Journal of Reproductive Immunology 90 (2011) 214219

Contents lists available at ScienceDirect

Journal of Reproductive Immunology

journal homepage: www.elsevier.com/locate/jreprimm

Interferon- expression in trophoblast cells in pregnant ewes

challenged with Chlamydophila abortus
S. Worrall a, , D.J. Sammin b , H.F. Bassett a , C.R. Reid a , J. Gutierrez a , P.X. Marques a ,
J.E. Nally a , J. ODonovan c , E.J. Williams a , A. Proctor a , B.K. Markey a
a
School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Beleld, Dublin 4, Ireland
b
Central Veterinary Research Laboratory, Department of Agriculture Fisheries and Food, Backweston, Cellbridge, Co. Kildare, Ireland
c
Regional Veterinary Laboratory, Department of Agriculture Fisheries and Food, Coosan, Athlone, Co. Westmeath, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: Pregnant ewes were challenged with Chlamydia abortus at 9198 days of gestation and
Received 27 November 2010 euthanised at 14, 21 and 28 days post-challenge. IFN mRNA labelling appeared to be
Received in revised form 15 March 2011 co-localised with Chlamydial lipopolysaccharide within trophoblast cells in discrete areas
Accepted 21 March 2011
lining the primary villi in the limbus and hilar zone of the placentomes from challenged
sheep on days 21 and 28 post-infection. The presence of IFN was also demonstrated by
Keywords: immunohistochemistry. No labelling was seen in tissues from the non-infected ewes. The
Chlamydophila abortus
presence of IFN in trophoblast cells from infected ewes may indicate an attempt to restrict
Interferon-gamma
the replication of the organism and be an important trigger for the inammatory responses
Trophoblast cells
Ovine enzootic abortion that develop on the fetal side of the placenta in enzootic abortion.
In situ hybridization 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction et al., 2006). Humans and rodents have a very inva-

Chlamydophila abortus, formerly the ovine sub-type
of Chlamydia psittaci, is a Gram-negative obligate intra-
cellular pathogen responsible for enzootic abortion of
ewes (EAE) (Entrican et al., 2001; Longbottom and
Coulter, 2003). In infected pregnant ewes the organism
invades the chorionic epithelium (trophoblast cells) of
the placenta where it replicates producing intracellu-
lar inclusions (Stamp et al., 1950; Buxton et al., 1990).
After 4872 h the cells rupture, releasing elementary bod-
ies that invade adjacent cells (Moulder, 1991). Pregnant
ewes infected with C. abortus develop a severe placen-
titis associated with an innate fetal immune response
and an acquired maternal immune response (Sammin
Corresponding author at: School of Agriculture, Food Science and Vet-
erinary Medicine, Veterinary Science Centre, University College Dublin,
Beleld, Dublin 4, Ireland. Tel.: +353 01 7166154; fax: +353 01 7166165.
E-mail address: sheila.worrell@ucd.ie (S. Worrall).
sive haemochorial placenta with direct contact between
placental trophoblast cells and maternal circulation. In
contrast, the ovine cotyledonary synepitheliochorial pla-
centa (Wooding, 1992) has limited trophoblast invasion of
maternal tissue found only in the placentomes (Gogolin-
Ewens et al., 1989). Protection of the allogenic fetus from
rejection by the maternal immune system is achieved
partly by lack of expression of major histocompatibility
complex (MHC) class I antigens on the ovine trophoblast
cells (Gogolin-Ewens et al., 1989). In addition there are
fewer lymphocytes present in the placentomes com-
pared with interplacentomal areas (Gogolin-Ewens et al.,
1989), and secretion of lymphocyte-inhibitory uterine
milk proteins may induce a local immune suppres-
sion (Skopets and Hansen, 1993; Peltier and Hansen,
2001).
Interferon- (IFN) is a cytokine produced by acti-
vated T lymphocytes and natural killer (NK) cells and is a
potent activator of macrophages in both innate and adap-
tive cell-mediated immune responses. It also stimulates the

