Combrink_113-117.qxd


INTRODUCTION

The South African bivalent unfrozen redwater vac-
cine containing Babesia bigemina and Babesia
bovis infected blood was sold until April 1998, when
it was finally discontinued and replaced by the two
frozen monovalent African (Babesia bigemina) and
Asiatic (Babesia bovis) redwater vaccines. 

Diminazene aceturate has been shown to be highly
effective against B. bigemina at levels from 0.5 to

3.0 mg/kg (Kutler 1981). Therefore, a third (1.16
mg/kg) of the prescribed dose of the antibabesial
drug diminazene has long been used to block-treat
the unfrozen redwater vaccine reactions on day 7
with no known adverse effects to the organisms or
to the development of protective immunity in the
animal (De Waal 1996). The attenuated organisms
used in the unfrozen redwater vaccine are suscep-
tible for longer periods to the residual effects of
diminazene and imidocarb dipropionate than the vir-
ulent field strains (F.T. Potgieter & M.P. Combrink,
unpublished observations 1982). In addition, the
inhibitory effect, especially of imidocarb, is more
pronounced in reactions following the administra-

113

Onderstepoort Journal of Veterinary Research, 71:113–117 (2004)

Effect of diminazene block treatment on
live redwater vaccine reactions

M.P. COMBRINK1 and P.C. TROSKIE

Parasitology Division, Onderstepoort Veterinary Institute
Private Bag X05, Onderstepoort, 0110 South Africa

ABSTRACT

COMBRINK, M.P. & TROSKIE, P.C. 2004. Effect of diminazene block treatment on live redwater
vaccine reactions. Onderstepoort Journal of Veterinary Research, 71:113–117

One third of the manufacturer’s prescribed dose of diminazene has long been used to block treat the
South African unfrozen Babesia bigemina and Babesia bovis (redwater) vaccine reactions, with no
known adverse effects. It is known that the inhibitory effect of antibabesial drugs is more pronounced
in animals inoculated with the frozen vaccine than those with the unfrozen vaccine. Reports of vac-
cine failures in some animals in which diminazene was used for block treatment of the reactions fol-
lowing inoculation with frozen South African redwater vaccine led us to reinvestigate the required
waiting period before treatment and the reduced dose necessary for successful treatment and devel-
opment of immunity. Results from febrile reactions in cattle following vaccination indicated day 7 as
the optimal day for administering block treatment. Treatment of B. bigemina vaccine reactions in cat-
tle on day 7 at a level of 0.35 mg/kg (1/10 fraction of the normal dose) diminazene killed all the para-
sites while B. bovis vaccine parasites survived treatment using diminazene at levels between 0.35
mg/kg and 1.16 mg/kg. However, various other factors, such as the degree of natural resistance of
different cattle breeds and individual animals, the accuracy of diminazene content according to the
manufacturer’s label claim and the accuracy of the drug dose administered, all influence the suc-
cessful immunization of animals. Consequently block treating of Babesia vaccines with diminazene
on day 7 after vaccination is not recommended.

Keywords: Babesia bigemina, Babesia bovis, babesiosis, diminazene, live redwater vaccine

1 E-mail: mike@moon.ovi.ac.za

Accepted for publication 10 November 2003—Editor



tion of the frozen South African B. bovis and B.
bigemina vaccines than in those of the unfrozen
vaccine (Combrink, Troskie & De Waal 2002). In a
recent study on 19 different brands of diminazene
obtained from 11 African countries, it was found
that approximately one out of every three samples
tested fell outside the ± 10 % tolerance limit of the
manufacturer’s label claim on the content of dimi-
nazene aceturate (Tettey, Atsriku, Chizyuka & Sling-
enberg 2002).

Based on these findings and reports of vaccine fail-
ures in some animals in which diminazene was used
for the block treatment of vaccine reactions (Com-
brink, personal observations 2001), it was decided
to reinvestigate the duration of the interval between
vaccination and treatment as well as the optimum
dosage rate of diminazene necessary for success-
ful treatment and development of immunity.

