3.Eg9pt.Vet.Med.Ass. 57,No.1:363-386 (1997).
CLINICAL AND LABORATORY STUDIES ON RIFT
VALLEY FEVER VACCINES OF SHEEP
By
EL-SAWALHY, A.A.; ILAMODA, F.K.; KHAIRAT ABDEL MEGID A. ELIAN" and GEHAN, M. KAMAL*
Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University,
Bertha Branch.
' Rift Valley Fever Vaccine Production Department, Serum and Vaccine Research
Institute, abbassia, Cairo
SUMMARY:
This study was conducted on two field experiemnts. The first experiment was
carried out on 32 pregnant ewes to evaluate the clinical aspect and immunological response of sheep to
inactivate and attenuated RVF vaccine and to follow up the duration of maternal immunity in lambs.
Clinical findings of vaccinated and in-contact non vaccinated sheep showed only a significant rise in
body temperature after 2 days post vaccination (in sheep vaccinated with live vaccine), but there was no
abortion as specific signs. Results of vims reisolation revealed detection of RVF virus (vaccinal strain) in
tissue culture (BHK) from sera (only during third day post vaccination), these samples gave negative
results in inoculated mice. The acquired maternal immunity of lambs from vaccinated or non vaccinated
pregnant in-contact ewes was studied. The influence of vaccination on some hcpato•renal functions and
some biochemical
R.ecxtved:30.10.1996
tt-Sawaffiy et a[.
parameters revealed non significant alterations in AST, ALT, total proteins, globulins, albumin, cholesterol, total bilirubin, creatinine, urea, catcium and phosphorus.
The second experiment was done on 24 adult rams. Vaccinated rams showed non significant alterations in
the values of some semen characteristic as ejaculate volume, motility %, live sperm %, sperm cell count
%, and total sperm cell abnormalities%.
Resutls of the present study indicate that the locally produced live attenuated vaccine can be safely used to
immunize pregnant ewes. The immune response, as measured by RVFvirus specific antibodies developed
earlier and were more consistent than those produced by the formalin inactivated vaccine. The
immunogenicity, safety and requirements for only a single inoculation suggested that the locally produced
live attenuated vaccine may be effective and practical alternative to the killed vaccine for use in sheep
vaccination.
INTRODUCTION
Rift Valley Fever (RVF) is a mosquito borne viral disease, affecting animals and humans. It is
widely distributed in different localities of Africa, where periodic epizootics and epidemics
occured causing heavy losses
among lambs and calves. The disease is caused by arbovirus, RNA, single stranded virus,
belonging to family Bunyaviridae, Kahrs, (1981);
t
i
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Conical and Laboratory
WHO, (1983), Olaleye et al., (1992); Mariner and Yedloutschning
(1992) and radostits et al., (1994). 
In Egypt, RVF appeared as an acute febrile dangue like illnes affecting man as well as animals in
1977 (Imam and Darwish, 1977 and WHO 1977). Further outbreaks appeared in 1979 and 1980
(Seller et al., 1982 and Allah 1987) respectively. Finally Arthur et al. (1993); Gabery et al., (1994)
and Abou Zaid a al., (1995) described the last epidemic which extended from May 1993 up to
September 1994 in Aswan, Sharkia and some other governorates in Egypt. These workers
summarized the clinical signs of RVF in Egypt as an ocular form in humans and abortion in cattle
buffaloes and sheep.
Control of RVF in Egypt depends mainly on periodical vector control and vaccination of
susceptible animals. Vaccines already existing for veterinary use are inactivated and the live attenuated vaccines which are lo-
cally produced by the Rift Valley Fever Vaccine Production Department, Serum and Vaccine
research Institute, abbassia, Cairo.
A formaline inactivated RVF vaccine was prepared and studied by Abdel
Gaffar et al. (1979); El Nimr et al. (1981) and Eman (1990). This vac- ~ .
cine was used for vaccination of cattle and sheep in Egypt. Although safty of the vaccine was
proved, but its drawbacks are expensive in production, the duration of protection is short in
addition to colostral transfer of immunity and protection of the fetus are uncertain.
