Download Foot-and-mouth disease virus causes transplacental infection and death in foetal lambs

Appendix 60
Foot-and-mouth disease virus causes transplacental infection and death in foetal
lambs
Eoin Ryan1,2*, Jacquelyn Horsington1, Harriet Brooks2, Joe Brownlie2, Zhidong Zhang1.
1
Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey, GU24 0NF,
UK.
2
Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK.
*[email protected]
Abstract:
Introduction: There are field reports of foot-and-mouth disease virus (FMDV) causing
severe abortion in sheep and yet there are no published studies of experimental infection
of pregnant sheep. This would appear to be the first reported investigation of experimental
FMDV pathogenesis in foetal lambs.
Materials and Methods: Using FMDV type O/UKG/2001/34, we infected pregnant sheep at
two stages of pregnancy, 45 days and 75 days, and sequentially killed the dams postinoculation. Foetal lambs, the dams and foetal fluids were examined for virus using viral
isolation and real time reverse-transcription polymerase chain reaction. Foetal tissues were
also examined for the major cytokines (IFN, IFN, IFN, TNF, IL-1). In situ hybridisation
(ISH) was used to visualise FMDV RNA in foetal tissues.
Results: Infectious virus and viral RNA were isolated from amniotic fluid and foetuses from
2 dpi onwards. At 7 dpi, generalised systemic infections had been established in most
foetuses. At 18 dpi in the 75-day foetuses, 5 of 7 foetuses were infected; of these, 4 had
died in utero. There was an acute anti-viral cytokine response (IFN, IFN) at 2 and 4 dpi,
with the pro-inflammatory cytokines (IFN, TNF and IL-1) increasing from 7 dpi. Using ISH,
viral RNA was visualised in tongue epithelium, myocardium and skeletal muscle.
Discussion: The results show FMDV caused transplacental infection in foetal lambs, and
was associated with the deaths of some foetuses. There was an excellent degree of
correlation between the increase in cytokines and foetal deaths, and increased cytokine
levels were particularly noticeable in the myocardium. The presence of infectious virus in
foetal fluids at 18 dpi demonstrates the potential for FMDV-induced abortion to cause
further disease transmission, an epidemiological factor previously overlooked.
Introduction:
Foot-and-mouth disease (FMD) in sheep is milder than in cattle or pigs, with viraemia
present for up to three days before the appearance of vesicular lesions, as reviewed by
Alexandersen et al (2003) . During this time the sheep may be pyrexic and distressed with
lameness spreading through the flock. Agalactia may occur in ewes. Vesicular lesions occur
in the interdigital cleft, along the coronary bands and on the bulb of the heels. Oral lesions
are less common but can occur on the dental pad, tongue and gums (Barnett and Cox,
1999, Hughes et al., 2002, Pay T. W, 1988). Death may occur in young lambs (Ryan,
2004).
There are field reports of FMD causing severe outbreaks of abortion in sheep (Geering,
1967, Nazlioglu, 1972) yet there are no published studies of experimental infection of
pregnant farm animals. Oppermann (1921), cited by Littlejohn (1970), reported that in the
1920 epizootic in Germany the average abortion rate was 3-10% but was sometimes up to
37%, and as high as 100% in isolated cases. Abortion occurred in ewes at various stages
of gestation and usually began 10 days to six weeks after the start of the outbreak.
Parakin (1961), cited by Littlejohn (1970), described an outbreak in Russia in which 230 of
a flock of 754 ewes aborted, and mentioned that abortion may occur as early as the first to
second month of gestation. During the 2001 outbreak in the UK, large numbers of
abortions were reported in sheep in Dumfries within 24 hours of the onset of lameness in
the flock (Reid, 2002). Andersen and Campbell (1976) reported inoculation of pregnant
mice with an attenuated FMDV type O strain, and concluded that “the placenta serves as
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an active site of infection for FMDV in pregnant mice, but the foetus is relatively resistant
to infection”.
The absence of previous experimental data regarding the possible transmission of FMDV
transplacentally, its role in causing abortion and the potential of the foetus and associated
fluids to act as a reservoir of infection has hindered understanding of the disease. The
epidemiological significance of a uterine reservoir of virus has not previously been
examined. For these reasons, we carried out a series of experiments to address these
issues. In the present study, we demonstrated that FMDV caused transplacental infection
and foetal death.
