Download The length of BTV-8 viraemia in cattle according to infection doses

Research in Veterinary Science 91 (2011) 316–320
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Research in Veterinary Science
journal homepage: www.elsevier.com/locate/rvsc
The length of BTV-8 viraemia in cattle according to infection doses
and diagnostic techniques
L. Di Gialleonardo, P. Migliaccio, L. Teodori, G. Savini ⇑
Istituto G. Caporale Teramo, Via Campo Boario, 64100 Teramo, Italy
a r t i c l e
i n f o
Article history:
Received 17 September 2010
Accepted 10 December 2010
Keywords:
Bluetongue serotype 8
Viraemia
Real time RT-PCR
Virus isolation
a b s t r a c t
Four groups of BTV free Frisian and cross bred calves were used to determine the length of viraemia following infection with different doses of BTV-8 Italian isolate. The first group of five animals was infected
with 10 TCID50 of BTV-8, the second group of four animals with 103 TCID50 and the third group, which
also included four animals, was infected with 106 TCID50. A placebo containing uninfected tissue culture
medium was given to the four animals of the fourth group. The viraemia was evaluated by real time RTPCR and virus isolation. In all infected groups, virus isolation was able to detect infectious virus up to
39 days post infection (dpi) while RT-PCR was positive up to 151–157 dpi. Infectious dose did influence
neither the length nor the pattern of BTV-8 viraemia and confirmed that real time RT-PCR remains positive although no circulating virus is detectable in the peripheral circulation.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Bluetongue is a non-contagious, infectious, vector-borne viral
disease of domestic and wild ruminants caused by Bluetongue
virus (BTV), a member of the Reoviridae family, Orbivirus genus
(Mertens et al., 2004). The virus is transmitted by haematophagous
midges of the genus Culicoides and their presence is essential to
determine the occurrence of the disease and/or the virus circulation. BTV has been reported from latitudes 35°S to 53°N (Gibbs
and Greiner, 1994; OIE, 2009). Several species of Culicoides may
act as BTV vectors (Mellor, 1992). C. imicola is the main vector in
southern Europe (Mellor and Wittmann, 2002), while species of
the Obsoletus and Pulicaris complexes, and C. dewulfi are responsible for the BTV diffusion in the central and northern parts of the
continent (Wilson and Mellor, 2009). BTV is a double strand RNA
virus with a segmented genome consisting of 10 segments which
encode for seven structural (VP1–VP7) and four non-structural
proteins (NS1–NS3 and NS3A). Twenty-four serotypes have
been formally identified (Erasmus, 1990; Gibbs and Greiner,
1994) and a likely 25th has been recently suggested (Hofmann
et al., 2008).
The disease is characterised by various clinical forms, ranging
from acute to chronic. Acute clinical signs include fever, ulceration
of the mucosa, facial oedema and limp, while chronic forms are in
⇑ Corresponding author. Address: Dept. of Virology, OIE Reference Laboratory for
Bluetongue, Istituto G. Caporale Teramo, Via Campo Boario, 64100 Teramo, Italy.
Tel.: +390861 332440; fax: +390861 332251.
E-mail address: [email protected] (G. Savini).
0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rvsc.2010.12.017
general characterised by weakness, emaciation, muscle stiffness,
fragile fleece with consequent alopecia (Verwoerd and Erasmus,
2004).
Not long ago BT was included in the World Organization for
Animal Health (OIE) list A of diseases. Although now BT is among
the notifiable diseases, affected countries or provinces are still
banned to move ruminant livestock. This restriction has severe
economical repercussions on national and international trade. It
is well established that, at least within the European scenario, most
of the economical importance of BT is related to animal movement
restrictions rather than to the direct costs caused by animal disease
and production losses.
In 1998, BTV entered in Europe and, between 1998 and 2006,
caused an unprecedented epidemic with the incursions of six different serotypes into several European Countries around the Mediterranean basin. In August 2006, BTV-8 occurred for the first time
in Northern Europe affecting the most important European exporting Countries. Outbreaks were reported in Holland, Belgium,
Germany, Luxembourg and France (Mellor et al., 2009; Saegerman
et al., 2008). European BTV-8 is a particular virulent strain which,
unlike other field isolates, is capable of passing the placental barrier and infecting foetal tissues (Dal Pozzo et al., 2009). An animal
to be safely moved from an infected area must not be viraemic.
