Final Report RSF 06 394

DAFF Ref: RSF 06 394
Research Stimulus Fund
Final Report
Immunopathogenesis studies and the development of novel diagnostic methodologies with
application in the targetted surveillance of reproductive loss in sheep due to Toxoplasma
gondii and Chlamydophila abortus
DAFF Project Ref No: RSF06 394
Start date: 01/12/06
End date: 28/02/10 (3 month extension approved to 31/5/10)
Principal Coordinator: Dr. Bryan Markey, School of Agriculture, Food Science and
Veterinary Medicine, University College Dublin, Dublin 4
Email: bryan.markey@ucd.ie
Other Principal Collaborating Researchers: Dr. Hugh Bassett (UCD), Dr. Jarlath Nally (UCD), Dr.
Donal Sammin (Central Vet. Research Lab.), Dr Maire McElroy (CVRL), Colm Brady (CVRL), John
Fagan (Regional Vet. Lab., Athlone), Mícheál Casey (RVL, Sligo), Dr. David Buxton (Moredun
Research Institute, Edinburgh), Dr. Stephen Maley (MRI, Edinburgh)
Please tick below the appropriate area on the research continuum where you feel this project
fits
BASIC/FUNDMENTAL APPLIED/PRE COMMERCIAL
Key words: (max 4) ovine abortion, Chlamydia, toxoplasmosis
X

1. Rationale for Undertaking the Research
This project was aimed at: 1) Increasing our understanding of the pathogenesis of the two
leading causes of ovine abortion in Ireland, Toxoplasma gondii and Chlamydophila abortus; 2)
Improving our diagnostic capability regarding these two pathogens; 3) Providing information on
the infectious causes of ovine reproductive loss in Ireland at this time.
Figures are not available for the economic losses incurred by Irish sheep farmers due to these
infections but annual losses due to enzootic abortion of ewes (EAE) of £15 to £20 million per
annum have been estimated for the United Kingdom with a similar level of loss due to
toxoplasmosis considered likely. The zoonotic nature of these infections with regard to
pregnant women places an additional and significant responsibility on veterinary surgeons and
diagnostic laboratories to accurately diagnose and deal with these infections. Enzootic abortion
of ewes is a serious and potentially life-threatening zoonosis affecting pregnant women after
contact with lambing ewes, while a primary infection with T. gondii during pregnancy can cause
serious, sometimes life-threatening, problems in the unborn child.
The mechanisms employed by these intracellular microorganisms in producing disease and
evading the immune response of the host are not fully understood. Basic morphological studies
of the pathology of toxoplasma and chlamydial abortion have been carried out many years
previous. However, this project was timely in permitting serial experimental infection studies
to be carried out using the latest immunological, immunohistochemical and molecular
techniques available. In addition, this project developed novel diagnostic assays for the
detection of these two pathogens. This was achieved by taking advantage of recent advances
in DNA amplification technologies permitting direct identification from clinical specimens and,
in the case of chlamydiae, differentiation of pathogenic and non-pathogenic species.
Fluorimeter-based, real-time sequence detecting technologies are rapidly replacing gel-based
conventional PCR assays due to their enhanced speed, sensitivity and specificity. Multiplexing,
permitting the detection of the signature sequences of multiple pathogens, is possible by the
use of different fluorophors. The development during this project of a multiplex real-time PCR
assay capable of detecting C. abortus and T. gondii provided a reliable and sensitive means to
detect these two pathogens in clinical samples. It also provided an ideal means to quantify the
burden of infection in a range of tissues. This information provided insights into the
dissemination of the organisms during infection, permitting assessment of the levels of
infection commensurate with lesion production and indicating the samples of choice for
diagnosis.
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2. Research Approach
The objectives of the proposed project and the methodologies employed to achieve them
were:
1. The comprehensive description in terms of pathology, histopathology, immunology and
molecular biology of the sequential development of lesions in the placenta and foetus of
pregnant ewes experimentally infected with Chlamydophila abortus and Toxoplasma gondii.
Pregnant ewes were infected at about day 90 of gestation and sacrificed sequentially in order
to provide the necessary samples (Tasks 1 and 2).
2. The comparison of foetal and maternal immune responses to T. gondii following
experimental challenge infection of pregnant sheep. Humoral immunity was assessed using
commercial assays while cellular immunity was assessed using lymphocyte transformation
assays (Task 4).