0165-0378/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jri.2011.03.011
 S. Worrall et al. / Journal of Reproductive Immunology 90 (2011) 214219
expression of MHC class II molecules on antigen presenting
cells (APC), especially macrophages, and this may amplify
T-cell responses (Schroder et al., 2004). Consequently, the
expression of pro-inammatory cytokines such as IFN is
generally regarded as detrimental to pregnancy as these
cytokines may induce maternal T-cell responses, leading
to rejection of the fetal allograft (Wegmann et al., 1993;
Raghupathy, 1997). IFN has also been shown to restrict
the growth of C. abortus in vitro in a dose-dependent man-
ner (Byrne et al., 1986; Brown et al., 2001). It is associated
with persistence of C. abortus in non-pregnant ewes follow-
ing primary infection, where it is believed to be capable of
restricting the growth of the replicative form of the bacte-
ria by tryptophan starvation (Entrican et al., 1998; Brown
et al., 2001).
As part of a multidisciplinary investigation into ovine
fetal and maternal inammatory responses to C. abortus
in sheep, this study examined the expression pattern of
IFN mRNA within the placentomes of sheep experimen-
tally infected with C. abortus.
2. Materials and methods
2.1. Experimental animals
Eighteen adult crossbred ewes, seronegative for C. abor-
tus, were sourced from ocks with no known history of
enzootic abortion (Sammin et al., 2006). Twelve ewes were
inoculated intravenously with the C95-27 Irish strain of C.
abortus propagated in a yolk sac suspension and six were
inoculated with uninfected yolk sac suspension at 9198
days of pregnancy. Groups of four challenged and two
control ewes were euthanised at 14, 21 and 28 days post-
infection. Sections of placenta were xed in 4% buffered
formal saline, dehydrated, cleared and embedded in paraf-
n wax. As far as possible serial sections were used in the
following methods.
2.2. In situ hybridisation
A linearised cDNA template for ovIFN (436 bp; kindly
provided by the Moredun Research Institute, Scotland)
(McInnes et al., 1990), was used to transcribe digoxigenin
(DIG)-labelled riboprobes. No signicant homologies were
detected when the probe sequence was compared with
the complete genome sequence of C. abortus S26/3 (Ref-
seq: NC 004552) by BLAST (http://www.ncbi.nlm.nih.gov).
Using T7 and SP6 RNA polymerases both sense and anti-
sense riboprobes incorporating digoxigenin UTP were
produced using a DIG RNA labelling kit (SP6/T7; Roche
Diagnostics, West Sussex, UK). The concentration of
each labelled probe was determined by dotblot visu-
alisation and a NanoDrop spectrophotometer. Pairs of
serial sections (7 m) from formalin-xed, parafn-
embedded placentomes were placed onto positively
charged slides (Superfrost Plus, Menzel-Glsser, Braun-
schweig, Germany) and air dried prior to incubation at 57 C
for 1 h. Sections were de-waxed in xylene, rehydrated in
ethanol, then treated with 200 mM HCl for 20 min. Pre-
treatment to optimise mRNA labelling involved placing
slides in a solution of 0.01 M sodium citrate buffer, pH 6.0
215
for 20 min in a 700 W microwave (Lan et al., 1996). The
remainder of the protocol was performed as described by
Anderson et al. (2001) using the probes at a concentration
of 1 ng/l. A comparison was carried out on a number of
slides using an alternative pre-treatment with proteinase
K (Roche Diagnostics) at a concentration of 25 g/ml in
1 M CaCl, 1 M Tris-HCl pH 8.0 for 15 min at 37 C. Negative
controls were probed with sense riboprobes. In addition a
number of sections were pre-treated with RNAse overnight
at 37 C to degrade the mRNA prior to hybridisation with
antisense probe. Ovine tuberculous lymph node (Mycobac-
terium bovis infected sheep) was used as positive control
tissue (Malone et al., 2003).
2.3. Immunohistochemistry
Sequential sections of placenta were immunolabelled
to conrm the presence of chlamydial inclusions and
IFN protein. Mouse monoclonal antibodies for chlamydial
lipopolysaccharide clone AC1-P (IgG3 ; Progen, Heidelberg,
Germany) and IFN, CC302 (IgG1, MCA 1783B), a mouse
anti-bovine IFN (Serotec, Oxfordshire, UK) were used.
Detection was performed using the EnVisionTM System
reagents (DakoCytomation, UK). Parafn sections (5 m)
were de-waxed in xylene and rehydrated through graded
alcohol concentrations to water. Antigen retrieval was
carried out in 0.01 M sodium citrate buffer pH 6.0 in
a 700 W microwave oven, 20 min for IFN, and 10 min
for chlamydial LPS. Endogenous peroxidase was blocked
using a peroxidase blocking solution (DakoCytomation,
UK) for 15 min at room temperature, and non-specic
binding was minimised with 2.5% normal goat serum
(DakoCytomation, UK) in phosphate buffered saline for
15 min at room temperature. Slides were incubated with
primary antibody diluted in antibody diluent (DakoCy-
tomation, UK). Mouse anti-IFN was used at 1:100 dilution
(2.5 g/ml) and incubated overnight at room temperature,
whereas mouse anti-chlamydial LPS was used at 1:20 dilu-
tion (2.5 g/ml) and incubated overnight at 4 C. Negative
control sections were incubated with Mouse N-Universal
Negative Control reagent (DakoCytomation, UK) for the
same times. Slides were then incubated for 30 min at
room temperature with a link antibody (horseradish per-
oxidase for IFN, and alkaline-phosphatase for chlamydial
LPS) and developed using chromogen substrate solutions,
3,3 -diaminobenzidine (DAB) (DakoCytomation) for IFN
and Fast Red (DakoCytomation) for chlamydial LPS. Slides
were counterstained in haematoxylin and coverslipped.
Ovine tuberculosis lymph node sections (Mycobacterium
bovis infected sheep) that exhibited labelling of cells for
IFN mRNA (Malone et al., 2003) were used as a positive
control.
2.4. Evaluation
Sections were examined by light microscopy to evaluate
the pattern and intensity of the labelling reaction. Slides
were graded using a semi-quantitative method where: 0
represented no labelling; + weak labelling; ++ intermediate
labelling; and +++ strong labelling.
216 S. Worrall et al. / Journal of Reproductive Immunology 90 (2011) 214219