J.Egypt.Vet.SledA.ssoc. 2,1,t997
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tL-Sawathy et at.
.sue
Inepizootic breaks out of RVF, the use of live attenuated vaccine is rc t: M
ommended (WHQ 1"33). This vaccine was reviewed by Coakley et (17) and Kathryn et al
(1991). The study of live attenuated vacci trials in vaccination program in Egypt has been
documented by Taha, al. (1994), I3otot al. (1995); Eisa (1995); Gihan et al., (1996) a:
`/
v
Ibrahim (1996).
The aim of this study was to compare between the locally produced
attenuated and formaline inactivated RVF vaccine with respect to:1-Clinical studies of vaccinated and contact non vaccinated ewes
2- Studies on immune response of sheep vaccinated by those vaccines
3- Trials for reisolation of the vaccinal virus from either vaccinated or i contact non
vaccinated sheep.
4- To follow up the acquired colostral immunity
5- To study some serum biochemical parameters and semen characteri tic of rams of
vaccinated sheep.
MATERIAL AND METHODS
I- Aniamis
A- Pregnant ewes and their lambs:Thirty two, Rahmany and Osemy pregnant ewes, at about 3-4 months
pregnancy, there was no previous history of RVF vaccination. These a
366
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Clinical[ and [laboratory
mals were kept within the flock (250 sheep) in the farm of Faculty £o
Agriculture at Moshtohour, Benha. They were clinically normal and free from external and
internal parasites, no insecticides used during this work. These aniamis were divided into 4 gorups
each of eight animals.
I- Group I (GI) ewes were Vaccinated with the live attenuated RVF vaccine (Icc s/c). Ten lambs were born (LG1), after vaccination of their
dams by 18-40 days.
2- Group 2 (02) ewes were vaccinated with the inactivated RVF vaccine (Ice s/c), followed by a
live attenuated RVF vacine after 3 weeks (Icc s/c.) . Six lambs were born from this group
(LG2) after the second vaccination by 15-18 days. Some other born lambs were excluded from
the experiment since their time of birth was unsuitable (born after second vaccination by 6-13
days).
3- Group 3 (G3) ewes were vaccinated with inactivated RVF vaccine (1 cc s/c). Ten lambs were
born to their dams (LG3) after vaccination by
16-30 days.
4- Group 4 (G4) ewes were non vaccinated and kept in-contact with the vaccinated sheep. Eleven
lambs were born to their dams (LG4).
B- Adult rams:- Twenty four adult rams were used to study the effect of vaccination on some
semen characteristics. These rams were divided
into two subgroups (12 of each), group I was vaccinated with a live
J•Egypt.Vet.MedAssoc.5j,1,t997
367
EL-Sawathy et at.
attenuated RVF vaccine and subgroup 2 vaccinated with a formalin activited RVF vaccine
using the same previously described protoc The animals were housed together with the rams of
the flock rams).
C- Mice: Suckling swiss albino mice were used for virus isolation.
II- Samples:-
1- Colostral smaples were taken from ewes directly on the same day parturition and after 1 and 2
days post parturition and kept at -70 for virus reisolation.
2- Blood samples were collected from the jugular vein to separate sera different intervals before
and after vacciantion of ewes and tha lambs. All ewes in the three goups were bled before and
after vacs nation at I week, 2 weeks, 3 weeks, 15 months, 2.5 months, 3 months, 4.5 months
5.5. months and 6.5 months. Also sera sampl were similarly collected from non vaccinated
ewes at the same inu vals. All lambs at the first and second weeks after parturition and lal
every month until 6.5 months post lambing. Trials whenever possit to take blood samples from
the newly born lambs immediately afi parturition and before taking colostrum to see if there
was intrauteru transfer of antibodies or infection by estimating of neutralizing an bodies in
precolostral lamb sera.
368 J•Egvpt.Vet.Med,Assoc.L7,
CLtnU;at and Laboratory
3- Swabs:- Ocular, nasal, oral, and rectal swabs were collected at 3rd and 10th day post
vaccination in transport media and stored at -70°C for virus isolation from both vaccianted and
in-contact non vaccinated ewes. In addition serum, semen and colostrum/or milk smaples were
also collected.