3. The correlation of immune responses in the pregnant ewe with morphological changes
occurring in foetal and maternal components of the placenta and in specific foetal organs.
Evaluation of the cellular immune response at the site of placental lesions was carried out by
immunohistochemical labelling with monoclonal antibodies against cluster of differentiation
(CD) cell surface proteins CD3, CD4, CD8, CD68, CD79 and a gamma delta (γδ) epitope to
estimate the relative contributions of five lymphocyte subset types and macrophages (Task 3).
In situ hybridization techniques were used to assess the levels of mRNA of the proinflammatory
cytokines interferon gamma and tumour necrosis factor alpha in the placenta
(Task 5).
4. The development of novel diagnostic molecular assays for the detection and identification of
C. abortus and T. gondii in clinical material from ovine abortion cases. A multiplex real time PCR
assay was developed for the detection and quantification of infection levels of C. abortus and T.
gondii (Tasks 6 and 8).
5. The validation and application of real time technology in the field investigation of ovine
reproductive loss. The novel multiplex real time PCR assay was applied to diagnostic clinical
material and performance compared with that of standard methods of diagnosis (Task 9).
6. The provision of meaningful and up-to-date information on the infectious causes of ovine
reproductive loss in Ireland at this time. A standardised protocol for ovine abortion flock
investigations was developed and utilised by the participating Regional Veterinary Laboratories.
Several detailed flock investigations were carried out (Task 10).
7. The identification of proteins expressed preferentially during infection with C. abortus and T.
gondii in pregnant ewes. 1-D and 2-D gel electrophoretic studies were carried out in tandem
with immunoblotting studies to determine the most immunoreactive proteins expressed during
infection (Task 7). Proteins of interest were identified using mass spectrometry. Difficulty in
culturing and purifying sufficient quantities of toxoplasma tachyzoites was circumvented by the
purchase of commercially available preparations.
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3. Research Achievements
Task 1: Pregnant ewes were successfully infected with Chlamydophila abortus and a wide range
of clinical material obtained following serial sacrifice.
Task 2: Pregnant ewes were successfully infected with Toxoplasma gondii and a wide range of
clinical material obtained following serial sacrifice.
Task 3: A detailed comparison of the pathologies and cellular immune responses elicited by C.
abortus ansd T. gondii in pregnant ewes has been documented. In both experimental infections
infiltration with CD3+ T-lymphocytes was associated with the presence of lesions, and was
more pronounced at the later time points and/or with increasing lesion severity. Infiltration
with lymphocytes was more pronounced in the C.abortus lesions and may reflect the more
severe inflammatory response observed. In C. abortus lesions the most numerous Tlymphocyte
subsets were CD8+ and γδ+ T cells. Low numbers of CD4+ lymphocytes were
present in some of the sections. The lesions caused by the two pathogens differed in terms of:
a) Location: In the placentome, C.abortus lesions were predominantly located adjacent to
the hilus i.e. in the limbus (junction of the intercotyledonary chorio-allantoic membrane with
the placentome) and/or within the haematophagous organs. In contrast T.gondii lesions
were distributed within the interdigitating maternal septa and foetal villi of the placentome
and early lesions were centred on the maternal septa.
b) Inflammatory response: C. abortus lesions in placentomes were generally neutrophil-rich
and leukocytoclastic where ulceration of the infected chorionic epithelium had occurred. In
non-ulcerated lesions there was an increase in CD3+ T cells, (consisting of CD8+, γδ+ and/or
CD4+ T cells), in the foetal mesenchymal stroma of the hilus and this response was directed
towards infected chorionic epithelial cells containing intracytoplasmic C. abortus inclusions.
In contrast, T. gondii infection in the placentome presented as small focal or focally
extensive necrotic lesions associated with mild to moderate non-suppurative cellular
infiltration in the foetal mesenchymal stroma of the adjacent villi and hilus. The infiltrate
was predominantly composed of CD3+ T cells, with subtypes consisting chiefly of CD8+ cells
as well as some γδ+ T cells.
Task 4: There was a disappointing lack of responsiveness by peripheral blood lymphocytes to
the chlamydial antigen. In the case of T. gondii, lymphocyte transformation assays showed a
rise in stimulation index over the course of the trial, reaching a peak at 28 days post infection.