3. Results

3.1. In situ hybridisation

Labelling for IFN mRNA was detected in trophoblast

cells within discrete areas lining the primary villi from
infected ewes at 21 days post infection and 28 days post
infection (Table 1). Labelling appeared as intense, dark
intracellular deposits of chromogen in trophoblasts located
at the limbus and hilar zone of placentomes (Fig. 1A). In
these positively labelled sections no labelling was detected
in the connective tissue stroma sub-adjacent to the labelled
trophoblast cells despite the presence of a leucocytic inl-
trate. This fetal-derived leucocytic inltrate has previously
been characterised and consisted predominantly of neu-
trophils and macrophages with relatively few lymphocytes
(Sammin et al., 2006). Both microwave and proteinase K
pre-treatment produced similar distribution patterns of
labelled cells, but microwave pre-treatment resulted in
greater numbers of labelled cells with a greater intensity
of labelling. In the control sections of ovine tubercu-
lous lymph node there was distinct intracellular labelling
within lymphocytes that encircled foci of necrosis (Fig. 2A).
Labelling for IFN mRNA was not detected in placentomes
of non-infected ewes at any time point. No labelling was
evident in placentomes of infected ewes at 14 days post
infection. Labelling for IFN mRNA was not detected in sec-
tions hybridised with a sense probe, or in sections treated
with RNAse prior to hybridisation.

