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EvaluationofEpizooticHaemorrhagicDisease
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Veterinary Research Communications, 29 (2005) 447^451
# 2005 Springer. Printed in the Netherlands
Short Communication
Evaluation of Epizootic Haemorrhagic Disease Virus
Infection in Sentinel Calves from the San Joaquin Valley of
California
I.E. Aradaib1, R.A. Mederos2 and B.I. Osburn2
1
Molecular Biology Laboratory (MBL), Department of Medicine, Pharmacology and
Toxicology, Faculty of Veterinary Medicine, University of Khartoum, PO Box 32,
Khartoum North, Sudan; 2Veterinary Teaching and Research Centre, Tulare and School of
Veterinary Medicine, University of California, Davis, CA, USA
*Correspondence: [email protected]
Aradaib, I.E., Mederos, R.A. and Osburn, B.I., 2005. Evaluation of epizootic haemorrhagic disease virus
infection in sentinel calves from the San Joaquin Valley of California. Veterinary Research Communications,
29(5), 447^451
Keywords: diagnostics, EHDV, epidemiology, RT-PCR
Abbreviations: ds, double-stranded; EHDV, epizootic haemorrhagic disease virus; RT-PCR, reversetranscription polymerase chain reaction
INTRODUCTION
Epizootic haemorrhagic disease virus (EHDV), an arthropod-borne double-stranded
(ds) RNA virus, is a member of the genus Orbivirus in the family Reoviridae (Borden et
al., 1971). EHDV infects domestic, captive and free-ranging ruminants, of which the
white-tailed deer (Odocoileus virginianus) is the most seriously a¡ected with the disease
(Shope et al., 1960). EHDV serotypes 1 and 2 are recognized in the United States
(Gorman, 1992). Because it is di¤cult to obtain more than one blood sample from the
same wild animal under ¢eld conditions, routine surveillance of sentinel herds for virus
infection and associated vector transmission of EHDV is the backbone of the
epidemiological studies on EHDV in a particular geographical location. In the
absence of clinical haemorrhagic disease, sentinel cattle herds provide the most
e¡ective approach for monitoring animal health and obtaining EHDV isolates
including recovery of new serotypes of EHDV from a particular location. The
objectives of the present study were to determine the incidence of EHDV serotypes
and their association with clinical haemorrhagic disease in sentinel cattle from the San
Joaquin valley of California, and also to determine the interval after natural infection
when EHDV could be detected in blood of infected calves, using conventional virus
isolation and RT-PCR ampli¢cation technology.
447
448
MATERIALS AND METHODS
A sentinel calf herd was maintained at the San Joaquin Valley of California. Eighteen
calves of approximately 6^8 months of age were selected, ear-tagged, and housed as a
group from March 1993 to February 1994. The animals were observed daily for signs
of clinical haemorrhagic disease throughout the experimental period. The sentinel
calves were fed a ration of concentrates and hay with free access to water. Processing of
the blood samples for virus isolation (VI) was as described previously (Aradaib et al.,
1995). The multiplex EHDV RT-PCR for simultaneous detection of EHDV serogroup
and identi¢cation of EHDV-1 and EHDV-2 has been described previously (Aradaib et
al., 1998).
RESULTS AND DISCUSSION
No signs of clinical haemorrhagic disease were observed in any of the sentinel calves.
The incidence rate of EHDV in the sentinel calves as determined by virus isolation was
found to be 3/18 (17%) (Table I). The incidence rate of EHDV infection as determined
by RT-PCR was found to be 6/18 (34%) (Table II). EHDV infections were reported
during late summer and early fall, which coincided with high activity of the insect
vector, Culicoides imicola. The infection with EHDV-2 was found to be more prevalent
than EHDV-1.
TABLE I
Results of virus isolation on BHK-21 cell line
Months
March
April
May
June
July
August
September
October
November
December
January
February
EHDV-1
EHDV-2
^
^
^
^
^
^
+
^
^
^
^
^
^
^
^
^
+
^
+
^
^
^
^
^
449
TABLE II
Results of serogroup^speci¢c EHDV and serotype^speci¢c EHDV-1 and EHDV-2 PCR-based
detection assays
Months
March
April
May
June
July
August
September
October
November
December
January
February
EHDV serogroup
EHDV-1
EHDV-2
^
^
^
^
+
+
++
++
^
^
^
^
^
^
^
^
^
^
+
^
^
^
^
^
^
^
^
^
+
^
+
++
^
^
^
^
Despite the extended nature of the study, only a few EHDV isolates were recovered.
A lack of success in isolating EHDV from the blood samples of sentinel calves has been
reported previously (Shope et al., 1960; Pearson et al., 1992; Aradaib et al., 1994, 1995).
This could be attributed to viraemia being missed because of infrequent sampling. In
experimentally infected calves, viraemia may be detected for 10^14 days post infection
by conventional virus isolation, whereas PCR-based assay detected the viral nucleic
acids for at least 28 days (Aradaib et al., 1994). In the present study, blood samples
were collected from the sentinel calves on a monthly basis. More EHDV isolates could
have been recovered if the animals had been bled at relatively shorter intervals.
Even in the absence of clinical haemorrhagic disease, certi¢cation of animals free of
EHDV infection by virus isolation or serology is necessary for import/export
regulations (Pearson et al., 1992). Currently, only the rudiments of the epidemiology
of the disease are known in domestic or wild ruminants. To advance beyond the current
knowledge of the epidemiology of EHDV, we have evaluated EHDV infection in
sentinel calves from the San Joaquin Valley, California. To our knowledge, this is the
¢rst time a sentinel cattle herd has been evaluated for EHDV infection in the United
States.
Infections of cattle with EHDV in local areas of endemicity are usually associated
with indirect losses such as drop in milk production, loss of body weight and condition,
and poor subsequent reproductive performance. (Mohammed et al., 1996).
Nevertheless, a few reports have shown that the virus may also cause clinical
haemorrhagic disease in North American cattle (Metcalf et al., 1991).
450
Application of these PCR-based detection assays for simultaneous serogroupspeci¢c detection and serotype-speci¢c identi¢cation of EHDV-1 and/ or EHDV-2 in
blood samples from the sentinel calves provides a simple and rapid method for
detection of EHDV infection, saves time and, above all, saved on cost. This is
because the samples were tested once instead of individual testing against each
individual serotype of EHDV serogroup. Since the EHDV PCR assays are extremely
sensitive procedures, the point should be stressed that PCR-negative animals would
more than ful¢l the present requirements for negative status for the purpose of export
(Mohammed et al., 1996; Aradaib et al., 2003).
It is worth mentioning that PCR-positive and culture-negative results, from the same
clinical sample, are not uncommon. This is because virus isolation requires the
presence of infectious virus, whereas PCR-positive results can be obtained from
clinical samples containing incomplete virion or viral nucleic acid as well as intact
infectious virus.
The data presented in this communication indicate that EHDV-1 and EHDV-2 were
being transmitted among sentinel calves in the San Joaquin valley of California as
determined by virus isolation and RT-PCR technology. In addition, the infections were
only transient and no clinical haemorrhagic disease was reported among the sentinel
calves. Moreover, infected calves can amplify the virus and will provide infectious virus
for insect transmission to more susceptible animals. The importance of virus isolationnegative calves, despite their PCR-positive status, in the epidemiology of the disease is
yet to be investigated.
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