One of the two control animals showed a high level of response to the toxoplasma antigen at
some of the time points, but the erratic pattern of this response suggested that it was not the
result of exposure to T. gondii. Serological titres were generally low in response to both
pathogens reflecting the fact that the animals were sacrificed prior to abortion. An in-house
immunoblotting assay for C. abortus performed better than the commercial assays evaluated.
Task 5: In the case of C. abortus infection of cultured AH-1 ovine trophoblast cells, evidence
was found for an increase in expression of mRNA for the pro-inflammatory cytokines
interleukin (IL)-1beta, TNF alpha and IL-6 between 3 and 12 hours post infection. These
findings are consistent with in vivo studies that have demonstrated a characteristic
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inflammatory response in the chorioallantois in response to chlamydial infection and suggest a
significant role for the trophoblast layer in initiating this inflammatory response. In situ
hybridization studies of mRNA expression for interferon gamma and tumour necrosis factor
alpha in C. abortus and T. gondii infected placental tissue produced some unexpected findings:
1. No clear association between mRNA expression for tumour necrosis factor alpha and
infection by either pathogen could be detected. 2. The most striking difference between the
two pathogens was the difference in expression of interferon gamma mRNA. In the case of C.
abortus there was a clear association with infection (no evidence of expression in the control
animals) and expression became more marked during the later time points of the experimental
infection. It is also note-worthy that the expression of interferon gamma was found to colocalise
with the presence of chlamydial inclusions in the trophoblast layer. The interferon
gamma mRNA produced in infected trophoblast cells may indicate an attempt to restrict
multiplication of the organism as well as an important trigger for the inflammatory responses
which develop on the foetal side of the placenta in enzootic abortion. In contrast, no interferon
gamma mRNA expression was detected in the T. gondii infected placental tissue.
Task 6: Protocols for real time PCR assays for C. abortus and T. gondii were optimised and
applied to the harvested tissues. The sample of choice for diagnosis by real time PCR of EAE is
placenta. Infection may be missed if only foetal tissues are available for testing. Placental
tissue is also the most reliable sample for the diagnosis of toxoplasmal abortion by real time
PCR. In the absence of placental tissue, brain and foetal lung are the next preferred samples.
Task 7: Proteins of C. abortus identified as highly immunoreactive were polymorphic outer
membrane proteins, major outer membrane protein, macrophage infectivity potentiator,
inorganic pyrophosphatase, elongation factor, ABC transporter, membrane transport protein,
lipoprotein, cysteine-rich outer membrane protein and heat shock-related exported protease.
The macrophage infectivity potentiator protein was found to be particularly immunoreactive
and has been cloned and expressed. A large number of immunoreactive T. gondii proteins were
detected including the major surface antigen (designated gp30 or SAG 1) which is highly
immunogenic. Protein fractionation using detergent phase partition technology would be
required in future studies to permit identification of individual proteins by mass spectrometry.
The foetal fluids of infected foetuses were found to frequently contain specific antibodies to
the infecting pathogen. The source of this antibody is unclear. It is most likely that it is derived
from the maternal circulation, particularly in the early stages of infection when there is no
evidence of pathogen-specific antibody in the foetal circulation. However, there may be some
foetal contribution during the later stages of infection.
Task 8: A novel, multiplex real time PCR capable of detecting both C. abortus and T. gondii has
been developed and evaluated. The assay is both highly sensitive and robust.
Task 9: The real time PCR assay was shown to be highly sensitive and capable of detecting
additional cases of EAE and toxoplasmosis on farms that would have been missed if standard
methods of diagnosis had been relied upon solely. A total of 486 samples, representing
abortion cases from 66 farms, were tested using the real time multiplex PCR. Application of
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this diagnostic assay resulted in the detection rate for EAE rising from 11 to 17% of farms
investigated, from 12 to 14% for toxoplasmosis and for dual infection from 3 to 6%.
Task 10: Criteria for the diagnosis of EAE and toxoplasmosis using the real time PCR assay were
determined using a combination of results from the experimental trials and from field material
from abortion outbreaks where the results of other diagnostic techniques were available.