3.2. Immunohistochemistry

3.2.1. IFN
At 14 days post infection, one placentome from an
infected ewe (567A, Table 1) showed weak intracellu-
lar labelling for IFN in trophoblast cells. At 21 and 28
days post infection all of the placentomes from infected
ewes showed intracellular immunolabelling for IFN in
trophoblast cells. The most intense labelling occurred
in trophoblast cells containing variably sized basophilic
chlamydial inclusions (Fig. 1B). Adjacent trophoblasts with
no obvious inclusions also showed labelling of lower inten-
sity that extended in some sections into the deep stroma
of the placentome. There was also labelling of individual
cells underlying trophoblasts in some areas of the fetal villi.
In some placentomes cells within the stroma in the hilar
zone adjacent to blood vessels also demonstrated medium
intensity labelling. No immunolabelling for IFN was seen
in any sections from non-infected ewes. Fig. 1. Sections of placentome from a C. abortus infected ewe at 21days
In the control ovine tuberculous lymph node section a post infection 200. In situ hybridisation labelling of trophoblast cells
lining the primary villi with antisense probe for IFN mRNA (sense probe
distinct intralesional labelling pattern was evident (Fig. 2B). inset) (A). Immunolabelling with Mab mouse anti-bovine IFN (CC302,
MCA 1783B) and mouse negative control antibody (inset) (B). Immuno-
3.2.2. Chlamydial LPS labelling for chlamydial lipopolysaccharide (Clone AC1-P, Progen) and
At 14 days post infection a small number of trophoblast mouse negative control antibody (inset) (C).
cells within the hilar zone of one placentome (567A) were
immunolabelled for chlamydial LPS. At 21 days post infec- trophoblast cells mirrored the labelling pattern of IFN
tion, foci of chlamydial LPS immunolabelled trophoblasts mRNA using in situ hybridisation. At 28 days post infection
cells were seen at primary villi in placentome sections immunolabelling for chlamydial LPS was evident in tro-
from all infected ewes of which three exhibited labelling of phoblast cells in placentomes from all of the infected ewes
intermediate intensity in the hilar zone (Fig. 1C). The loca- examined. Two of the placentomes (1046B, 1047B) showed
tion and distribution of immunolabelled, inclusion-bearing a severe inammatory inltrate and inclusions in these sec-
 S. Worrall et al. / Journal of Reproductive Immunology 90 (2011) 214219 217

Table 1
Summary of in situ hybridisation and immunohistochemistry results for interferon- and chlamydial LPS in sections of placentomes from pregnant ewes
experimentally infected with Chlamydophila abortus.a

Fetus IDb IFN mRNA IFN immunohis- C. abortus

in situ tochemistry LPS
hybridisation

14 days 599A (uninfected control) 0 0 0

562B (uninfected control) 0 0 0
564A 0 0 0
564B 0 0 0
565A 0 0 0
566A 0 0 0
566B 0 0 0
567A 0 + +

21 days 816A (uninfected control) 0 0 0

819A (uninfected control) 0 0 0
820B ++ ++ ++
821A ++ ++ ++
821B + + +
822A + + +
822B ++ ++ ++
823A + + +
823B + + +

28 days 1041A (uninfected control) 0 0 0

1043B (uninfected control) 0 0 0
1044B + + +
1045B + + +
1046A + + +
1046B + + +
1047A ++ + ++
1047B + + +
a
0 no labelling, + weak labelling, ++ intermediate labelling, +++ strong labelling.
b
Numerical code relates to ewe (dam ID), and lettering to fetus, e.g. 564B indicates twin fetus A&B from ewe 564.