Viraemia and particularly its duration are crucial for BTV transmission. Amongst susceptible species, cattle can harbour infectious
virus in the blood for up to 2 months. They play an important
epidemiological role by acting as BTV reservoirs for Culicoides
(MacLachlan, 1994). The OIE recommends that a BTV free Country
imports animals upon the respect of certain conditions which
L. Di Gialleonardo et al. / Research in Veterinary Science 91 (2011) 316–320
basically ensure the absence of the virus in the blood of the moving
animal. This should result negative when tested for the presence of
BTV antibodies or for the presence of virus in the blood. Today
most of the labs use molecular technology to detect virus in blood
or tissue samples. RT-PCR and, more recently, real time RT-PCR assays provide a versatile system able to give information on virus
serogroup and serotype within a few hours. They are also highly
sensitive and capable of detecting very low concentration of viral
RNA. In addition the real time assay is able to quantify the viral
genome. Because of all these advantages, in most labs these new
techniques are preferred to the classical viral detection techniques
which requires 3–4 weeks to be completed. However, RT-PCRs are
actually not able to distinguish whether the RNA detected in the
animal is part of an infectious virus or just RNA of degraded virus
not any longer able to infect the vector. The classical virus isolation
is still the only method able to reveal the presence of infectious
virus in an animal. This information becomes crucial when an animal from an infected area has to be moved. Several studies have
confirmed that, at least for some BTV serotypes, the RNA could
be detected in the blood of susceptible species longer than the
infectious virus (Bonneau et al., 2002; MacLachlan et al., 1994;
Singer et al., 2001). For BTV-8, the duration of viraemia as detected
by RT-PCR or viral isolation still needs to be elucidated (Dal Pozzo
et al., 2009). Also, it is critical to know how long an animal can
maintain an infectious BTV-8 in the blood and if this period is affected by the infection dose.
This study was aimed to evaluate the relationship between
BTV-8 infection dose and viraemia pattern in cattle by using classical virus isolation method (OIE, 2008) and real time RT-PCR (Polci
et al., 2007).
2. Materials and methods
2.1. Virus
The isolate of BTV serotype 8 used in this trial is a field strain
isolated at the Virology Department of the Istituto G. Caporale
Teramo from an Italian cow belonging to a herd in which numerous animals were imported yearly from France. Before use, the
virus was propagated on Vero cells (African green monkey kidney)
cultivated in minimum essential medium (MEM) (Eurobio, France)
containing L-glutamine, antibiotics (penicillin 100 IU/ml (Sigma,
Munich), streptomycin 100 lg/ml (Sigma), gentamicin 5 lg/ml
(Sigma), nystatin 50 IU/ml (Sigma)) and 3% foetal calf serum
(FCS) (Sigma). Virus titration was carried out on Vero cells using
standard microtitration assay (Savini et al., 2004). The virus titre
was calculated using the Reed and Muench formula (Reed and
Muench, 1938) and expressed as TCID50/ml.
2.2. Animals and infection
Frisian and crossbred calves of 3 months of age, free of respiratory, digestive, umbilical and osteo-articular disease were used in
the trial. The animals originated from BTV-free herd located in a
BTV-free area and resulted negative for BTV-antibodies, when
tested by competitive ELISA.
The animals were divided into four groups, one of 5 animals
(Group A), and 3 of 4 animals each (Groups B, C and D). Animals
of group A were infected subcutaneously with 1 ml suspension
containing 10 TCID50 of the BTV-8 virus, while those of group B
and Group C with 1 ml containing 103 TCID50, and 106 TCID50,
respectively. One milliliter suspension containing uninfected tissue
culture medium was inoculated to the animals of the group D as
placebo.
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2.3. Sampling and laboratory tests
From each animal, ethylenediaminetetraacetic acid (EDTA)
blood and serum samples were taken before the infection and
three times a week for 10 weeks. Serum samples were tested for
the presence of BTV-8 antibodies by serum neutralisation (Savini
et al., 2004). Blood samples were tested for the presence of BTV
by using both the classic virus isolation method (Savini et al.,
2004, 2005; OIE, 2008) and real time RT-PCR (Polci et al., 2007).
In group C the viraemia was assessed using only the classic virus
isolation. Intravenous egg inoculation followed by two blind passages in VERO cells was used to isolate BTV-8 from EDTA blood
samples, according to the method described by Savini et al.
(2005). Virus titres were determined in the blood of viraemic animals as follows: the blood cells were washed three times in phosphate buffer saline (PBS) containing antibiotics. After the last
washing, the sample was re-suspended in MEM with antibiotics
(1/10 v/v) and sonicated. Four 10-fold dilutions of each sample suspension (from 1:10 to 1:10,000) were inoculated into a 96 flat bottomed microtitre plate wells, following the method described by
the OIE (2008). Four replicates were made for each dilution.