There was a good correlation between the presence of characteristic histopathological lesions
and high numbers of genome copy in the case of both pathogens. It was found that C. abortus
was present on 54% of farms but often at a low level suggesting that infection could be present
but did not always give rise to abortion (absence of characteristic pathological lesions). In the
case of Toxoplasma gondii, there was evidence of infection on 28% of farms although evidence
of disease was only demonstrated on 20% of farms. Genotyping of the toxoplasmosis cases has
shown that the majority of infections are caused by genotype 2 of T. gondii. Bacterial species
detected in foetal abomasal contents included Campylobacter spp. (9% of farms), Escherichia
coli (4.5%), Listeria sp. (1.5%) and Salmonella sp. (1.5%).
4. Impact of the Research
Scientific community
There is currently intense interest in the status of the maternal immune response to a) the
normal foetus; b) the infected foetus. The findings of this project will inform other researchers
regarding the ovine immune responses at the level of the placenta to these two important
intracellular pathogens as well as providing more general research information regarding the
immune capability of the pregnant dam. The application of real time PCR technology has
permitted a very accurate picture to be constructed of the dissemination and shedding patterns
of these two pathogens. This information is useful with regard to epidemiological studies and
the development of diagnostic assays. The data generated regarding the causes of ovine
abortion on Irish farms give an accurate, up to date snap-shot of the current situation on Irish
farms and indicate that the importance of EAE was probably underestimated in the past.
Industry
The knowledge obtained regarding the role of the immune response in the pathogenesis of
these diseases will inform applied research into the development of improved vaccines for
these abortifacient pathogens. In many cases placental tissue is not submitted by farmers
along with aborted foetal material. In such cases brain is the preferred organ for
histopathological examination for the characteristic toxoplasma lesions. Useful information has
been provided by this study regarding the optimal brain sections to examine for diagnosis. In
addition, the exquisite sensitivity of the real time PCR assay will permit detection of these
pathogens at very low levels in foetal tissues, increasing the chances of detection of these
pathogens in cases where placental tissue is not provided as part of the diagnostic submission.
The detection of specific antibody in the foetal fluids of infected foetuses suggests that
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abomasal contents (similar in composition to amniotic fluid) may be a useful sample to test for
diagnostic purposes.
Regulatory authorities
The real time multiplex PCR assay provides a very valuable additional tool for diagnostic
laboratories permitting rapid and sensitive detection of toxoplasmosis and C. abortus in flocks.
Laboratory confirmation by the Regional Veterinary Laboratories of such cases is essential for
the proper implementation of control programmes, particularly vaccination programmes. The
incidence of infection for these two pathogens is much higher than expected although the
presence of infection did not equate with disease in all cases. However, the risk of infection is
clearly high suggesting an urgent need to increase levels of awareness in the industry and to
advise accordingly with regard to vaccination.
Consumers
The real time multiplex PCR assay is both accurate and reliable having been validated using field
material. It ha s provided valuable epidemiological data regarding these two important
zoonotic diseases in Ireland.
5. Exploitation of the Research
A highly immunoreactive protein of C. abortus has been identified, cloned and expressed.
Funding to develop a diagnostic assay based on this protein was sought from Enterprise Ireland.
This application was not approved. However, with regard to the novel real time multiplex PCR
for detection of C. abortus and T. gondii an Invention Disclosure entitled “Multiplex diagnostic
real time PCR test for ovine abortions” was registered by Nova UCD on the 10 th March 2010
(reference number is 2005402). The technology has since been licensed to Bofinn Diagnostics.
The results of a comparison of serological tests including commercial ELISAs for C. abortus and
for T. gondii produced by ID-Vet Innovative Diagnostics (Montpelier, France) were supplied to
the company.
6. Summary of Research Outputs
(a) Intellectual Property applications/licences/patents
1. Invention Disclosure entitled “Multiplex diagnostic real time PCR test for ovine abortions”
registered by Nova UCD on the 10 th March 2010 (reference no. 2005402)
2.
(b) Innovations adopted by industry
1. Standardised protocol for investigation of abortion outbreaks in sheep flocks
2.
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(c) Number of companies in receipt of information
1. ID-Vet Innovative Diagnostics (Montpelier, France)
(d) Outcomes with economic potential
1. Development and validation of real time multiplex PCR for simultaneous detection of C.
abortus and T. gondii.
2. Identification of novel immuno-reactive C. abortus proteins
(e) Outcomes with national/ policy/social/environmental potential
1. Higher incidence of infection with C. abortus and T. gondii than expected, emphasising the
importance of A) a coordinated control programme for these pathogens in sheep; B) education
regarding the zoonotic nature of these agents.