Materials and methods:
21 ewes, 7 pregnant 45 days and 14 pregnant 75 days, were used. Three ewes, one
pregnant 45 days and two pregnant 75 days, were killed as negative controls and negative
control samples of serum were taken from all remaining ewes prior to inoculation. The
ewes were inoculated in the coronary band with FMDV O UKG34/2001, as described
previously (Alexandersen and Donaldson, 2002), each animal receiving 5.9 log10 TCID50
ml-1. Serum samples were collected daily, temperatures were measured and clinical signs
noted. Two ewes were killed from each pregnancy group at two, four and seven days post
inoculation (dpi). Of the remaining six sheep at day 75 of pregnancy, three were killed at
17 dpi and three at 18 dpi (see table 1). Serum samples were tested for the presence of
antibodies to FMDV by an enzyme-linked immunosorbent assay (ELISA) (Ferris, 1987;
Ferris et al., 1990). At post-mortem examination, tissue samples were taken and put in
RNAlater (Ambion) for RNA extraction, 50% (v/v) glycerol in M25 phosphate buffered
saline and 10% formalin for histological examination. Amniotic fluid samples were taken
and stored at -80oC. Tissue samples in RNAlater were stored at -20oC. Due to their smaller
size and less developed stage, 8-11 different tissue samples were taken from each foetus
at day 45 of gestation, compared to 14 samples per foetus at day 75 of gestation.
Automated RNA extraction from samples was performed using a MagNA Pure LC robotic
workstation (Roche) and the accompanying nucleic acid isolation kits (Roche), according to
the manufacturer’s instructions, as described previously (Quan et al., 2004). Extracted
RNA was stored at -80oC until used. The level of viral RNA in samples was quantified by
real-time RT-PCR as described previously (Alexandersen et al., 2001, Reid et al., 2001,
Zhang and Alexandersen, 2003), using a Taqman probe and primers specific for FMDV
type O strain UKG 34/2001 as described previously (Quan et al., 2004). Similarly, levels of
IFN, IFN, IFN, TNF and IL-1 were quantified by real-time RT-PCR using specific Taqman
probes and primers, as described previously (Zhang et al., 2006). Cytokine levels were
normalised to the housekeeping gene GAPDH. PCR assays were performed on a Stratagene
MX4000 machine. Statistical analysis was carried out using Minitab version 14. The
infectivity of tissue samples was determined by inoculation of monolayers of primary
bovine thyroid (BTY) cells, essentially as previously described (Snowdon, 1966).
Serotyping ELISA was used to confirm the presence of FMDV in the supernatant of the
positive tubes (Ferris and Dawson, 1988).
In situ hybridisation (ISH) was carried out using a digoxenin-labelled RNA probe to the 3D
region of the FMDV genome. ISH was performed on formalin-fixed, paraffin-embedded
sections using an mRNAlocator ISH kit (Ambion). An RNA ISH probe for SVDV was used as
a negative control on all sections, and ISH was also performed on tissues from control
foetuses as a further negative control.
Results:
No vesicular lesions observed on foetuses: None of the 11 foetuses at day 45 of gestation
at inoculation had grossly visible pathological changes. In the group of day 75 of gestation
at the start of the experiment, of the 19 foetuses, 10 had gross abnormalities, which
included multiple cutaneous petechial haemorrhages, reddened peritoneal fluid,
subcutaneous oedema, ascites and epicardial petechiae (table 1). Two of these foetuses
had no detectable viral RNA and were negative for virus isolation. Three of the foetuses at
17 dpi and one at 18 dpi were not freshly dead at post-mortem examination and were
showing signs of autolysis. However, no vesicular lesions were visible on any foetuses. No
381
significant pathological lesions were visible on histological examination of foetal and
placental tissues.
Table 1: The gestational age and gross pathological lesions in foetuses.