tions appeared larger, paler and more diffuse with some
chlamydial LPS staining evident in cell debris. Immunola-
belling for chlamydial LPS was not seen in the placentomes
of non-infected ewes.
4. Discussion
The nding that IFN mRNA is expressed in ovine tro-
phoblast cells in placentomes from ewes infected with
C. abortus at 21 and 28 days post infection is novel and
unexpected and to our knowledge has not previously
been reported. Not all trophoblast cells were positive and
the expression pattern was strongly suggestive of co-
localisation with chlamydial inclusions. The absence of
IFN mRNA expression in the placentome from one chal-
lenged ewe at 14 days post infection (567A) in which a
small number of trophoblast cells were immunolabelled
for chlamydial LPS may be due to section geometry, which
poses a limitation in sequential section labelling when
the target is present in only a small number of cells.
The cDNA template used in this study was previously
used to label IFN mRNA in ovine skin infected with
orf virus (Anderson et al., 2001). In that study cytokine
cDNA transfected Chinese hamster ovary cells express-
ing IFN, tumour necrosis factor-alpha and interlukin-4
mRNA were used as positive controls for in situ hybridi-
sation (Anderson et al., 2001). In the present study, lymph
node tissue from M. bovis-infected sheep in which cells
expected to be expressing IFN exhibited labelling with the
riboprobe. A BLAST (http://www.ncbi.nlm.nih.gov) search
for sequence homologies between the probe and the
C. abortus genome returned no signicant hits (Refseq:
NC 004552).
Immunohistochemical labelling was carried out using
a mouse anti-bovine IFN monoclonal antibody stated
to react with sheep. The labelling pattern mirrored the
mRNA expression in the trophoblasts conrming tran-
scription, and no labelling was detected in the absence of
primary antibody. Labelling was also detected in under-
lying stromal cells, which may indicate the location of
secreted IFN. Interpretation of this staining pattern war-
rants caution, however, since a comparative study of a
number of monoclonal and polyclonal antibodies to human
IFN described a large variation in staining patterns when
applied to unxed frozen sections (van der Loos et al.,
2001).
IFN mRNA was not detected in any leucocytes
inltrating the placenta of the infected ewes at the time-
points studied. A previous study using the same tissues
characterised the inammatory response in the (fetal)
chorioallantois as comprising predominantly neutrophils
and macrophages and relatively few lymphocytes (Sammin
et al., 2006). This may be a reection of the local immune
environment where suppression of T-cell proliferation may
be operating to prevent rejection of the fetus (Gogolin-
Ewens et al., 1989). Buxton et al. (2002) reported a different
pattern of IFN mRNA expression in placentomes from
ewes experimentally infected with C. abortus. In that study
sparse numbers of positive cells, lymphoid in appearance,
and associated with or adjacent to areas of arteriolitis
218 S. Worrall et al. / Journal of Reproductive Immunology 90 (2011) 214219
Fig. 2. Section of ovine lymph from a sheep infected with M. bovis 100.
In situ hybridisation labelling of ovine IFN mRNA using DIG labelled
antisense riboprobe (sense probe inset) (A). Immunolabelling with Mab
mouse anti-bovine IFN (CC302, MCA 1783B) and mouse negative control
antibody (inset) (B).
or arteritis, expressed IFN mRNA. However, that study
looked at placental tissue voided by ewes after lambing or
abortion and it is likely that inclusion-bearing trophoblasts
were not present, since this layer undergoes rapid degen-
erative change during the process of placental separation
(Steven, 1975).
Some previous studies have described IFN production
by trophoblast cells. In humans the villous syncytiotro-
phoblast and extravillous interstitial trophoblast produce
IFN during the rst trimester of normal pregnancy with
levels declining up to term (Paulesu et al., 1994). In pigs,
the implantation of the conceptus is accompanied by
an intense secretion of IFNs (IFN and IFN) that ends
around 15 days gestation. The IFN protein secreted by
the conceptus is antigenically identical to its leucocytic
counterpart, but it differs in glycosylation and C-terminal