2.
(f) Peer-reviewed publications, International Journal/Book chapters.
Acceptable Format: Walsh, D.R., Murphy, O., Cosgrave, J. (2008). Echinococcosis - an
international public health issue. Research in Veterinary Science 774, 891-902.
1. Sammin D, Markey B., Bassett H. and Buxton D. (2009). The ovine placenta and placentitis –
a review. Veterinary Microbiology, 135: 90-97.
2. Gutierrez J., O’Donovan J., Williams E., Proctor A., Brady C., Marques P.X., Worrall S.,
Nally J.E., McElroy M., Bassett H., Sammin D., Buxton D., Maley S. and B.K. Markey (2010)
Detection and quantification of Toxoplasma gondii in ovine maternal and foetal tissues from
experimentally infected pregnant ewes using real-time PCR. Veterinary Parasitology, 172, 8-15.
3. Marques P.X., Souda P., O’ Donovan J., Gutierrez J., Williams E.J., Worrall S., McElroy M.,
Proctor A., Brady C., Sammin, D., Bassett, H.F., Whitelegge J.P., Markey B.K. and Nally J.E.
(2010) Identification of immunologically relevant proteins of Chlamydophila abortus using sera
from experimentally infected pregnant ewes. Clinical and Vaccine Immunology, 17, 1211-1216.
4. Gutierrez J., Williams E., O’Donovan J., Brady C., Proctor A., Marques P.X., Worrall S.,
Nally J.E., McElroy M., Bassett H., Sammin D. and B.K. Markey (2010). Monitoring clinical
outcomes, pathological changes and shedding of Chlamydophila abortus following
experimental challenge of periparturient ewes utilizing the natural route of infection.
Veterinary Microbiology, in press doi:10.1016/j.vetmic.2010.06.015
5. Marques P.X., Souda P., O’ Donovan J., Gutierrez J., Williams E.J., Worrall S., McElroy M.,
Proctor A. , Brady C., Sammin, D., Bassett, H.F., Whitelegge J.P., Markey B.K. and Nally J.E.
(2010) Amniotic and allantoic fluids from experimentally infected sheep contain
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immunoglobulin specific for Chlamydophila abortus. Veterinary Immunology and
Immunopathology, in press doi:10.1016/j.vetimm.2010.11.002
(g) Scientific abstracts or articles including those presented at conferences
1. Marques P.X., Williams E.J., O’Donovan J., Brady C., Worrall S., Sammin D., Bassett H., Markey
B.K. and Nally J.E. (2008). Identification of Chlamydophila abortus antigens expressed during
infection of pregnant ewes. Association of Veterinary Teachers and Research Workers, Spring
Scientific Meeting, Central Veterinary Research Laboratory, Backweston Laboratory Campus,
March 12 th , 2008.
2. Worrall S., Sammin D., Bassett H., Brady C., Marques P.X., Nally J., O’Donovan J., Proctor A.,
Williams E.J. and Markey B.K. (2008). Interferon gamma expression in the placenta of
Chlamydophila abortus infected sheep. Association of Veterinary Teachers and Research
Workers, Spring Scientific Meeting, Central Veterinary Research Laboratory, Backweston
Laboratory Campus, March 12 th , 2008.
3. Williams E., O’Donovan J., Marques P.X., Sammin D., Brady C., Worrall S., Nally J.E., Bassett H.
and Markey B.K. (2008). Detection of Chlamydophila abortus in ovine maternal and foetal
tissues using real-time PCR. Association of Veterinary Teachers and Research Workers, Spring
Scientific Meeting, Central Veterinary Research Laboratory, Backweston Laboraotry Campus,
March 12 th , 2008.
4. Williams E., O’Donovan J., Marques P., Sammin D., Brady C., Worrall S., Nally J., Bassett H.,
Sachse K. and Markey B. (2008). Quantification of Chlamydophila abortus in ovine maternal
and foetal tissues using real-time PCR. Proceedings of the Sixth Meeting of the European Society
for Chlamydia Research, Aarhus, Denmark. p. 316.
5. Marques P., Williams E., O’Donovan J., Brady C., Worrall S., Sammin D., Bassett H., Markey B.
and Nally J. (2008). Characterisation of antigens reactive with IgG from amniotic and allantoic
fluids of pregnant ewes experimentally infected with Chlamydophila abortus. Proceedings of
the Sixth Meeting of the European Society for Chlamydia Research, Aarhus, Denmark. p. 315.