Foet
us ID
Gestation
al age*
0
2
2
Gross
abnormalities at
post-mortem
examination
None
None
None
FA17
FB17
FA11
75
75
75
2
2
4
45
2
None
FB11
75
4
FA56
FB56
FA57
45
45
45
4
4
4
None
None
None
FA12
FB12
FA09
75
75
75
4
4
7
FB57
45
4
None
FB09
75
7
FA58
45
7
None
FA13
75
7
FB58
45
7
None
FB13
75
7
FA59
45
7
None
FA07
75
17
FB59
FA05
45
75
7
0
None
None
FA08
FA10
75
75
17
17
FB05
75
0
None
FB10
75
17
FA06
75
0
None
FA14
75
18
FA16
75
2
None
FA15
75
18
FB16
75
2
None
FA18
75
18
Foetu
s ID
Gestati
onal
age*
dp
i†
FA53
FA54
FA55
45
45
45
FB55
*
†
dpi
†
Gross
abnormalities at
post-mortem
examination
None
None
Dead before 0
dpi
Cutaneous
petechial
haemorrhages
None
None
Cutaneous
petechiation
Cutaneous
petechiation
Cutaneous
petechiation,
reddened
peritoneal fluid
None
Dead, autolyzed,
reddened
subcutaneous
oedema
None
Dead, autolyzed,
swollen abdomen
Dead, autolyzed,
swollen abdomen
Cutaneous
petechiation,
ascites,
epicardial
haemorrhages
Cutaneous
petechiation
Dead, autolyzed
The gestational age of foetuses at the day the ewes were inoculated.
dpi: days post inoculation at the day the ewes were killed.
Viral RNA and infectious virus in foetal tissues during acute infection: Viral RNA levels in
tissues from foetuses which were at 45 days gestation at maternal inoculation are shown
in table 2. Low levels of viral RNA were detected in a few tissues at 2 and 4 dpi, but at 7
dpi viral RNA was detected in every tissue sample examined from all four foetuses
although there was no detectable viraemia in ewes at this time. Among the tissues
examined from these foetuses, heart and tongue samples contained the highest level of
viral RNA.
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Table 2 Viral RNA in foetal tissues, 45 days gestation
Viral RNA load
dpi
foetus
ID
IV
†
0
FA53*
FA54
2
FA55
FB55
FA56
FB56
4
FA57
FB57
FA58
FB58
7
FA59
FB59
N
N
N
P
N
N
N
N
P
P
P
P
ND
0
0
0
0
0
1.80
0
5.55 6.05 7.36
intestine
0
0
0
0
0
heart
0
0
0
5.75 9.01 8.92
0
0
0
0
0
kidney
0
0
0
5.64 6.81 6.18
0
0
0
0
0
leg
0
0
0
3.67 7.73 7.42
0
0
0
0
0
liver
1.74
0
0
4.81 5.93 5.19
0
0
0
0
0
lung
1.66
0
0
6.60 7.02 7.56
0
0
ND
0
mouth
3.41 1.82
0
1.94
7.65 8.47 8.03
soft
ND
ND
ND
ND
ND
0
ND
ND
palate
7.27 7.73 7.69
ND
0
ND
ND
ND
0
0
0
spleen
5.87 5.67 7.61
ND
ND
ND
ND
0
ND
ND
tongue
0
9.20 8.99 8.16
* uninfected foetus was killed as control and tissues collected for analysis.
† viral RNA loads in tissues were quantified by real-time RT–PCR and expressed as log10
copy number per g tissue
ND: no samples taken.
IV: infectious virus in tissue samples. P: positive, N: negative
dpi: days post inoculation at the day the ewes were killed
Viral RNA levels in tissues from foetuses which were at 75 days gestation at maternal
inoculation are shown in table 3. No viral RNA was detected at 2 dpi, and only low levels at
4 dpi. At 7 dpi, although there was no detectable viraemia in ewes at this time, viral RNA
was detected in every tissue sample from three of the foetuses. Among the tissues
examined, muscle and tongue samples contained the highest level of viral RNA. At 17 dpi,
three of the four foetuses contained viral RNA. All of them were dead and autolyzed at
post-mortem examination. At 18 dpi, two of three foetuses were positive for viral RNA.
One foetus, dead and autolyzed at post-mortem examination, had viral RNA in all tissue
samples at a higher level than those in the second foetus, which had survived to this
timepoint and which had viral RNA in 8 of 14 tissue samples examined.
As shown in tables 2 and 3, infectious virus was isolated from foetuses at both stages of
pregnancy. In the 45 day gestation group (n=11), of eight foetuses which were positive for
viral RNA, five tested positive for infectious virus and three negative. In the 75 day
gestation group, among foetus tissues examined (n=12), virus was only isolated from
tissues collected at 7 dpi from the three foetuses (n=3) in which viral RNA had been
detected. It was not possible to perform virus isolation on samples from 17 and 18 dpi due
to inadvertent sample deterioration during processing.