4- The semen samples of rams under investigation were collected using
artificial vagina before vaccination and on days 1, 4, 7, 10 and IS post
vacciantion for studying the effect of vaccination of semen qualities.
III- Vaccines used: B oth types of vaccine were kindly supplied from
Rift Valley Fever Vaccine Production Department, Serum and
Vaccine Research Institute, Abbassia, Cairo.
I- A formalin inactivated vaccien was prepared at the serum and Vaccine Research Institute, Rift
Valley Fever Vaccine Production Department,
Abbassia, Cairo (Batch No. 130).
2- Live attenuated vaccine was locally produced at rift Valley Fever Vaccine Production
Department, Serum and Vaccine Research Institute, abbassia, Cairo (Batch No. 2). Its a
Lyophilized RVF live attenuated vaccine produced from Smithburn neurotropic strain at the
104th mouse brain passage and then 2 passage in BHK. Each vial has to be reconstituted in 100
nil saline and Icc injected immediately subcutanously in each ewes.
J.Egypt.Vet.MedAssoc. 57.1.1997
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tt-Sawathy et at.
IV- Clinical examination:- The body temperature of the pregnant ew
was recorded before vaccination, and daily after vaccination for
days for all experimental groups as well as incontact non vaccinau
ewes. In addition all animals were examined daily for abortion,
any clinical abnormalities.
V- Serological technique:- All collected sera were tested for the d( tion
of the RVF specific antibodies by agar gel precipitation
(AGPT) according to the method described by Ayoub and AI
(1981); serum neutralization test according to Walker, et al., (1S
neutralizing index was calculated according to Reed and Mu
(1938).
VI- Virus reisolation:- Nasal, oral, ocular, rectal swabs and serum.
men and colostral/or milk smaples were examined using baby
and BHK according to El-Nimr (1980); Taha (1982) and Dig
and Peters, (1989).
VII- Semen examination:Ejaculate volume, sperm motility %,
sperm%, sperm cell concentration and total sperm cell aborma
were estamlted for all collected semen samples accordi
Zemjanis (1970).
VIII. Serum biochemical analysis: Liver and kidney function t
extimated in serum sampels of all gorups of dams before
tion and 1, 6 weeks post vacciantion. Asparate aminotran;
370 J.Egypt.Vet.Med.Assoc.
CLLnicaL and laboratory
(AST), and alanine aminotransaminase (ALT) according to Reitman and Frankel, (1957);
serum total proteins were extimated according to Doumas et al. (1981); serum albumin level
was estimated according to Doumas and Biggs (1972); globulins values were
obtained by subtraction of total protein and albumin; serum total
bilirubin were estimated according to Bartholomeu and Delong, (1966); and creatinin levels
were estimated according Schirmeister et al. (1964). The obtained results were statistically
analyzed using student's "t" test as explained by Snedecor and Cochran (1969).
RESULTS and DISCUSSION
Rift Valley fever is one of the serious viral zoonotic disease. In Egypt the virus was isolated since
1977 - 1978, and 1993 - 1994. therefore it is important to vaccinate all susceptible animals with a
safe, potent, and highly immunogenic vaccine. Recently live attenuated vaccine was introduced
to vaccination program of animals beside inactivated one in Egypt. So, this field experiment was
performed to compare and evaluate both types of vaccine in pregnant ewes as well as in rams.
Ewes in group I vaccinated with live attenuated vaccine in the late third
of gestation period, as well as contact sheep revealed no clinical signs related to RVF, except the
body temperature were significantly elevated on the second day post vaccination (P < 0.05)
compared to the body
temperature before vaccination. The increase in body temperature was
J.Egypt.Vet.bted.4ssoc. z7.1,1997
371
EC-SawatFty et a[.
considered a normal regime after vaccination by live attenuated RVF vaccine due to short
period of viremia (Kathryn et al., 1991; Hassan 1994; and Ibrahim 1996). As shown in table 1.