MATERIALS AND METHODS

Animals

Fifty-four fully susceptible 6- to 12-month-old intact
Ayershire and Friesian cattle, procured as calves
and raised in quarantine stables, were used in a
trial to determine the waiting period required before
administering diminazene after inoculation with the
frozen vaccine. The animals were selected random-
ly and were divided into two groups (Table 1). 

The efficacy of using reduced dosage rates of dim-
inazene to treat frozen vaccine reactions was deter-
mined in eight fully susceptible 20- to 30-month-old
splenectomized Hereford cattle born and bred in
the stables of the Parasitology Division of the Onder-
stepoort Veterinary Institute. They were divided into
four groups of two animals each (Table 2).

The effect of using reduced dosage levels of dimi-
nazene to block-treat frozen vaccine reactions was
assessed using twenty-four fully susceptible 12- to
22-month-old intact Hereford and Friesian cattle born
and bred in the stables of the Parasitology Division.
The animals were placed into six experimental, two
treated and two untreated control groups (Table 3).

All the animals were housed under tick-free condi-
tions before and for the duration of the experiments.

Drug administration, vaccination and
heterologous challenge

Accurately determined reduced doses of commer-
cially available diminazene (Berenil RTU, Intervet

SA) were administered to those experimental ani-
mals indicated in Tables 2 and 3.

The frozen Babesia vaccines that were used con-
tained the B. bovis South African “S” strain (De Vos
1978; Callow, Mellors & McGreggor 1979) and the
B. bigemina Australian “G” strain (Dalgleish, Callow,
Mellors & McGreggor 1981; De Vos, Combrink &
Bessenger 1982). The vaccines were thawed by
placing them directly from liquid nitrogen storage
onto melting ice in which they were kept for 4 h be-
fore intramuscular inoculation of 1 ml volumes into
those experimental animals indicated in Tables 1, 2
and 3.

The frozen heterologous blood stabilates that were
used to challenge the cattle were the B. bovis South
African “F” strain ( De Vos 1978) and the B. bigem-
ina South African unmodified “P” strain (De Vos et
al. 1982). At 120 days post vaccination, these frozen
stabilates were thawed directly in a container with
water at 37 °C and administered intramuscularly, in
quantities that contained 5 x 107 parasites before
freezing, into those animals indicated in Table 3.

Monitoring redwater reactions

Rectal temperatures, haematocrit levels and blood
smears were monitored daily (De Waal & Potgieter
1987). Antibodies against Babesia were determined
in sera collected before and 30 days after vaccina-
tion or challenge, using the indirect fluorescent anti-
body test technique (Gray & De Vos 1981).

Evaluation of infectivity and reactions

The criteria used to evaluate successful infectivity
were either a positive blood smear diagnosis or pos-
itive seroconversion following vaccination or chal-
lenge. Evaluation of the vaccine or challenge reac-
tions was based on a total reaction index score
determined during the reaction period for each ani-
mal, by adding one point scored for every 1 % para-
sitaemia, 1 % decline in packed red blood cell vol-
ume (De Vos 1978) and 1 °C in total temperature
rise above the mean pre-inoculation normal tem-
perature of the animal (Combrink, De Waal & Tros-
kie 1997). Five points were scored for other clinical
signs and ten points for every antibabesial drug
treatment, blood transfusion and death.

The unpaired t-test was used to determine whether
results obtained for the mean total reaction indexes
of the vaccinated and challenged groups differed
significantly (P < 0.05).

114

Diminazene block treatment on live redwater vaccine reactions



RESULTS AND DISCUSSION

Start of vaccine temperature reactions

Results from febrile responses in 12 of the 28 B. bi-
gemina vaccinated animals of group 1 (Table 1),
indicated day 7 as the mean starting time of tem-
perature rise > 39.5 °C. As some of the temperature
reactions to B. bovis in group 2 animals began as
early as day 6 and due to the fact that the two vac-
cines are genarally inoculated simultaneously, it was
decided that the waiting period required before block
treating of vaccine reactions should remain at 7
days. None of the animals in the two groups showed
any clinical signs of disease.