Viral RNA and infectious virus in amniotic fluid samples: Data on viral RNA load and
infectious virus in amniotic fluid samples is summarised in table 4. Viral RNA was detected
in 5 of 11 amniotic fluid samples taken from the group at day 45 of gestation at the start
of the experiment and in 7 of 19 amniotic fluid samples taken from the group at day 75 of
gestation at the start of the experiment. As shown in table 4, amniotic fluid associated with
foetuses which were free of viral RNA (FA54, FB57) could contain detectable level of viral
RNA, while the other amniotic fluid samples did not contain detectable viral RNA, although
the foetuses associated with them were positive for viral RNA (FA58, FB59) (table 2).
Infectious virus was isolated from amniotic fluid samples collected from both groups. The
supernatant from all positive virus isolation tubes was confirmed as FMDV type O by
serotyping ELISA.
383
6.87
8.77
6.68
7.51
6.57
7.20
7.80
8.28
6.68
8.29
Table 4 Quantification of viral RNA in amniotic fluid*
Foetus ID
Viral load †
IV
Foetus ID
Viral load †
IV
#
FA53
0
N
FA17
0
N
FA54
4.96
P
FB17
0
N
FA55
0
N
FA11
0
N
FB55
0
P
FB11
0
N
FA56
0
N
FA12
0
N
FB56
0
P
FB12
0
N
FA57
5.18
P
FA09
0
N
FB57
7.29
P
FB09
0
P
FA58
0
N
FA13
3.51
P
FB58
10.37
P
FB13
3.68
P
FA59
6.06
P
FA07
4.69
P
FB59
0
P
FA08
0
N
#
FA05
0
N
FA10
4.25
N
FB05#
0
N
FB10
4.28
N
FA06#
0
N
FA14
1.71
P
FA16
0
N
FA15
0
N
FB16
0
N
FA18
6.58
P
*Amniotic fluid samples were collected from both the group at day 45 of gestation at the
start of the experiment and the group 75 days pregnant at the start of the experiment.
†
The viral RNA levels were quantified by real-time RT–PCR and expressed as log10 copy
number per ml sample.
#
uninfected foetuses as controls
IV: infectious virus in tissue samples. P: positive, N: negative
Cytokine responses in foetuses at day 75 of gestation: Levels of IFN and IFN increased in
the cervical lymph nodes (LN) of infected foetuses at 2 and 4 dpi by a factor of 3-4 times
control levels. By 7 dpi, levels were increased in all tissues by factors ranging from 3.4
(spleen) to 24 (kidney) times control levels, while the levels in skin increased by 319 and
252 times for IFN and IFN respectively. At 17/18 dpi, levels in all tissues increased by
factors ranging from 703 (skin) to 18,552 (heart). When ANOVA was carried out, the
difference in IFN response between the infected and uninfected foetuses was found to be
significant (p<0.05) in the coronary band, intestine, heart, liver, lung, soft palate, spleen
and tongue. For IFN, a significant (p<0.05) difference was found in all tissues except skin
and tongue. Scatterplots of log10 fold increases in levels of IFN are shown in fig. 1 and IFN
in fig. 2.
Levels of IFN did not increase at 2 and 4 dpi, but there was an increase at 7 dpi in some
tissues, with cervical LN and tonsil having 11 times control levels and skin showing an
increase of 51 times control levels. At 17/18 dpi, levels in all tissues increased by factors
ranging from 235 (mandibular LN) to 14,013 (heart) (see fig. 3). A significant (p<0.05)
difference was found in the IFN response between infected and uninfected foetuses in the
cervical LN, heart, liver, muscle and spleen.
Levels of TNF increased in the cervical LN by a factor of 10 at 2 dpi, but not in other
tissues. At 4 dpi, levels in the cervical LN were increased by 24 times, while levels in some
other tissues increased by factors of 2 (tonsil) to 8 (intestine), with no increase in coronary
band or muscle. At 7 dpi, there was an increase of 905 times control levels in skin, with
increases in other tissues ranging from 3 times (spleen) to 41 times (cervical LN) control
levels. By 17/18 dpi, levels in all tissues increased by factors ranging from 112
(mandibular LN) to 20,933 (heart) (see fig. 4). The differences in the TNF responses in the
cervical LN, heart, lung and spleen were found to be significant (p<0.05).
Levels of IL-1 did not increase in any tissues, and showed a slight decrease in some
tissues, at 2, 4 and 7 dpi. At 17/18 dpi, levels were increased in all tissues by factors
ranging from 25 (coronary band) to 3717 (liver) times control levels (see fig. 5). All tissues
except kidney, liver, mandibular LN and tonsil showed a significantly (p<0.05) different IL1 response in the infected foetuses.