post-translational processing (Cencic and La Bonnardiere,
2002). In mice IFN is present in cycling and pregnant uteri
and weak IFN mRNA signals have been detected in giant
trophoblast cells on day 6 of gestation (Platt and Hunt,
1998).
In vitro studies have shown that IFN is capable of
restricting the multiplication of the replicative form of
C. abortus in ovine broblasts over a range of concentra-
tions in a dose-dependent manner (Graham et al., 1995;
Brown and Entrican, 1996; Entrican et al., 1998). The nd-
ings of this study suggest that once C. abortus reaches
the placenta even local expression of IFN does not sig-
nicantly restrict the growth of the organism or modify
the pathogenesis of EAE. Secreted IFN may however,
be responsible for the early inammatory responses with
macrophage-like cells aggregating subjacent to inclusion-
bearing trophoblast cells (Sammin, et al. 2006). It may be
priming adjacent macrophages to produce other cytokines,
such as tumour necrosis factor-alpha (TNF) as part of
an innate inammatory response to external chlamydial
LPS or ruptured trophoblast cells. The resulting severe
inammatory response would contribute to tissue damage
and vasculitis, eventually leading to abortion (Wheelhouse
et al., 2009).
In the present study, infected ovine trophoblast cells
have been identied as a source of IFN mRNA in ovine
placentomes infected with C. abortus. The results are sig-
nicant in that they highlight the capacity of trophoblast
cells to act as part of the immune response in sheep
infected with C. abortus. Expression of IFN may constitute
an innate intracellular immune response to the intracel-
lular phase of C. abortus acting to contain or restrict the
growth of the inclusion without provoking a substantial
maternal immune response in the chorioallantois. How-
ever, the capacity of Chlamydia-infected ovine trophoblast
cells to elaborate IFN may be self-defeating for the con-
ceptus if it triggers the severe inammatory response on
the fetal side of the placenta that characterises enzootic
abortion. Further investigations to determine if IFN mRNA
expression in ovine trophoblast cells is unique to C. abor-
tus infection or if it occurs with other abortion-causing
ovine pathogens are now warranted. In addition, further
functional studies looking for expression of IFN receptors
on trophoblast cells to investigate the potential signalling
between infected and uninfected cells will form the basis
of further studies.
4.1. Conict of interest statement
None of the authors has a nancial or personal rela-
tionship with other people or organisations that could
inappropriately inuence or bias the paper entitled
Interferon-gamma expression in trophoblast cells in preg-
nant ewes challenged with C. abortus
Acknowledgements
This work was supported by funding from the Research
Stimulus Fund of the Irish Department of Agriculture, Fish-
eries and Food. We also acknowledge the support of COST
(European Cooperation in Science and Technology). Frank
Malone, Department of Agriculture and Rural Development
for Northern Ireland, is thanked for providing the ovine M.
bovis-infected lymph node blocks. The authors also thank
Brian Cloak for photographic work and Natasha Nally for
expert technical assistance.
 S. Worrall et al. / Journal of Reproductive Immunology 90 (2011) 214219
References
Anderson, I.E., Reid, H.W., Nettleton, P.F., McInnes, C.J., Haig, D.M., 2001.
Detection of cellular cytokine mRNA expression during orf virus
infection in sheep: differential interferon-gamma mRNA expression
by cells in primary versus reinfection skin lesions. Vet. Immunol.
Immunopathol. 83, 161176.
Brown, J., Entrican, G., 1996. Interferon- mediates long-term persistent
Chlamydia psittaci infection in vitro. J. Comp. Pathol. 115, 373383.
Brown, J., Howie, S.E.M., Entrican, G., 2001. A role for tryptophan in
immune control of chlamydial abortion in sheep. Vet. Immunol.
Immunopathol. 82, 107119.
Buxton, D., Barlow, R.M., Finlayson, J., Anderson, I.E., Mackellar, A., 1990.
Observations on the pathogenesis of Chlamydia psittaci infection of
pregnant sheep. J. Comp. Pathol. 102, 221237.
Buxton, D., Anderson, I.E., Longbottom, D., Livingstone, M., Watteged-
era, S., Entrican, G., 2002. Ovine chlamydial abortion: characterization