Approximately 104 cells, in a volume of 100 ll of MEM plus antibiotics and 3% FCS, were added per well and the plates incubated in
an incubator at 37 °C under 5% CO2. The plates were examined
after 6 days and the TCID50 calculated by using the Reed and
Muench formula (Reed and Muench, 1938).
The real time RT-PCR used was that described by Polci et al.
(2007) and adapted for BTV-8 (unpublished data). The method is
able to detect 101.55TCID50/ml of BTV-8 and shows a very strong
linear correlation between the virus concentration and the Ct value
(R2 = 0.994). The efficiency of the method is also high (91.43%).
To avoid the possible presence of inhibitors and to prevent false
negative results, an Internal Control (IC), was included in the real
time RT-PCR assay used in this trial. The Internal Control is a
non-infectious synthetic RNA representing a fragment of the
NS5-2 region of West Nile Virus (HNY1999) acquired from Ambion
diagnostics. For the IC amplification specific primers and a probe
labelled with VIC at the 50 -end and TAMRA at the 30 -end were used.
Samples with a threshold cycle (Ct) value of less than 39 cycles
were considered as positive.
3. Results
Fig. 1 shows the viraemia and neutralising antibody titre patterns obtained when the blood and serum samples of cattle infected with different doses of BTV-8 were tested by using the
classic virus isolation and the serum neutralisation assay, respectively. Except for one animal of the group A which resulted negative to both the real time RT-PCR and the virus isolation for the
entire experimental period, all infected animals had viraemic titres
after infection. Conversely, no viraemia was detected in the animals of the control group.
In groups A and B, BTV was detected from day 5 post infection
(p.i.) to day 39 p.i. In group C, BTV was firstly detected on day 2 p.i.
but the last day of viraemia was the 39th day p.i. as the other
groups. In all infected groups the highest viraemic titre (between
105.3 and 105.5 TCID50/ml) was reported on day 12 p.i.. Animals of
group A showed first neutralising titres on day 5 p.i. whereas those
of the other two groups on day 12 p.i. (Fig. 1).
The same animals which resulted viraemic by classical virology
were reactive to the real time RT-PCR. As in the virus isolation,
either in animals of group A or in those of group B the viraemia
started on day 5 p.i. In calves infected with the 10 TCID50 dose,
the real time RT-PCR resulted positive until day 157 p.i. whereas
in those infected with the 103 TCID50 dose, the BTV RNA was
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L. Di Gialleonardo et al. / Research in Veterinary Science 91 (2011) 316–320
Fig. 1. Average viraemic and neutralising titers in cattle infected with different doses of European BTV-8.
Fig. 2. RT-PCR Ct values in cattle infected with 103 TCID50 of BTV-8 European strain.
detected up to 151 p.i. Both groups showed similar viraemic trends
with threshold cycle (Ct) values ranging from 22 to 38 (Figs. 2 and
3). No correlation was demonstrated between the Ct values and the
presence of infectious virus.
4. Discussion
The major problem related to the BTV infection is the ban of animal trade from BTV-infected to BTV-free areas or countries. When
BTV-8 affected Northern European Countries where cattle export
represents a significant source of income, the trade restriction
was difficult to sustain. Vaccination was proposed as a practical
solution for eluding the trade limitation. In Europe, most of the
BTV vaccination campaigns were implemented to allow safe movement of animals. Vaccine elicits cellular and/or humoral immunity
which should prevent animals from viraemia in case of infection.
Not all vaccinated animals, however, are protected from viraemia
following infection and therefore animals should be checked serologically and virologically for BTV before moving. Many techniques
are now available to detect the presence of virus in the blood of the
L. Di Gialleonardo et al. / Research in Veterinary Science 91 (2011) 316–320
319
Fig. 3. RT-PCR Ct values in cattle infected with 10 TCID50 of BTV-8 European strain.