384
Viral RNA visualistion in tissues using in situ hybridisation: Positive ISH signal was
detected at the highest levels in the skeletal muscle of the tongue and leg, in the cardiac
muscle and in the orbital muscles. Positive signal was only occasionally seen in the
epithelium of the tongue. Viral RNA was visualised in the cytoplasm of the infected cells.
Figure 6 shows positive ISH signal in these tissues.
Discussion:
FMDV infection in foetuses: Although all ewes developed clinical FMD and viraemia
following inoculation, not all foetuses became infected. In some cases, one twin became
infected while a co-twin did not. The reason for this phenomenon remains unclear. The
results do not demonstrate a minimum maternal viraemia level necessary for
transplacental infection, as the ewe with the lowest amount of viral RNA in its serum still
transmitted FMDV to its foetus.
The gross abnormalities in the foetuses included multiple cutaneous petechial
haemorrhages, reddened peritoneal fluid, subcutaneous oedema, ascites and epicardial
haemorrhages. These are non-specific findings, and viral replication has not been shown to
be directly responsible for them. The finding that two uninfected foetuses exhibited these
changes supports this. One foetus killed at 7 dpi (foetus FB13) was positive by real time
RT-PCR and virus isolation, yet no gross abnormalities were recognised. No placental
abnormalities were recognised.
In the infected foetuses at 2 and 4 dpi, a low level of viral RNA was detected in epithelial
tissues such as the coronary band, tongue, skin, soft palate and mouth. These are tissues
that are the sites of initial virus replication in adult infections (Alexandersen et al., 2003).
The virus replicated at these sites and spread throughout the body of the foetus.
Transplacental infection occurred at 2-4 dpi, the time of peak maternal viraemia. By 7 dpi,
the infection had become systemic, with viral RNA detected at high levels throughout the
foetus.
Amniotic fluid samples associated with infected foetuses tested positive by virus isolation
and real-time RT-PCR. The volume of amniotic fluid associated with a sheep foetus is
variable and is influenced by the stage of gestation and whether the foetus is a singleton,
twin or triplet (Wintour et al., 1986). We estimated the average volume per foetus in this
study at day 45 to be 200-300 ml and at 75 days to be 500-700 ml. Thus if an infected
foetus and associated fluids were expelled from the ewe after FMD-related abortion, a
large amount of FMDV would be released into the surrounding environment. This could
have profound effects on the spread of the disease. A pregnant sheep could be infected
with FMDV but not show severe clinical signs and thus escape detection. We have shown
that up to 18 days later, such a sheep, now fully recovered from disease and showing no
clinical signs, may be carrying a foetus and associated foetal fluids containing large
amounts of virus.
Cytokine response to FMDV infection: The increase in levels of mRNA of the anti-viral
cytokines IFN and IFN and the pro-inflammatory cytokines IFN, TNF and IL-1 during foetal
FMDV infection provides an insight into the pathogenesis of the disease. The initial type 1
interferon (α and β) response occurred at 2 and 4 dpi, when the virus was crossing the
placenta and establishing an infection. FMDV has been shown to inhibit type 1 interferon
expression by L proteinase blocking both translation and transcription in cell culture
(Chinsangaram et al., 1999, de Los Santos et al., 2006). This has not been shown in vivo,
but a reduction in type 1 interferon production may occur to some degree. Other work has
shown an up-regulation of IFN mRNA during FMD in cattle (Brown et al., 2000). The data
presented here agrees with that, showing an acute innate immune anti-viral response to
FMDV.
This initial type 1 interferon response was followed with a pro-inflammatory response from
7 dpi. Although it has been proposed that the ovine foetal immune system has a Th2 bias
due to placental progesterone (Entrican, 2002), the strong Th1 response shown here
suggests this may not be correct. IFN has been shown to be an inhibitor of persistent
FMDV infection (Zhang et al., 2002), but it also stimulates TNF release. The responses to
TNF and IL-1 in foetal lambs have been studied as a model for premature babies (Ikegami
et al., 2003, Mulrooney et al., 2004). IL-1 was associated with chorioamnionitis,
385
bronchopulmonary dysplasia and premature parturition, while TNF was a potent inducer of
a generalised inflammatory response. The strong correlation between increases in these
cytokines and foetal death, particularly in the heart tissue, suggest that cytokine-mediated
viral-induced inflammation was a factor in the in utero deaths.