There was no abortion of vaccinated or in-contact non vaccinated ewes. All the dams and their
offspring and the control sheep housed in close contact with those receiving the live attenuated
vaccine, were normal during the experimental period (6.5 months) and slid not exhibit any rise
in body temperature or showed any signs of RVF virus infection. This might be contributed to
the safety of the locally produced vaccine, in contrast to most previous works done on imported
Smithburn live attenuated vaccine, when a high percentage of abortion was observed by Eisa
(1995) and Ibrahim (1996). However Baskervillae et al. (1992) reported that abortion is not a
common sequev lac after vaccination of pregnant ewes with a live attenuated smithburn RVF vaccine.
Studies on the immunogenicity and duration of the immune response following immunization
of pregnant ewes with both locally produced formalin inactivated and live attenuated RVF
vaccines under field condition as demonstrated in Table (2) similar results were observed in
both 01 (vaccinated with live attenuated vaccine) and G2 (sheep vaccinated with formalin
inactivated vaccine followed by live attenuated one after 3 weeks). In both gorups the peak of
RVF neutralization index were obtained in the first sample taken after 3 weeks (1.5 months)
was 3.95 log and 4.2 log in G, and 02 respectively and nealry remained constant after
parturition until the end of the experiment 6.5 months where it was 2.5 long and 2.6 long for Gt
and G2 respectively. Similar results were
372 J.Egypt.Vet.bled.Assoc. 2,1,1997
CC6nt.onl and Laboratory
obtained in sheep of both gorups using agar gel precipitation test. It is
At
evident from the results of both groups the,antibody titers remained protective during the
experiment (neutralizing index more than 1.2 log.) In this respect Walker eta!., (1970) and Pini et
at., (1973) considered that one long serum neutralization index is the protective titer for aniamls.
In contrast 03 (vaccinated with formalin inactivated vaccine only) showed a neutralizing index of
2.75 log and 1.5 log after 1.5 and 2.5 months respectivley then declined gradually till the end of
the experimental period which became non protective (0.9). These results go parallel with these
obtained by Coackley et al. (1967); El-Nitner (1980); Eisa, (1995) and Ibrahim (1996). In non
vaccinated in-contact group G4 the mean SNI was detectable after 3 weeks until 3.5 months. This
low level of antibodies NI of 0.3 - 0.6 log in control may be attributed to a low dose of live vaccinal virus transmitted
to this gorup without evidence of reversion to virulence virus (Turell and Rossi 1991), in addition
to this experiments were done during the period of low insect activities (November-May).
A
The results of maternal immunity of lambs born to RVF vaccianted pregnant ewes and their incontact vaccinated are shown in table (3) LGI lambs born from dams vaccinated with live
attenuated vaccine and L02 lambs born from dams vaccianted with killed vaccine followed by live
attenuated one. The antibody level was higher and remained protective for a period from 3.5 - 4.5
months (more than one log protective level). Contrary to LG3 (dams only vaccinated with killed
vaccine only). The antibody level is low and remained protective for a short period (2.5 months).
No antibodies were observed in lambs of 04 (born from non
J.Egypt.Vet.MedAssoc.7,1,1997
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C6intcaL and Laboratory
vaccinated in-contact dams). T_he.r, ults of this study are similar to those
previously reported by WHO (1983); Nawal (1984) and Eisa (1995). The
present results demonstrate that non RVF antibodies could be detected in
sera of lambs collected directly at birth before colostrum intake this means
that the only was for maternal immunity of the lambs were from the
colostrum and there is non intrauterine transmission of antibodies from dam
to fetus via placenta. Simialr results were obtained by Eisa (1995).
The results of RVF virus reisolated from vaccinated and control in-contact
pregnant ewes in tissue culture (Table 4), clearly demonstrate that only 2
and I serum sample from Gt and GZ respectively gave positive CPE. On the
other hand sera samples which gave positive results in tissue culture when
inoculated in mice were negative (no signs of RVF virus infection).