animals. Amongst these, the real time RT-PCR is undoubtedly one
of the most sensitive and commonly used diagnostic test. It is characterised by having a very high sensitivity and specificity. It could
then represent the optimal solution to prevent trading of viraemic
animals which is likely the major source of BTV spreading. However, even if the RT-PCR technique detects viral RNA with a very
high level of sensitivity, it is not capable of determining whether
the sample contains infectious virus or not. The classical virus isolation conversely detects only infectious virus. In this study real
time RT-PCR and the classical virus isolation were used to assess
the length of BTV-8 viraemia in cattle following infection with various doses of BTV-8. Similarly to what found with other BTV serotypes (Bonneau et al., 2002; MacLachlan et al., 1994; Singer et al.,
2001), positivity by real time RT-PCR in blood lasted longer
(>150 dpi) than the period over which infectious virus could be isolated (39 dpi). Concerning the maximal predicted duration of viraemia in BTV infected cattle, quite a few data are available in the
literature. It has been observed that the length of viraemia depends
on the BTV serotype involved, the natural or the experimental
infection protocol, the animal’s age and the method of isolation
(Dal Pozzo et al., 2009). According to Bonneau and MacLachlan
(2004), only ruminants whose blood contains BTV as determined
by virus isolation were able to infect the vector. Therefore, calves
infected with BTV-8 in this study would have been able to infect
Culicoides until 39 dpi. After that period they should have been
considered epidemiologically safe although still positive to RTPCR. Any evidence proving a correlation between Ct-values and
the animal infectious status could represent a possible solution
to the real time RT-PCR limitations. However, in this study no correlation was found between the real time RT-PCR Ct values and the
presence of infectious virus. At the beginning of infection, BTV was
isolated from samples with Ct values above 30 whereas, after
39 dpi, it was not possible to isolate virus despite the Ct values
found in the samples were below 30. In the early phase of infection
larger part of RNA virus detected in the blood is likely to derive
from live virions as opposed to later phases where most of the
RNA was probably deriving from degraded virions, no more capa-
ble of infecting vectors. The impact of virus dose on the outcome
of infection is generally poorly understood. A strong correlation
has been described between dose and length of incubation period,
severity of clinical disease and efficiency of transmission. It is generally believed that the higher the dose, the shorter the incubation
period and the time from inoculation to onset of viraemia and clinical signs (Alexandersen et al., 2003; Howey et al., 2009; Quan
et al., 2004). In this study, the size of the inoculum did not influence the kinetics of viraemia as shown for hepatitis B virus (Asabe
et al., 2009), and thus probably also having no effect on the capability of transmission and diffusion of BTV in the environment.
The only difference was in animals infected with the 106 dose, in
which viraemia started earlier, suggesting that they were able to
transmit the virus earlier than the other two groups. However,
the length and the intensity of viraemia were similar in all experimental groups. As in other viral infections the clearance of Bluetongue virus and a reduction in viral titres occur at the time of
seroconversion confirming that neutralising antibodies prevent
viraemia and further replication of viruses (Stott and Osborn,
1992). In the present experiment, the immune system of cattle responded more quickly to a low antigen dose than to the higher
doses, suggesting that higher BTV infection doses affected the immune response. Although we do not have specific data to explain
this unexpected findings, other authors have observed a transient
immune-suppression and lympho-cellular depletion in infected
lymph nodes during BTV infection (Barratt-Boyes et al., 1992;
Odeon et al., 1997; Quist et al., 1997a,b; Lacetera and Ronchi,
2004; Owens et al., 2004).
Nonetheless, apart from an earlier presence of the virus in the
blood of animals infected with the highest dose, no other outcomes
resulted from this different response. In all experimental groups
there was a quite long period during which high neutralising antibodies and viraemic titres were present at the same time. This is in
line with other studies and further confirms the role played by the
red blood cell membranes in protecting bluetongue virus from the
attack of the neutralising antibodies (Brewer and MacLachlan,
1994).
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L. Di Gialleonardo et al. / Research in Veterinary Science 91 (2011) 316–320
It could be concluded that real time RT-PCR is still the technique
of choice for detecting infection because it is a very sensitive test
and therefore useful as screening to detect BTV. Virus isolation,
however, is needed to assess if the sample contains infectious virus
or not. The duration of BTV positivity by RT-PCR in blood lasts
longer than the period over which infectious virus can be isolated.
Consequently, to avoid blocking trading of safe animals, care
should be taken in interpreting positive results. The OIE terrestrial
code (2009) states that the infectivity period for bluetongue virus
shall be 60 days. The infectious period of all infected animals included in this trial was much less then 60 days and this occurred
despite the challenge dose used. Regarding the host-virus interaction, apart from either a delay in the immune response observed in
the two highest dose groups or an earlier presence of circulating
BTV in animals infected with 106 dose, no other effects were found
in the viraemia and neutralising response when varying the infectious dose.
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