Viral RNA visualistion in tissues using in situ hybridisation: The localisation of viral RNA
chiefly in the skeletal and cardiac muscle shows that FMDV in foetuses has a different
tissue tropism than in adults, and also explains the lack of epithelial vesicular lesions. We
have previously reported viral RNA in the myocardium of infected young lambs (Ryan,
2004), but not in the myocytes of the tongue, leg and orbital muscles.
This study is the first we are aware of to demonstrate the ability of FMDV to cause
infection and death in foetal lambs. It establishes that amniotic fluid may contain FMDV up
to 18 dpi - a possible mechanism by which infected but recovered pregnant sheep may
cause fresh outbreaks of FMD by release of aborted foetuses and associated fluids.
Conclusions:
•
FMDV causes transplacental infection and death in foetal lambs.
•
Foetal pathology and death was associated with a pro-inflammatory cytokine
response and virus localisation in skeletal and cardiac muscle.
•
Infected foetuses and associated fluids can be infectious up to 18 dpi.
Recommendations:
•
Further research into infection at different stages of gestation.
•
Research on transplacental transmission in vaccinated sheep.
•
Research on epidemiological risk posed by uterine reservoir of infection.
References:
Alexandersen, S., and A. I. Donaldson. 2002. Further studies to quantify the dose of
natural aerosols of foot-and-mouth disease virus for pigs. Epidemiol Infect
128:313-323.
Alexandersen, S., M. B. Oleksiewicz, and A. I. Donaldson. 2001. The early
pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative
time-course study using TaqMan RT-PCR. J Gen Virol 82:747-755.
Alexandersen, S., Z. Zhang, A. I. Donaldson, and A. J. M. Garland. 2003. The
pathogenesis and diagnosis of foot-and-mouth disease. J Comp Pathol 129:1-36.
Andersen, A. A., and C. H. Campbell. 1976. Experimental placental transfer of foot-andmouth disease virus in mice. Am J Vet Res 37:585-9.
Barnett, P. V., and S. J. Cox. 1999. The role of small ruminants in the epidemiology and
transmission of foot-and-mouth disease. Vet J 158:6-13.
Brown, C. C., J. Chinsangaram, and M. J. Grubman. 2000. Type I interferon
production in cattle infected with 2 strains of foot-and-mouth disease virus, as
determined by in situ hybridization. Can J Vet Res 64:130-3.
Chinsangaram, J., M. E. Piccone, and M. J. Grubman. 1999. Ability of foot-and-mouth
disease virus to form plaques in cell culture is associated with suppression of
alpha/beta interferon. J Virol 73:9891-8.
de Los Santos, T., S. de Avila Botton, R. Weiblen, and M. J. Grubman. 2006. The
leader proteinase of foot-and-mouth disease virus inhibits the induction of beta
interferon mRNA and blocks the host innate immune response. J Virol 80:1906-14.
Entrican, G. 2002. Immune regulation during pregnancy and host-pathogen interactions
in infectious abortion. J Comp Pathol 126:79-94.
386
Ferris,
N. P., and M. Dawson. 1988. Routine application of enzyme-linked
immunosorbent assay in comparison with complement fixation for the diagnosis of
foot-and-mouth and swine vesicular diseases. Vet Microbiol 16:201-9.
Geering W, G. 1967. Foot and mouth disease in sheep. Aust Vet J.
Hughes, G. J., V. Mioulet, R. P. Kitching, M. E. Woolhouse, S. Alexandersen, and A.
I. Donaldson. 2002. Foot-and-mouth disease virus infection of sheep:
implications for diagnosis and control. Vet Rec 150:724-7.
Ikegami, M., T. J. Moss, S. G. Kallapur, N. Mulrooney, B. W. Kramer, I. Nitsos, C. J.
Bachurski, J. P. Newnham, and A. H. Jobe. 2003. Minimal lung and systemic
responses to TNF-alpha in preterm sheep. American journal of physiology. Lung
cellular and molecular physiology 285:L121-9.
Littlejohn, A. 1970. FMD in sheep - part 1. State Veterinary Journal 25:3-12.
Mulrooney, N., A. H. Jobe, and M. Ikegami. 2004. Lung inflammatory responses to
intratracheal interleukin-1alpha in ventilated preterm lambs. Pediatr Res 55:682-7.