of the inammatory immune response in placental tissues. J. Comp.
Pathol. 127, 133141.
Byrne, G.I., Lehmann, L.K., Landry, G.J., 1986. Induction of tryptophan
catabolism is the mechanism for gamma-interferon-mediated inhibi-
tion of intracellular Chlamydia psittaci replication in T24 cells. Infect.
Immun. 53, 347351.
Cencic, A., La Bonnardiere, C., 2002. Trophoblastic interferon-gamma: cur-
rent knowledge and possible role(s) in early pregnancy. Vet. Res. 33,
139157.
Entrican, G., Brown, J., Graham, S., 1998. Cytokines and the protective host
immune response to Chlamydia psittaci. Comp. Immunol. Microbiol.
Inf. Dis. 21, 1526.
Entrican, G., Buxton, D., Longbottom, D., 2001. Chlamydial infection in
sheep: immune control versus fetal pathology. J. R. Soc. Med. 94,
273277.
Gogolin-Ewens, K.J., Lee, C.S., Mercer, W.R., Brandon, M.R., 1989. Site-
directed differences in the immune response to the fetus. Immunology
66, 312317.
Graham, S.P., Jones, G.E., MacLean, M., Livingstone, M., Entrican, G., 1995.
Recombinant ovine interferon gamma inhibits the multiplication of
Chlamydia psittaci in ovine cells. J. Comp. Pathol. 112, 185195.
Lan, H.Y., Mu, W., Ng, Y.Y., Nikolic-Paterson, D.J., Atkins, R.C., 1996. A
simple, reliable, and sensitive method for nonradioactive in situ
hybridization: use of microwave heating to improve hybridization
efciency and preserve tissue morphology. J. Histochem. Cytochem.
44, 281287.
Longbottom, D., Coulter, L.J., 2003. Animal chlamydioses and zoonotic
implications. J. Comp. Pathol. 128, 217244.
219
Malone, F.E., Wilson, E.C., Pollock, J.M., Skuce, R.A., 2003. Investigations
into an outbreak of tuberculosis in a ock of sheep in contact with
tuberculous cattle. J. Vet. Med. 50, 500504.
McInnes, C.J., Logan, M., Redmond, J., Entrican, G., Baird, G.D., 1990. The
molecular cloning of the ovine gamma-interferon cDNA using the
polymerase chain reaction. Nucleic Acids Res. 18, 4012.
Moulder, J.W., 1991. Interaction of chlamydiae and host cells in vitro.
Microbiol. Mol. Biol. Rev. 55, 143190.
Paulesu, L., Romagnoli, R., Cintorino, M., Ricci, M.G., Garotta, G., 1994. First
trimester human trophoblast expresses both interferon-gamma and
interferon-gamma-receptor. J. Reprod. Immunol. 27, 3748.
Peltier, M.R., Hansen, P.J., 2001. Immunoregulatory activity, biochemistry,
and phylogeny of ovine uterine serpin. Am J Reprod Immunol. 45,
266272.
Platt, J.S., Hunt, J.S., 1998. Interferon-gamma gene expression in cycling
and pregnant mouse uterus: temporal aspects and cellular localiza-
tion. J. Leukoc. Biol. 64, 393400.
Raghupathy, R., 1997. Th 1 type immunity is incompatible with successful
pregnancy. Immunol. Today 18, 478482.
Sammin, D.J., Markey, B.K., Quinn, P.J., McElroy, M.C., Bassett, H.F., 2006.
Comparison of fetal and maternal inammatory responses in the ovine
placenta after experimental infection with Chlamydophila abortus. J.
Comp. Pathol. 135, 8392.
Schroder, K., Hertzog, P.J., Ravasi, T., Hume, D.A., 2004. Interferon-gamma:
an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75,
163189.
Skopets, B., Hansen, P.J., 1993. Identication of the predominant proteins
in uterine uids of unilaterally pregnant ewes that inhibit lymphocyte
proliferation. Biol. Reprod. 49, 9971007.
Stamp, J.T., McEwen, A.D., Watt, J.A., Nisbet, D.I., 1950. Enzootic abortion
in ewes; transmission of the disease. Vet. Rec. 62, 251254.
Steven, D.H., 1975. Separation of the placenta in the ewe: an ultrastruc-
tural study. Q. J. Exp. Physiol. Cogn. Med. Sci. 60, 3744.
van der Loos, C.M., Houtkamp, M.A., de Boer, O.J., Teeling, P., van der Wal,
A.C., Becker, A.E., 2001. Immunohistochemical detection of interferon-
gamma: fake or fact? J. Histochem. Cytochem. 49, 699710.
Wegmann, T.G., Lin, H., Guilbert, L., Mosmann, T.R., 1993. Bidirectional
cytokine interactions in the maternal-fetal relationship: is successful
pregnancy a TH2 phenomenon? Immunol. Today 14, 353356.
Wheelhouse, N., Wattegedera, S., Stanton, J., Maley, S., Watson, D., Jep-
son, C., Deane, D., Buxton, D., Longbottom, D., Baszler, T., Entrican,
G., 2009. Ovine trophoblast is a primary source of TNF[alpha] during
Chlamydophila abortus infection. J. Reprod. Immunol. 80, 4956.
Wooding, F.B.P., 1992. The synepitheliochorial placenta of ruminants: bin-
ucleate cell fusions and hormone production. Placenta 13, 101113.