Samples from Gt which were taken on third day post vaccination and for G"
of the same date post vacciantion with live attenuated vaccine. All other
collected samples (from colostrum, milk, serum, nasal, ocular, oral, fecal,
and semen) were negative for CPE after 3 successive blind passages in
tissue culture. These results might reveale that live attenuated RVF virus is
not excreted in feces, nasal, ocular discharges
and colostrum or semen. These findings might be contributed to a short
period of viremia (2-3 days) post vaccination and this might lower the
chance of virus excretion and transmission but hazard infection has been
reported by Kathryn et al. (1991). Similar findings were reported by Hassan
(1994) and Ibrahitn (1996).
J.Egrpt.Vet.Med..4ssoc.;7,l,1997
377
Cttnicat and laboratory
The results of the effect of RVF vaccines on the hepato-renal function and some serum
biochemical values are demostrated in Table (5). The resalts reveale a non significant increase in total proteins, globulins, albumin, total bilirubin, and
urea at I week and 1.5 months post vaccination for Gt, 03 and 02 after inoculation of the live
attenuated vaccine. In addition non significant changes in AST, ALT, cholesterol, creatinine, calcium and phosphorus were found. Results are in contrast with those obtained by Abo 'Laid et al.
(1995) who worked on ewes naturally infected with RVF virus, and thes`differences might by
attributed to the severe systemic disturbances during the acute attack of the disease. However,
Botros et al. (1995) stated that biochemical assays performed on sheep
receiving the live attenuated smithburn vaccine and in-contact sheep did not show significant
changes throughout the 28 days follow up period. Moreover similar results were reported in calves
vaccinated with tissue culture rindcrpest vaccine by Hanan et al. (1994). In general there is no
published data available about the kidney and liver function disturbance
in sheep vaccinated by both types of RVF vacciens.
As shown in Table (6) there was a non significant variation in all semen
characteristics recorded in the present investigation before and after vacciantion of rams with
killed RVF vaccine. Moreover, there was a non significant variation in ejaculate volume and
sperm cell concentration following injection of live attenuated RVF vaccine thai before
vacciantion.
However, the injection of rams with live attenuated RVF vaccines leads to a non significant
reduction in sperm motility (69.42, 73.16 and 68.15%); sperm livability (73.54, 76.38 and
71.31%a) and a non signifiJ.Egypt.VetAledAssoc. .1,1997
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EL-Sawathy et at.
cant increase in total sperm cell abnormalities (19.45, 22.47 and 18.16%) (luring the 4 th, 7th and
1011 day after vacciantion followed by a non significant variation, in sperm motility (78.47%),
sperm livability ($1.64%), and total sperm cell abnormalities (16.37%) of 15th day post
vaccination that that recorded for the sperm motility (83.88% & 80.17), sperm livability (83.18 &
82.46) and total sperm cell abnormalities (17.11 & 15.42%) before vaccination and during the ls t
day after injection of livef attenuated vaccines respectively. These non significant changes in live
attenuated RVF vaccinated gorup may be attributed to a slight elevation of body temperature•in
contrast to Chenoweth and Burgen (1972) who observed mid piece abnormalities in semen of
bulls naturally infected by ephemeral fever virus. Generally data on the effect of vacciantion on
semen picture especially RVF are lacking.
In summary the results of clinical examination after vaccination, seroconversion, virus
reisolation, liver and kidney functions, some biochemical serum values, and some semen
characteristics in rams indicate that the locally produced live attenuated RVF vaccine can be
safely used to immunize pregnant ewes and breeding rams. The immune response indicated that
RVF virus specific antibiodies developed earlier and were more consistent than those produced
by the formalin inactivated vaccine. The immunogenicity, safety and requirements for only a
single inoculation suggest the the locally produced live attenuated RVF vaccine may be effective
and practical alternative to the killed vaccine for RVF sheep vaccination. Application of similar
studies on other species of domestic livestock is highly recommend to generate more reliable
estimates on the
382 J•Egypt.Vet.hled.Assoc.2,1,1997
C16nicaL and laboratory
safety and immunogenicity of RVF live attenuated vaccine for different domestic aniamis.
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