Nazlioglu, M. 1972. Fmd in Sheep and Goats. Bulletin Office International des Epizooties
77:1281-1284.
Pay T. W, F. 1988. FMD in sheep and goats: a review. FMD Bulletin 26:2-13.
Quan, M., C. M. Murphy, Z. Zhang, and S. Alexandersen. 2004. Determinants of early
foot-and-mouth disease virus dynamics in pigs. J Comp Pathol 131:294-307.
Reid, H. W. 2002. FMD in a parturient sheep flock. Vet Rec 150:791.
Reid, S. M., N. P. Ferris, G. H. Hutchings, Z. Zhang, G. J. Belsham, and S.
Alexandersen. 2001. Diagnosis of foot-and-mouth disease by real-time
fluorogenic PCR assay. Vet Rec 149:621-623.
Ryan, E., Durand, S., Brownlie, J., Alexandersen, S. 2004. Presented at the Research
Group of the Standing Technical Committe of the European Commission for the
Control of Foot-and-Mouth Disease, Chania, Crete, Greece.
Snowdon, W. A. 1966. Growth of foot-and mouth disease virus in monolayer cultures of
calf thyroid cells. Nature 210:1079-80.
Wintour, E. M., B. M. Laurence, and B. E. Lingwood. 1986. Anatomy, physiology and
pathology of the amniotic and allantoic compartments in the sheep and cow. Aust
Vet J 63:216-21.
Zhang, Z., J. B. Bashiruddin, C. Doel, J. Horsington, S. Durand, and S.
Alexandersen. 2006. Cytokine and Toll-like receptor mRNAs in the nasalassociated lymphoid tissues of cattle during foot-and-mouth disease virus
infection. J Comp Pathol 134:56-62.
Zhang, Z. D., and S. Alexandersen. 2003. Detection of carrier cattle and sheep
persistently infected with foot-and-mouth disease virus by a rapid real-time RTPCR assay. J Virol Methods 111:95-100.
Zhang, Z. D., G. Hutching, P. Kitching, and S. Alexandersen. 2002. The effects of
gamma interferon on replication of foot-and- mouth disease virus in persistently
infected bovine cells. Arch Virol 147:2157-2167.
387
Fold increase in log10 IFNalpha mRNA levels vs. control
Fold increase in log10 IFNbeta mRNA levels vs. control
Fold increase in log10 IFNgamma mRNA levels vs. control
Fig. 1: Scatterplot of log10 IFNalpha vs dpi
Gestational stage = 75 days
0
CB
10
20
Cervical LN
0
10
Heart
20
Intestine
5.0
2.5
0.0
5.0
Kidney
Liver
Lung
Mandibular LN
muscle
skin
Soft palate
Spleen
status
infected
not infected
2.5
0.0
5.0
2.5
0.0
Tongue
5.0
Tonsil
0
10
20
2.5
0.0
0
10
20
dpi
Panel variable: tissue type
Fig. 2: Scatterplot of log10 IFNbeta vs dpi
Gestational stage = 75 days
0
CB
10
20
Cervical LN
0
10
Heart
20
Intestine
4
2
status
infected
not infected
0
Kidney
Liver
Lung
Mandibular LN
muscle
skin
Soft palate
Spleen
4
2
0
4
2
0
Tongue
Tonsil
0
4
10
20
2
0
0
10
20
dpi
Panel variable: Tissue
Fig. 3: Scatterplot of log10 IFNgamma vs dpi
Gestational stage = 75 days
0
CB
10
20
Cervical LN
0
Heart
10
20
Intestine
4
0
Kidney
Liver
Lung
Mandibular LN
muscle
skin
Soft palate
Spleen
status
infected
not infected
-4
4
0
-4
4
0
Tongue
Tonsil
4
-4
0
10
20
0
-4
0
10
20
dpi
Panel variable: Tissue
Figures 1-3: Scatterplots showing fold increases compared to controls in IFN, IFN and IFN
levels in infected and uninfected foetuses on a log scale.
