futhermore,
may be conferred
a g a i n s t homologous but not a g a i n s t heterologous B. bovis p a r a s i t e s .
Thus the
failure
only
to
attributed
induce to
experiment w i t h the
later
protective
a poor
immunity
in
these
immune response by them.
12 animals,
it
was shown t h a t
two
animals
However,
in
both the i n i t i a l
heterologous challenge produced only mild r e a c t i o n s
Further studies on the e f f e c t
of i r r a d i a t i o n
can
repeating infection in a l l
be this and
animals.
on B. bovis are warranted.
REFERENCES Brocklesby, D.W., P u r n e l l , R.E. and s e l l w o o d , S.A., 1972. The e f f e c t of i r r a d i a t i o n on i n t r a e r y t h r o c y t i c stages of Babesia major. Br. Vet. J . , 1 1 8 : i i i v. Callow, L . L . , 1971. The c o n t r o l of babesiosis w i t h a h i g h l y i n f e c t i v e , a t t e n u ated vaccine. Proc. 19th World Vet. Congr., 1:357-360. Goodger, B.V. and Mahoney, D.F., 1974. Evaluation of the passive haemagglutina t i o n t e s t f o r the diagnosis of Babesia a r g e n t i n a i n f e c t i o n in c a t t l e . Aust. Vet. J . , 50:246-249. Halacheva, M. and K a r a r i z o v a , L . , 1977. E f f e c t of i o n i z i n g r a d i a t i o n on the v i r u l e n c e and the immunogenic p r o p e r t i e s of Babesia o v i s . Vet. Med. Nauki, 14:32-38. Lewis, D., P u r n e l l , R.E. and Brocklesby, D.W., 1979. Babesia divergens: the immunisation of splenectomised calves using i r r a d i a t e d p i r o p l a s m s . Res. Vet. S c i . , 25:220-222. Mahoney D.F., 1972. Immune response to hemoprotozoa. I I . Babesia spp. In: Immunity to Animal P a r a s i t e s . E.J.L. Soulsby ( E d i t o r ) , Academic Press, New York, pp.-30-1---]~T~. Mahoney, D.F., K e r r , J . D . , Goodger, B.V. and Wright, I . G . , 1979. The immune response of c a t t l e to Babesia bovis (syn. B. a r g e n t i n a ) . Studies on the nature and s p e c i f i c i t y of p r o t e c t i o n . I n t . J. P a r a s i t o l . 9:297-306. Mahoney, D.F. and Saal, J . R . , 1961. B o v i ~ a - b - e - s i o s i s : t h i c k blood f i l m s f o r the d e t e c t i o n of p a r a s i t a e m i a . Aust. vet. J. 37:44-47. Mahoney, D.F, and Wright, loG., 1976. Babesia a r g e n t i n a : immunization of c a t t l e w i t h a k i l l e d antigen a g a i n s t i n f e c t i o n w i t h a heterologous s t r a i n . Vet. P a r a s i t o l . 2:273-282.
601 Mahoney, D.F., Wright, I.G., and Ketterer, P.J., 1973. Babesia argentina: the i n f e c t i v i t y and immunogenicity of i r r a d i a t e d blood p ~ s for splenectomized calves. I n t . J. P a r a s i t o l , 3:209-217. Purnell, R.E., Brocklesby, D.W., Stark, A.J. and Young, E.R., 1977. Babesia divergens: reactions of splenectomized calves to the inoc u la t io n of t ~ r a t e d or i r r a d i a t e d piroplasms. Parasitology, 75:xxxi. Purnell, R.E., Brocklesby, D.W. and Stark, A.J., 1978. Protection of c a t t l e against Babesia major by the inoculation of i r r a d i a t e d piroplasms. Res. Vet. Sci. 25:388-390. Purnell, R.E. and Lewis, D., 1981, Babesia divergens: combinationof dead and l i v e parasites in an i r r a d i a t e d vaccine. Res. Vet. Sci. 30:18-21. Wright, I.G., Goodger, B.V, and Mahoney, D.F., 1980, The i r r a d i a t i o n of Babesia bovis. 2. The difference in pathogenicity between i r r a d i a t e d and non-irradiated populations. Z. Parasitenkde, 63:47-57. Wright,I.G., Goodger, B.V. and Mahoney, D,F., 1 9 8 1 . Virulent and a v i r u l e n t strains of Babesia bovis: the r e l a t i o n s h i p between parasite protease content and p a t h o ~ o ~ a l effect of the s t r a i n . J. Protozool, 28:118-120.