6. Worrall S., Sammin D., Bassett H., Brady C., Marques P., Nally J., O’Donovan J., Fagan J.,
Williams E. and Markey B. (2008). Interferon gamma expression in the placenta of
Chlamydophila abortus infected sheep. Proceedings of the Sixth Meeting of the European
Society for Chlamydia Research, Aarhus, Denmark. p. 305.
7. Markey B., O’Donovan J., Brady C., Gutierrez J., Marques P.X., Proctor A., Worrall S., Nally J.,
McElroy M., Sammin D., Bassett H. and Williams E. (2009). Use of quantitative real time PCR to
evaluate shedding and tissue distribution of Chlamydophila abortus during the course of the
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disease. Proceedings of the Association of Veterinary Teachers and Research Workers, 63 rd
Annual Meeting, Scarborough, UK. p. 18.
8. Proctor A., O’Donovan J., Gutierrez J., Williams E., Brady C., Marques P.X., Worrall S., Nally J.,
Bassett H., Sammin D. and and Markey B. (2009). Comparison of two serological assays for the
detection of antibodies to Toxoplasma gondii in experimentally infected sheep. Proceedings of
the Association of Veterinary Teachers and Research Workers, 63 rd Annual Meeting,
Scarborough, UK. p. 32.
9. Worrall S., Sammin D.J., Bassett H.F., Brady C., Marques P.X., Nally J.E. O’Donovan J., Fagan J.,
Williams E.J. and Markey B.K. (2009). Interferon-gamma expression in ovine placentomes from
sheep experimentally infected with Chlamydophila abortus. Proceedings of the Association of
Veterinary Teachers and Research Workers, 63 rd Annual Meeting, Scarborough, UK. p. 40.
10. Gutierrez J., O’Donovan J., Proctor A.F., Brady C., Marques P.X., Worrall S., Nally J.E.,
McElroy M., Bassett H., Sammin D., and Markey B.K.. (2009). Development and validation of a
multiplex real time PCR for the detection of Chlamydophila abortus and Toxoplasma gondii in
sheep. Proceedings of the First European Meeting on Animal Chlamydioses and Zoonotic
aspects, University of Murcia, Murcia Spain. June 14 th to 16 th .
11. Marques P.X., O’Donovan J., Williams E.J., Proctor A., Brady C., Worrall S., Sammin D.,
Bassett H., Markey B.K. and Nally J.E. (2009). Comparison of three serological methods for the
detection of Chlamydophila abortus infection in experimentally infected pregnant ewes. Society
for Applied Microbiology Summer Meeting, July 2009, Manchester, U.K.
12. Gutierrez J., O’Donovan J., Proctor A.F., Brady C., Marques P.X., Worrall S., Nally J.E.,
McElroy M., Bassett H., Sammin D., Buxton D., Maley S. and Markey B.K.. (2009). Detection and
quantification of Toxoplasma gondii in ovine maternal and foetal tissues using real-time PCR.
10 th International Congress on Toxoplasmosis, Kerkade, The Netherlands, June 19 th to 23 rd .
13. Marques P.X., O’Donovan J., Williams E.J., Proctor A., Brady C., Gutierrez J., Worrall S.,
Sammin D., Bassett H., Markey B.K. and Nally J.E. (2009). Detection of antibodies to Toxoplasma
gondii in allantoic and amniotic fluids and foetal stomach contents following experimental
infection of pregnant ewes. Association of Veterinary Teachers and Research Workers, Scientific
Meeting, Four Seasons Hotel, Carlingford, October 2 nd .
14. Gutierrez J., O’Donovan J., Proctor A.F., Brady C., Marques P.X., Worrall S., Nally J.E.,
McElroy M., Bassett H., Sammin D., and Markey B.K. (2009). Use of Real-Time PCR for targeted
surveillance of the two leading causes of ovine abortion in Ireland, Chlamydophila abortus and
Toxoplasma gondii. . Association of Veterinary Teachers and Research Workers, Scientific
Meeting, Four Seasons Hotel, Carlingford, October 2 nd .