Treatment efficacy of reduced diminazene doses

Results obtained in this study using 0.5 and 0.35
mg/kg doses of diminazene to treat B. bigemina
and B. bovis vaccine reactions in the splenecto-
mized cattle proved to be quite effective at para-
sitaemia levels higher than those which normally
prevail in intact animals on day 7 after vaccination.
None of the animals in the four groups required any
additional treatment (Table 2).

Effect of block treatment on the development
of immunity

Babesia bigemina

No parasites could be demonstrated in the blood
smears of the experimental animals in groups 1, 2

and 3 after treatment of vaccine reactions on day 7
with 1.16, 0.58 or 0.35 mg/kg doses of diminazene
and none of the animals seroconverted (Table 3).
Attempts to detect the organisms by subinoculating
100 ml blood from each individual animal into sus-
ceptible splenectomized animals also proved nega-
tive.

Heterologous challenge of experimental animals
produced clinical disease requiring treatment in
50 % of the animals. This contributed to significant
differences between challenge and vaccination re-
action indices, indicating no or inadequate immune
response to the killed vaccine parasites. Both the
vaccinated untreated control animals (group 4) were
positive on blood smear examination and serology,
and showed no difference between mean challenge
and vaccination reaction index results. 

None of the heterologous strain-infected animals of
the unvaccinated treated control group 5 developed
clinical signs that required treatment and the result-
ing mean reaction index also showed no significant
differences when compared to those obtained for
the vaccinated control and experimental groups.
Nevertheless, all other results clearly indicate that
block treatment with diminazene on day 7 of the B.
bigemina vaccine reaction cannot be recommended.

Babesia bovis 

Parasites could still be demonstrated in the blood
smears of all of the experimental animals in groups

115

M.P. COMBRINK & P.C. TROSKIE

TABLE 1 Start of temperature reactions in cattle vaccinated with the live frozen Babesia bigemina and Babesia bovis blood vaccines

Vaccine Group No. of No. of animals with No. of animals Mean start time of temp-
no. animals blood smear positive showing temperature erature reactions > 39,5 °C 

reactions > 39,5 °C (day ± sd)

Babesia bigemina 1 28 28/28 12/28 7.08 ± 3.26

Babesia bovis 2 26 26/26 11/26 10.55 ± 4.87

TABLE 2 Efficacy of 0.50 and 0.35 mg/kg diminazene as treatment dose of Babesia bigemina and Babesia bovis frozen vaccine
reactions in splenectomized cattle

Vaccine Group No. of Diminazene dosage Mean parasitaemia Recovered animals not
No. animals mg/kg (fraction of  at treatment requiring additional 

normal dose) (% ± sd) treatment

Babesia bigemina 1 2 0.50   (1/7) 1.6 ± 1.13 2/2
2 2 0.35 (1/10) 0.8 ± 0.14 2/2

Babesia bovis 3 2 0.50   (1/7) 3.2 ± 3.11 2/2
4 2 0.35 (1/10) 0.9 ± 0.14 2/2



116

Diminazene block treatment on live redwater vaccine reactions

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6, 7 and 8 after treatment of vaccine reactions on
day 7 with 1.16, 0.58 or 0.35 mg/kg doses of dimi-
nazene and all seroconverted (Table 3). None of
these, nor the animals in control group 9 required
any treatment during the heterologous challenge
reaction and there were no significant differences to
be found between the respective challenge and vac-
cination reaction index results, indicating adequate
protection. However, various factors, such as the
degree of natural resistance of different cattle breeds
and individual animals to Babesia parasites, the
infectivity of frozen vaccine being less predictable
than that of unfrozen vaccine (due to the death of
parasites during freezing and thawing), different
procedures of vaccine administration, the accuracy
of diminazene content according manufacturer’s
label claim and the accuracy of the drug dose
administered, all influence the successful immuni-
zation of animals. Consequently the block treating
of B. bovis on day 7 of the vaccine reaction is also
not recommended.

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117

M.P. COMBRINK & P.C. TROSKIE