388
Fold increase in log10 TNFalpha mRNA levels vs. control
Fold increase in log10 IL-1alpha mRNA levels vs. control
Fig. 4: Scatterplot of log10 TNFalpha vs dpi
Gestational stage = 75 days
0
CB
10
20
Cervical LN
0
10
Heart
20
Intestine
3
0
Kidney
Liver
Lung
Mandibular LN
muscle
skin
Soft palate
Spleen
status
infected
not infected
-3
3
0
-3
3
0
Tongue
-3
Tonsil
0
10
20
3
0
-3
0
10
20
dpi
Panel variable: Tissue
Fig. 5: Scatterplot of log10 IL-1alpha vs dpi
Gestational stage = 75 days
0
CB
10
20
Cervical LN
0
Heart
10
Intestine
20
5
0
5
Kidney
Liver
Lung
Mandibular LN
muscle
skin
Soft palate
Spleen
status
infected
not infected
-5
0
-5
5
0
Tongue
5
Tonsil
-5
0
10
20
0
-5
0
10
20
dpi
Panel variable: Tissue
Figures 4&5: Scatterplots showing fold increases compared to controls in TNF and IL-1
levels in infected and uninfected foetuses on a log scale.
389
6A
6B
6C
6D
6E
6F
6G
6H
Figure 6: ISH on sections from FMDV-infected foetuses. A: tongue from foetus
FA14, 4x magnification, and B: tongue from FA14, 40x, showing abundant
positive signal in myocytes. C: tongue from FB09, 40x, showing viral RNA in
basal layer of epithelium and in muscle. D: tongue from FB09, 63x, showing
cytoplasmic staining in multinucleated myocytes. E: heart from FB09, 40x, with
diffuse positive signal in myocardium. F: heart from FA58, 40x, with focal area
of viral RNA in myocardium. G: leg section from FA58, 40x, positive signal in
skeletal muscle. H: orbital muscle from FB58, 40x, positive cytoplasmic staining.
390
Table 3: Viral RNA in foetal tissues, 75 days gestation
* three uninfected foetuses were killed as controls and tissues collected for analysis.
†viral RNA loads in tissues were quantified by real-time RT–PCR and expressed as log10 copy number per g tissue.
Viral RNA load†
dpi
foetus ID
0
control*
2
FA16
FB16
4
FA17
IV
N
N
N
coronary
0
0
0
band
0
0
0
cervical LN
0
0
0
intestine
0
0
0
heart
0
0
0
kidney
0
0
0
liver
0
0
0
lung
mandibular
0
0
0
LN
0
0
0
muscle
0
0
0
skin
0
spleen
0
0
0
tongue
0
0
0
soft palate
0
0
0
tonsil
0
0
ND: no samples taken.
dpi: days post inoculation at the day the
IV: infectious virus in tissue samples. P:
7
17
FA08 FA10
FB10
FA14
18
FA15
FA18
ND
7.03
ND
6.99
ND
0
ND
0
ND
9.21
0
0
0
0
0
0
0
8.60
6.79
9.01
7.54
7.04
7.94
ND
7.83
7.08
8.71
7.80
6.73
7.34
ND
0
0
5.58
0
0
6.42
5.92
0
0
0
0
0
0
0
9.10
7.64
10.58
8.79
7.07
8.74
9.75
0
0
0
0
0
0
8.90
9.00
ND
8.58
9.32
ND
9.26
7.71
7.66
7.91
8.53
ND
7.00
5.92
0
8.41
6.39
6.93
0
0
0
0
0
0
10.81
9.08
8.28
10.33
10.34
9.89
FB17
FA11
FB11
FA12
FB12
FA09
FB09
FA13
FB13
FA07
N
0
N
0
N
0
N
5.20
N
0
N
4.79
N
0
P
9.77
P
9.86
P
9.13
ND
7.67
ND
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ND
0
0
0
0
0
ND
0
0
0
5.14
0
0
0
0
0
0
0
4.82
0
0
0.00
0
0.00
0.00
0.00
0
0
0
0
0
0
0
0
0
10.43
9.09
9.99
8.31
9.06
7.69
11.25
10.24
8.51
10.47
8.62
8.33
8.44
10.99
10.49
7.80
10.62
7.85
7.97
7.53
11.01
7.99
7.98
9.17
8.64
7.21
8.24
ND
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4.95
0
0
0
0
4.85
5.21
5.13
0
4.77
5.29
0
0
0
0
0
0
0
0
0
0
11.70
9.49
8.86
12.16
9.46
11.88
12.01
9.59
11.26
11.71
9.47
11.04
11.18
9.76
9.06
9.75
7.65
7.77
9.17
9.18
ND
0
ND
0
0
ND
11.42
10.83
10.92
ewes were killed
positive, N: negative
391