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15. Marques P.X., Puneet S., O’Donovan J., Gutierrez J., Williams E.J., Worrall S., M. McElroy,
Proctor A., Brady C., Sammin D., Bassett H., Whitelegge J.P., Markey B.K. and Nally J.E.. (2009).
Identification of antigens of Chlamydophila abortus expressed during infection of pregnant
ewes. Proceedings of the 64th Annual Conference of the Association of Veterinary Teachers and
Research Workers, York, March 29th-31st.
16. Marques P.X., O’Donovan J., Gutierrez J., Williams E.J., Worrall S., M. McElroy, Proctor A.,
Brady C., Sammin D., Bassett H., Maley S., Buxton D., Nally J.E. and Markey B.K. (2010).
Characterisation of immunoglobulins in the foetal fluids of pregnant ewes experimentally
infected with Chlamydophila abortus and Toxoplasma gondii. Proceedings of the 64th Annual
Conference of the Association of Veterinary Teachers and Research Workers, York, March 29th-
31st.
(h) National Report
(i) Popular non-scientific publications
1. Continuing education article: Williams E. and O’Donovan J. (2009). Ovine abortion: an
overview. Irish Veterinary Journal, 62: 342-346.
(j) Workshops/seminars/ open days at which results were presented (excluding those in
(g))
1. Poster presentation: Marques P.X., Williams E., O’Donovan J., Brady C., Sammin D., Bassett
H., Markey B. and Nally J. (2007) Characterisation of the proteome of Chlamydophila abortus
and antigens reactive with convalescent sheep sera. Microbial and Host Response Meeting,
15 th to 19 th September, Cold Spring Harbor, USA.
2. Poster Presentation: Marques P.X., Williams E., O’Donovan J., Brady C., Sammin D., Bassett
H., Markey B. and Nally J. (2007). Identification of antigens of Chlamydophila abortus
expressed during infection of pregnant ewes. UCD, School of Agriculture, Food Science and
Veterinary Medicine Research Day, 3 rd December.
3. Poster presentation: Marques P.X., Williams E., O’Donovan J., Brady C., Sammin D., Bassett
H., Markey B. and Nally J. (2008) Characterisation of IgG in amniotic and allantoic fluids
obtained from pregnant ewes experimentally infected with Chlamydophila abortus. 8th Annual
UCD Conway Institute Festival of Research, 25 th September.
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4. Poster presentation: Worrall S., Sammin D., Bassett H., Brady C., Marques P., Nally J.,
O’Donovan J., Fagan J., Williams E. and Markey B. (2008). Demonstration of the production of
the cytokine interferon-gamma by trophoblast cells in pregnant ewes infected with
Chlamydophila abortus. 8th Annual UCD Conway Institute Festival of Research, 25 th
September.
5. Poster presentation: Marques P.X., O’Donovan J., Williams E., Brady C., Worrall S., Sammin
D., Bassett H., Markey B. and Nally J. (2008). A comparison of three serological methods for
the identification of pregnant ewes infected with Chlamydophila abortus. UCD, School of
Agriculture, Food Science and Veterinary Medicine Research Day, 4 th December.
6. Poster presentation: Proctor A., O’Donovan J., Marques P.X., Gutierrez J., Sammin D., Brady
C., Worrall S., Nally J., Bassett H. and Markey B. (2008). Detection of antibodies to Toxoplasma
gondii in serum from experimentally infected pregnant ewes. UCD, School of Agriculture, Food
Science and Veterinary Medicine Research Day, 4 th December.
7. Poster presentation: Gutierrez J., Williams E., O’Donovan J., Proctor A., Brady C., Marques
P.X., Worrall S., Nally J., Bassett H., Sammin D. and Markey B. (2008). Development of Duplex
Real Time PCR for the detection of Chlamydophila abortus and Toxoplasma gondii in sheep.
UCD, School of Agriculture, Food Science and Veterinary Medicine Research Day, 4 th December.
8. Oral presentation: Marques P.X. (2009). An ovine model of Chlamydophila abortus
infection: characterization of immunoglobulin in amniotic and allantoic fluids. UCD Infection
Biology Research Day, 21 st September.
9. Proctor A., Williams E., Gutierrez J., Marques P.X., Brady C., Worrall S., Nally J., Bassett H.,
Sammin D. and Markey B. (2009). Cytokine expression in ovine trophoblast cells infected with
Chlamydophila abortus. UCD, School of Agriculture, Food Science and Veterinary Medicine
Research Day, 8 th December.
