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Theses and Dissertations--Veterinary Science Veterinary Science
2013
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Rafaela De Negri
University of Kentucky, [email protected]
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De Negri, Rafaela, "EQUINE SERUM ANTIBODY RESPONSES TO STREPTOCOCCUS EQUI AND
STREPTOCOCCUS ZOOEPIDEMICUS" (2013). Theses and Dissertations--Veterinary Science. 13.
https://uknowledge.uky.edu/gluck_etds/13
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The document mentioned above has been reviewed and accepted by the student’s advisor, on
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Rafaela De Negri, Student
Dr. John F. Timoney, Major Professor
Dr. Daniel Howe, Director of Graduate Studies
Tittle Page
EQUINE SERUM ANTIBODY RESPONSES TO STREPTOCOCCUS EQUI AND
STREPTOCOCCUS ZOOEPIDEMICUS
____________________________
THESIS
____________________________
A thesis submitted in partial fulfillment of the requirements
for the degree of Master of Science
in the College of Agriculture at the
University of Kentucky
By
Rafaela Sans De Negri
Lexington, Kentucky
Director: Dr. John F. Timoney,
Keeneland Professor of Infectious Diseases
Lexington, Kentucky
2013
Copyright © Rafaela De Negri 2013
Abstract
EQUINE SERUM ANTIBODY RESPONSES TO STREPTOCOCCUS EQUI AND
STREPTOCOCCUS ZOOEPIDEMICUS
Streptococcus zooepidemicus (Sz) and Streptococcus equi (Se) share 98% DNA
sequence homology, but display different pathogenic properties. Infection by one
organism does not cross-protect against the other. To better understand pathogenic
differences between these organisms and gain information about which proteins are
expressed in horses with respiratory or intrauterine Sz infection or with Se infection we
compared antibody specificities of convalescent sera using ELISA. These comparisons
were based on sets of 8 and 14 immunoreactive recombinant proteins of Se strain CF32
and Sz strain NC78, respectively. Sera from donkeys that were previously naturally
affected with strangles and later developed Sz pneumonia secondary to an experimental
influenza challenge were also included.
Serum antibody responses were quantitatively and qualitatively much greater
following recovery from strangles than following respiratory Sz infection. Increased
reactions to Se proteins IdeE2, Se75.3, Se46.8, Se18.9 and Se42.0 were observed for the
majority of strangles sera but not for sera from respiratory Sz infection cases. Reactions
of sera from Sz respiratory disease to Sz proteins varied greatly and were mostly to HylC
and ScpC. Interestingly, sera of donkey recovered from Sz bronchopneumonia did not
show increased antibody reaction to any of the proteins although these donkeys had
recovered from clinical strangles 6 months previously. Only 1/5 mares with Sz placentitis
showed increased serum antibody responses to MAP. In conclusion, adaptive immune
responses to Se of horses with strangles are stronger and involve a greater number of
proteins than adaptive immune responses to Sz infection of the lower respiratory tract.
In an effort to develop an improved vaccine against Se, the modified live strain of
EHV-1, RacH was constructed to express three recombinant antigens of Se SeM, IdeE
and Se18.9. Two groups of 10 and 2 ponies were vaccinated intramuscularly or
intranasally, respectively. Another group (n=6) vaccinated with empty RacH served as
controls. Sera from 2/3 ponies from each vaccination groups and 1/2 serum from IN
vaccinated ponies showed increased serum neutralizing antibodies to EHV-1. ELISA
detected no significant increase in antibodies to proteins. Only one IM and IN vaccinated
pony showed serum bactericidal activity post vaccination.
Keywords: Streptococcus equi subsp. equi, Streptococcus equi subsp. zooepidemicus,
serum antibody response, immunogen, vaccine vector, RacH.
Rafaela Sans De Negri
June, 27 2013.
EQUINE SERUM ANTIBODY RESPONSES TO STREPTOCOCCUS EQUI AND
STREPTOCOCCUS ZOOEPIDEMICUS
By
Rafaela Sans De Negri
John F. Timoney
Director of Thesis
Daniel Howe
Director of Graduate Studies
June 27, 2013.
Dedication
In Memoriam Maria De Lurdes Sans De Negri.
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Acknowledgments
I would like to express my sincere appreciation to Dr. John F. Timoney, Professor
and Chairman of my advisory committee, for giving me the opportunity to work in his
laboratory. I am very grateful for the time he dedicated to my learning as well as helping
me to become a better researcher. The guidance, patience, consideration, support and
suggestions provided by him during this research were crucial for the development of it
and are greatly appreciated. I am especially grateful for his assistance in correcting my
English during the development of this thesis.
I would like to thank members of my advisory committee, Dr. David Horohov,
Dr. Sarah D’Orazio and Dr. Allan Loynachan. I am grateful for their interest, time,
support and flexibility throughout the development of this Master’s thesis.
I would also like to acknowledge the contributions of laboratory members that
have assisted me during the development of my research. To Dr. Sergey C. Artiushin,
Research Assistant Professor, I am thankful for his explanations of laboratory techniques
that provided me a better understanding of molecular biology. I am also thankful to him
for providing me with some of the recombinant proteins used during my research. To Mr.
Michael Fettinger, I am very thankful for his technical and management assistance, but
foremost I am grateful for his friendship and willingness to help. To Dr. Sridhar Velineni,
Post-doctoral Scholar. I am thankful for his guidance as well as for providing the S.
zooepidemicus proteins for my research. To Dr. K. Natarajaseenivasan, Visiting
Scientist, I am appreciative for the guidance and support provided. To Dr. Suresh
Multhupulani, former Doctoral Student, I am grateful for his work which provided basis
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and material for the development of this research. Additionally, I would like to thank Dr.
Igor Canisso, Graduate Student of the Equine Reproduction Laboratory of the Veterinary
Science Department, for providing me with sera from S. zooepidemicus placentitis cases,
and to Dr. Osterrieder for providing the RacH constructs.
The prompt guidance and support of Dr. Dan Howe, Graduate Student
Coordinator of the Department of Veterinary Science is greatly appreciated. Additionally,
the guidance and support provided by Dr. Ernest Bailey is also greatly appreciated.
The support of the Department of Veterinary Science staff is greatly appreciated.
I am especially grateful to Ms. Felicia Kost, Mr. Lynn Ennis and the farm staff for
assisting with animal care and management.
I would like to acknowledge the Department of Veterinary Science for funding
the research assistantship provided to me during my graduate studies. Without this
financial support, it would have been impossible for me to afford this opportunity to
study.
I would like to express my gratitude for fellow graduate students that I had the
opportunity to interact during my studies. In special, I’d like thank Dr. Elizabeth
Woodward, which I was blessed to share an office with and to have found much more
than a colleague in her. She has been a great tutor and friend. I have been also blessed to
get to know Dr. Anthony Claes, Dr. Juliana Roberta Campos, Dr. Aline Pereira, Dr.
Macarena Sanz and Dr. Chong Liu. Their assistance and friendship helped me through
the difficult days. On a more personal level, I would like to express my deepest gratitude
to my dear family for their unconditional love and support. They have been
understanding, accommodating and have gone above and beyond to ensure I was able to
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complete my graduate work. I would also like to acknowledge Dr.. Elizangela Bertolli,
Mr. Renato Moreira, Ms. