J Vet Diagn Invest 13:462–467 (2001)
Bovine ephemeral fever in Taiwan
Fun-In Wang, A. M. Hsu, K. J. Huang
Abstract. Bovine ephemeral fever (BEF) is a vector-borne disease of cattle, spanning tropical and subtrop-
ical zones of Asia, Australia, and Africa, caused by Ephemerovirus of the Rhabdoviridae. Taiwan has had 3
BEF epizootics, occurring in 1989, 1996, and 1999, since the vaccination regimen was initiated in 1984, given
once a year in the spring with a single-dose formaldehyde-inactivated vaccine using the 1983 isolate as the
seed virus. This study evaluated the 1999 population immunity against BEF virus in Taiwanese dairy cows
with a neutralization test and whether the recent BEF virus isolates have mutated signiﬁcantly from the vaccine
virus. In March 1999, before vaccination, 94% of the animals studied were already seropositive, suggestive of
an endemic or persistent infection from the previous year. By June 1999, when 51% of herds had been vacci-
nated, the antibody level rose, and by September 1999, the serum-neutralizing antibody (SNA) level fell to a
minimum, preceding the outbreak of BEF in October 1999, during which the antibody levels of vaccinated
cows continued to decline while those of unvaccinated cows rose sharply. The results suggest that, in 1999,
vaccine-induced immunity was partially protective against BEF. Because the current single-dose vaccination
regimen resulted in minimal population immunity by September, a booster vaccination given in late summer
may be advisable for future disease control. Analysis of the glycoprotein gene of Taiwanese isolates between
1983 and 1999 showed a 97.4–99.6% homology, with an alteration of 4 amino acids in antigenic sites G1,
G3b, and G3c. Phylogenetic analysis of Taiwanese isolates revealed at least 2 distinct clusters: the 1983–1989
isolates and the 1996–1999 isolates. Both were distinct from 2 Japanese strains and the Australian BB7721
strain. Thus, at least 2 distinct BEF viruses, which had diverged before 1983, existed in Taiwanese dairy cows.
Bovine ephemeral fever (BEF) is a vector-borne vaccination with inactivated virus since 1984.4 The
viral disease of cattle, spanning tropical and subtrop- disease incidence rates in successive epizootics
ical zones of Asia, Australia, and Africa.1,3,16,18 The dropped from 26.6% in 1967 to 5.6% in 1999, while
virus is transmitted via insect vectors, wherein Culi- deaths and culls rose from 5.2% in 1967 to 21.9% in
coides spp., e.g. C. brevitasis Kieffer, Culex spp., and 1999 (Table 1). The disease course extended from a
Anopheles spp. are implicated. In cattle herds of usually acute self-terminating form3,12 to a more chron-
southern Taiwan, Culicoides oxystoma and Culicoides ic one11 in which the prognosis was grave and symp-
nipponensis are the prevalent species.9 The causative tomatic treatments with nonsteroidal antiinﬂammatory
ephemerovirus virus is bullet shaped, consists of a drugs and calcium borogluconate were ineffective in
negative-sense, single-stranded RNA genome and 5 curing the affected cattle. Sporadic occurrences in win-
nonstructural proteins, including a nucleoprotein (N), ter months3 are suggestive of an endemic status or per-
a polymerase-associated protein (P), a matrix protein sistent infection. This study evaluated the population
(M), a large RNA-dependent RNA polymerase (L), immunity in Taiwanese dairy cows in 1999 and wheth-
and a surface glycoprotein (G), which induces the er the recent BEF viruses have mutated signiﬁcantly
production of protective neutralizing antibody.22,24 from the vaccine virus, with a discussion in the context
Bovine ephemeral fever is characterized clinically by of previous epizootics.
the sudden onset of fever, stiffness, lameness, nasal
and ocular discharges, depression, cessation of ru- Materials and methods
mination, and constipation. The pathogenesis of this Sera sampling (Tables 2, 3; Fig. 1). In 1999, 5 surveys
disease is complex,19,20,27 although it seems clear that were conducted for serum-neutralizing antibody (SNA), for
host inﬂammatory responses, mediated by the release which samples were obtained from 2–10-year-old lactating
of cytokines, are involved in the expression of dis- cows, primarily Holstein, raised island wide. The ﬁrst sur-
ease.8,18,21 vey, done in March 1999, was to monitor the population
In the past 40 years, Taiwan has had 5 epizootics, immunity before vaccination. The vaccination regimen was
occurring in 1967, 1983, 1989, 1996, and 1999 (Table initiated in 1984, given once a year in March with a single-
1), despite the implementation of annual single-dose dose of formaldehyde-inactivated virus, using the 1983 iso-
late as the seed virus. The sample size (number of herds)
From the Department of Veterinary Medicine, National Taiwan needed for detection was determined by the disease inci-
University, 142 Chou-San Road, Taipei, 106 Taiwan. dence rate of 13.6% of the 1996 epizootic (Table 1) because
Received for publication September 14, 2000. the seroprevalence rate for 1998 was unavailable, with at
Bovine ephemeral fever 463
Table 1. Epizootics of BEF in Taiwan.*
Disease Number Month District
incidence Case Number of of virus of ﬁrst of ﬁrst
Year rate fatality abortions isolates outbreak outbreak
1967 26.6% 5.2% 31 None§ Late March Kaoshiung
(1,183/4,441)† (62/1,183)‡ (southern Taiwan)
1983–1984 20.1% 6.0% 189 4 October Chiayi
(5,650/28,117) (340/5,650) (southern Taiwan)
1989–1990 14.5% 5.0% NA 11 April Chiayi
(4,216/29,157) (210/4,216) (southern Taiwan)
1996 13.6% 11.3% NA 8 August Hsinchu City
(14,973/110,247) (1,685/14,973) (central Taiwan)
1999 5.6% 21.9% NA 2 October Kaoshiung
(538/9,593) (118/538) (southern Taiwan)
* Compiled from references 2, 3, 11-13, 17, and information from the National Institute for Animal Health, Council of Agriculture,
TAIWAN, R.O.C. NA not available.
† The number of cows affected/total number of cows in the affected districts.
‡ Includes the number of deaths and culls.
§ The epizootic was later conﬁrmed by serology.12,17
Includes the KS-1999 strain isolated by the authors (Table 4; Figs. 2, 3) and the TN-1999 (Tn88128) isolate sequenced by Yang et al.,
1999 (Gene Bank accession number Af208840).
95% conﬁdence interval in a population at risk found as in
Martin et al.14 The local veterinary ofﬁcers selected coop-
erative herds for sampling and, from each herd, selected 10
lactating cows for sampling. The second survey, done in
June 1999, was to monitor the population immunity after the
vaccination (Table 2). Because the ﬁrst and second surveys
revealed 98–100% seroprevalence rates by herds and 94–
99% seroprevalence rates by head, apparently a smaller scale
of sampling would be adequate for future surveys.14 Based
on results of the ﬁrst and second surveys, herd status was
categorized into 6 groups as follows: group 1; vaccinated
with high ( 1:32) SNA titer; group 2; vaccinated with in-
termediate (1:8–16) titer; group 3; vaccinated with low ( 1:
4) titer; group 4; unvaccinated with high titer; group 5; un-
vaccinated with intermediate titer; group 6; unvaccinated
with low titer. In September 1999, 8–10 head were sampled
from selected herds in each of the 6 groups. In October 1999,
sera were collected from 88 animals from 7 herds in 2 out-
break districts for which the disease incidence rate was 5.6%
based on the 538 clinically affected cows in a population of
9,593. The November 1999 survey was conducted 2–3
weeks after the outbreak to monitor the serological status in
herds affected in October 1999 and a few herds in neigh-
Serum neutralization antibody test. Sera, inactivated at 56
C for 30 minutes, were 2-fold serially diluted with serum-
Figure 1. Anti-BEFV serum-neutralizing antibody proﬁle of free minimal essential medium (MEM). Viral ﬂuid (0.1 ml)
Taiwanese dairy cows in 1999. The ordinate indicates the geometric containing 200 TCID50 was mixed with 0.1 ml of diluted
mean titer (GMT) using the product(s) of log2 to the SNA titer of serum and incubated at 4 C overnight. Then 0.1 ml of the
each animal in the target population as raw data for analysis before mixture was absorbed into baby hamster kidney (BHK-21
converted to GMT. The abscissa indicates the month in which the (C-13), CCRC 60041) cells at 37 C for 60 min before 1 ml
surveys were done. Total indicates the GMT of all animals tested of MEM was added to each well. Cytopathic effect (CPE)
(Table 2) regardless of the vaccination or infection status. The vac-
was observed 7 days later. The SNA titer was the reciprocal
cinated or unvaccinated indicates the GMT of only animals that had
records of with or without vaccination at the time of sample sub- of the highest dilution in which at least 1 of the 2 wells
mission. Note the rise in antibody level after the March vaccination. exhibited the inhibition of CPE. The virus used was a vac-
The decline to minimum in September precedes the October out- cine strain (Vacc-1983; Tables 1,4), a prototype of Taiwanese
break. isolates, isolated from buffy coat cells in 1983 and further
464 Wang, Hsu, Huang
Table 2. Survey for serum-neutralizing antibody against BEFV in Taiwanese dairy cows in 1999.
Parameter March June September October November
Number of districts 19 16 10 2 4
Total herds 164 137 45 7 42
(%) 98 100 75 100 100
Protected* herds (%) 37 64 0 43 88
(%, by herds) 26 51† 28 14† 0
herds (%) 42 64 0 0 0
Total cows 1,619 1,376 321 88 558
(%) 94 99 93 90 95
Protected cows (%) 53 71 0 20 87
* Seropositive titers are 1:2 and protective SN titers are arbitrarily assigned as 1:32.
† The rate in June may overlap with those in March and September. The rate in October may represent those boostered during the
adapted to BHK-21 cell for the production of inactivated one reverse transcription reaction,26 in which 7.5 l of viral
vaccine.2,4 RNA was incubated at 42 C for 45–60 minutes in 20 l of
Analysis of glycoprotein gene sequence. For virus isola- buffer containing 100 units of RNasin,c 0.01 M MgCl2, 0.1
tion, buffy coats of blood in anticoagulant (1 g/ml of EDTA) M KCl, 0.05 M Tris-HCl, pH 8.3, 0.01 M dithiothreitol, 0.03
were washed 3 times with phosphate-buffered saline before M -mercaptoethanol, 0.01 mM each of dNTPs, 0.5 M of
being inoculated into BHK-21 cells and cultured in MEM synthetic oligonucleotide primer (forward primer GF1), and
supplemented with 0.5% bovine serum albumin and 1% de- 25 units of avian myeloblastosis virus reverse transcriptase.d
ﬁned fetal bovine serum.a The cytopathic effect was ob- The reaction mixture was then incubated at 94 C for 10
served 4–6 days later, and the supernatant was passed 2 minutes or frozen and thawed to inactivate the enzyme. The
times in BHK-21 cells if the ﬁrst culture was negative. The cDNA was ampliﬁed by polymerase chain reaction (PCR),
history of other Taiwanese isolates identiﬁed by the year and in which 4 l of reverse transcription mixture was mixed
district of isolation is summarized in Table 1. The YHL (the with 46 l of buffer containing 0.05 M KCl, 0.01 M Tris-
Japanese prototype) and Jap-NIAH (designated by the au- HCl, pH 8.3, 2.5 mM MgCl2, 0.01% gelatin, 200 M of
thors) strains, originally from the National Institute of Ani- dNTPs, 1–2 units of Taq polymerase,c and 0.2 M of syn-
mal Health in Japan, were also studied for comparison. Each thetic oligonucleotide primers (reverse and forward primers,
virus was puriﬁed by 3 rounds of limited dilution, expanded, GF1 and GR1). Primers were designed following the pub-
and cleared by centrifugation at 2,000 g for 25 min at 4 lished sequences,23 where GF1 were 5 -ATGTTCA-
C. The supernatant containing viral particles was used for AGGTCCTCATAATTACC-3 (1–24) and GR1 were 5 -
RNA extraction by mixing 0.25 ml of virus suspension with TAATGATCAAAGAACCTATCATCAC-3 (1,847–1,871).
0.75 ml of Trizol .b Half of the pelleted RNA was used for The reaction mixture was denatured at 94 C for 4 minutes,
followed by 30 cycles of ampliﬁcation at 94 C for 45 sec-
onds, 52 C for 60 seconds, and 72 C for 90 seconds, with a
Table 3. Comparison of SNA titer between vaccinated and un-
ﬁnal extension at 72 C for 10 minutes. The PCR product
vaccinated cows before and during the 1999 outbreak.
was resolved in 1.5–2% agarose gel in 0.5 TBE buffer and
Month Vaccinated* Not vaccinated* P value stained with ethidium bromide. The PCR product was cloned
into the pcDNA3.1/V5-His-TOPO vector contained in the
September 10.49 6.471† 10.92 5.892† 0.552 TA cloning kit.e Nucleotide sequencing was performed us-
2.87 1.459‡ 3.08 1.227‡ 0.007
ing reagents provided in ABI PRISM BigDye Terminator
(n 158) (n 146)
October 7.58 6.266 19.16 4.815 1.4 10 8 Cycle Sequencing Ready Reaction Kit using an automated
2.32 1.455 3.17 1.774 0.058 ABI PRISM 377-96 DNA Sequencer.f The G genes of var-
(n 19) (n 63) ious BEF viruses (BEFVs) were analyzed utilizing the Clus-
tal method of MegAlign-expert sequence analysis software.g
* The vaccinated or not vaccinated indicates only animals that had
Statistical analysis. Student’s t-test with n 2 df was
records of with or without vaccination at the time of sample sub-
mission. Thus, the sum of vaccinated and not vaccinated is not nec-
performed to determine the signiﬁcance (P 0.05).h
essarily equal to that of the total number listed in Table 2.
† SNA titer(s) of each animal in the target population were used Results
directly for statistical comparison.
‡ The product(s) of log2 to the SNA titer of each animal in the The results of serological surveys are summarized
target population were used for statistical comparison. Each number in Table 2. In March 1999, before animals were vac-
indicates mean 1 standard deviation. n number of cows. cinated, 94% of the animals were already seropositive
Bovine ephemeral fever 465
Table 4. Amino acid variations in antigenic sites of Taiwanese BEFV strains.
Virus strains G1(487–503) G2(168–189) G3a(49–63) G3b(215–231) G3c(262–271)
Y———N499—–K FLT170———G Q———I K215–RSE220LN222D223K224–P I———Q271
Vacc-1983 N — — — A N E T R
HL-1989 N — — — G S E T R
KS-1989 N — — — E N E T R
PT-1989 N — — — E N E T R
TC-1996 S — — — E N E T Q
KS-1999 S — — — E N E T Q
TN-1999† S — — — E N E T Q
Jap-NIAH S — — — E N D T Q
YHL S N — N E N D T Q
* From reference 23 (GeneBank accession number M94266).
† Strain Tn88128 isolated and sequenced by Yang et al., 1999 (GeneBank accession number AF208840).
and 100% of the districts were seropositive. By June Glycoprotein gene sequences of various BEFVs
1999, after vaccination of 51% herds, 99% of the an- were compared (Tables 1, 4; Figs. 2, 3). Results
imals were seropositive, and the proportion of pro- showed a 97.4–99.6% homology between the 1983
tected animals (with SNA titer 1:32) rose from 53% and 1999 isolates, which led to alterations in 4 amino
to 71%, while the proportion of protected herds rose acids in antigenic sites G1, G3b, and G3c (Table
from 37% to 64%. By September 1999, the proportion 4).6,10,25 Results also showed a 96.7–97.3% homology
of protected herds and heads dropped to zero but rose between Taiwanese isolates and the Japanese strains
again in October and November of 1999 during and and a 90.3–90.6% homology with the BB7721 strain
after the outbreak. (Fig. 2). Phylogenetic analysis revealed 4 distinct clus-
The 1999 antibody proﬁle of the Taiwanese dairy ters: 1983–1989 Taiwanese isolates, 1996–1999 Tai-
cows is plotted in Fig. 1. The SNA level rose after the wanese isolates, the Japanese strains, and the Austra-
March 1999 vaccination, fell to a minimum by Sep- lian BB7721 strain (Fig. 3). Phylogenetic analysis of
tember 1999, rose and fell in different parts of the amino acids further divided the Taiwanese 1983–1989
population in October 1999, but rose sharply by No- cluster into 2 subgroups: Vacc-1983 and HL-1989, PT-
vember 1999, exceeding that of June 1999. 1989 and KS-1989 (data not shown). The amino acid
In September 1999, both vaccinates and unvaccin- sequence homologies were 97.4–99.4% among the
ates had similarly low levels (Table 3). During the Oc- Taiwanese isolates, 96.5–98.6% with the Japanese
tober 1999 outbreak, the antibody level of vaccinates strains, and 93.9–95.0% with the BB7721 strain.
continued to decline while that of unvaccinates rose
(Fig. 1), resulting in a signiﬁcantly higher level than Discussion
that of vaccinates (Table 3). The incidence rates observed in successive Taiwa-
nese epizootics have decreased, while the deaths and
culls have increased (Table 1). This is probably be-
cause 1) the epizootic patterns changed from a sweep-
ing epidemic3,12 into a slow epidemic11 and into local-
ized, compact-scale outbreaks such as seen in 1999
(Table 1), 2) more recent (1996–1999) epizootics may
have been caused by a BEFV distinct from that of the
1983–1989 epizootics (Fig. 3), 3) less likely, mixed
infection with other agents, such as Adelaide, Akaba-
ne, Ibaraki viruses, etc., may cause more severe illness
(but there is no proof), 4) cows were maintained on a
high-protein diet and a heavy lactation schedule, thus
predisposing them to higher susceptibility, and 5) the
monetary compensation program may have resulted in
Figure 2. Sequence homology of G gene of various BEFVs. inﬂated culling rates.
Refer to Table 4 for strain designation. The results also suggest that vaccine-induced im-
466 Wang, Hsu, Huang
tween infected cows cannot establish transmission. In
unvaccinated animals, there was a signiﬁcant reverse
linear relationship between the SNA titer and the am-
bient temperature and a positive linear relationship be-
tween the SNA titer and hours of sunlight in Septem-
ber 1999 (data not shown). Presumbably, these sea-
sonal factors favored the proliferation of insect vec-
tors, such as C. oxystoma and C. nipponensis, which
are most prevalent from May to October, coinciding
with the rainy season ( 108 mm monthly rainfall), the
25 C ambient temperature, and the neighboring wet
Figure 3. Phylogenetic relationship of G gene of various rice ﬁelds.9 Although these insects have not been
BEFVs. Note the 4 distinct clusters: 1983–1989 Taiwanese isolates, linked to the BEF epizootics, their prevalent seasons
1996–1999 Taiwanese isolates, Japanese strains, Australian BB7721 coincide with the usual epidemic seasons (Table 1).
strain. However, attempts to isolate BEFV and to detect
BEFV by PCR from Culicoides spp. (identiﬁed and
dissected under a microscope)15 trapped from the af-
munity was partially protective against BEF. This is fected herds in October 1999 failed to yield positive
because 1) a minimum antibody level in September results.
1999 preceded the outbreak in October 1999 (Fig. 1), Two distinct BEFV, diverging before the early
2) the antibody level of vaccinates continued to decline 1980s, exist in Taiwanese dairy cows, but it is unclear
during the outbreak (Fig. 1), suggesting that they re- whether both are from the same 1967 virus. Thus, the
mained protected despite the low SNA titer they had 1989 epizootic most likely originated from the residual
(Table 3), 3) vaccinates appeared to have been infected focus of the 1983 epizootic and the 1999 epizootic
later than the unvaccinates during the outbreak, as from that of the 1996 epizootic. The 2% divergence in
manifested by the late but sharp rise of their antibody 7 years (1996 vs. 1989) (Fig. 2) far exceeds the evo-
levels (Fig. 1). Although there is generally good cor- lutionary rate of 1/103–105 identiﬁed in vitro for
relation between SNA titer and protection, animals
BEFV,5 thus supporting the existence of at least 2 vi-
with low SNA titer can still be protected.7 In this re-
ruses. Because the divergence of these 2 viruses oc-
gard, the vaccine-induced low SNA titer or immunity
curred in the early 1980s, before the use of a vaccine,
seemed to be qualitatively different from the infection-
it can be speculated that the vaccine was effective in
induced low SNA titer (Table 3). Failure of the vaccine
suppressing 1 virus (1983–1989) while facilitating the
to protect is probably due to 1) the low average vac-
selection of the other (1996–1999). If this is correct,
cination rate, 51% by June (Table 2), 2) the inappro-
the change of seed virus for vaccine production is war-
priate time of vaccination (preferably 3 months prior
to the predicted time of outbreak), and 3) amino acid ranted. The distinction of Taiwanese isolates from the
alterations in antigen sites (Table 4). Occurrence of the Japanese and Australian BB7721 strains indicates the
lowest SNA titers in September 1999 suggests that importance of geography, in which the primary insect
SNA remained positive for approximately 5 months vectors prevail in the evolution of BEFV despite the
postvaccination (Fig. 1), which was within the accept- fact that cross-strait or cross-continent infections can
able range.4 Correct prediction of outbreak time or a be established.16
booster vaccination done in late summer would be bet- It has been concluded that at least 2 distinct BEFV
ter. strains have existed in Taiwanese dairy cows since the
The ﬁnding that the antibody level in unvaccinated early 1980s and that immunization with a vaccine
animals rose in June 1999 is intriguing. This is prob- made of an appropriate seed virus performed 3 months
ably due to 1) a subclinical wave of infection that may prior to infection at a vaccination rate of 70% should
have occurred before June 1999 and 2) some vacci- be effective in controlling outbreaks.
nates not being recorded as they should have been. Note. After the results of this study were revealed,
Because an epizootic occurred in 1996,11 it was in- the vaccination regimen for BEF in Taiwan was
teresting to see that, in most geographic regions of changed beginning in the year 2000 to 2 doses annu-
Taiwan, older cows ( 3 years old) had signiﬁcantly ally with formaldehyde-inactivated vaccine, with the
higher SNA titers than younger cows ( 3 years old) ﬁrst dose given in March and a booster dose given in
(data not shown), suggesting a persistent infection or late summer. Also, the Council of Agriculture TAI-
that older cows have been exposed to insect vectors WAN, R.O.C., is funding projects for developing BEF
longer than the younger ones since direct contact be- vaccine with a new seed virus—the 1999 isolate.
Bovine ephemeral fever 467
Acknowledgements cation of neutralizing epitopes on the G protein of bovine
ephemeral fever rhabdovirus. J Gen Virol 79:2573–2581.
The author thanks Dr. Yong-Hsiu Lu at the National Institute for 11. Liao YK, Inaba Y, Li NJ, et al.: 1998, Epidemiology of bovine
Animal Health (NIAH), Council of Agriculture, for sharing vaccine, ephemeral fever virus infection in Taiwan. Microbiol Res 153:
YHL and Jap-NIAH strains. This work was supported by grants 89- 289–295.
SA-1.4-IC-01(6) (NTU grant number 089A-2004) and 89-ST-6.3- 12. Lin CS, Inoue M: 1969, A study on the bovine inﬂuenza oc-
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