10. Marques P.X., O’Donovan J., Williams E.J., Proctor A., Brady C., Gutierrez J., Worrall S.,
Sammin D., Bassett H., Markey B.K. and Nally J.E. (2009). Detection of antibodies to Toxoplasma
gondii in allantoic and amniotic fluids and foetal stomach contents following experimental
infection of pregnant ewes. UCD, School of Agriculture, Food Science and Veterinary Medicine
Research Day, 8 th December.
DAFF Ref: RSF 06 394 12

7. Permanent Researchers
Institution Name Number of
Permanent staff
contributing to
project
DAFF Ref: RSF 06 394 13
Total Time
contribution (months)
UCD 4 25 6.3
CVRL 2 16.8 8.4
RVL 2 7 3.5
MRI 2 7 3.5
Total 10 55.8 5.6
8. Researchers Funded by RSF
Type of Researcher Number Total Time
contribution (months)
Post Doctorates 2 36 a 18
Contract Researchers
PhD postgraduates 2 48 b 24
Masters postgraduates 1 24 24
Temporary researcher
Other
Total 5 108 21.6
Average time
contribution per
permanent staff
member
Average time
a Two post doctorates (Erin Williams and Jorge Gutierrez), 18 months each
b RSF funded the first 12 months of PhD registration for James O’Donovan and 36 months of
PhD for Patricia Marques
9. Postgraduate Research
Total Number of PhD theses: __2__
Please include authors, institutions and titles of theses and submission dates. If not submitted
please give the anticipated submission date
Patricia Marques, UCD ‘Identification and characterisation of antigenic proteins expressed by
Chlamydophila abortus and Toxoplasma gondii during experimental infection of pregnant
sheep’ Submitted February 2010

James O’Donovan, UCD/CVRL ‘Comparative pathology of Toxoplasma gondii and
Chlamydophila abortus infections in pregnant sheep’
Anticipated submission date: December 2010
Total Number of Masters theses: __2__
Please include authors, institutions and titles of theses and submission dates. If not submitted
please give the anticipated submission date
Aisling Proctor, UCD ‘Ovine trophoblast cytokine expression elicited by Chlamydophila abortus’
Submitted February 2010
Sheila Worrall, UCD ‘Interferon gamma and tumour necrosis factor alpha expression in the
placenta of sheep experimentally infected with Chlamydophila abortus or Toxoplasma gondii’
Anticipated submission date: December 2010
10. Project Expenditure
Total expenditure of the project: €623,417.16
Total Award by RSF €624,989
Other sources of funding (specify) €
Breakdown of Total Expenditure
Category
Contract staff
Temporary staff
UCD
Post doctorates 144,149.94
Post graduates 138,218.11
Consumables 143,600.96
Travel and
subsistence
15,184.54
Sub total 441,153.55
Durable
equipment
22,982.54
Other 26,935
Overheads 132,346.07
Total 623,417.16
Name
Institution 2
DAFF Ref: RSF 06 394 14
Name
Institution 3
Name
Institution 4
Total

11. Future Strategies
One of the project members (JN) participated in the NovaUCD’s Campus Company
Development Programme and has investigated the feasibility of a research and development
company, Inviver, which will provide novel diagnostics and vaccine products to the veterinary
health industry. As part of a feasibility study, a diagnostic assay for EAE (“EAEdetect”) was one
of three products/services assessed. Funding has been applied for from Enterprise Ireland
through the Proof of Concept Commercialisation Fund. The funding is required to optimise and
validate an ELISA based on the recombinant protein of C. abortus generated as part of the
project. Unfortunately the original application and a subsequent amended application have
been turned down.
The transfer of the multiplex real time PCR technology to the Regional Veterinary Laboratories
has been hampered by the absence of the necessary equipment in the individual laboratories.
One machine has since been bought and is due to be allocated to one of the Regional
Laboratories.
12. Industry Collaboration
Discussions with a representative of a leading French veterinary diagnostic test manufacturer
ID-Vet Innovative Diagnostics (Montpelier, France) resulted in the provision free of charge of
ELISAs for C. abortus and T. gondii. A comparison of in house and commercial diagnostic assays
for these two pathogens were subsequently relayed to the company and also reported at
scientific meetings.
DAFF Ref: RSF 06 394 15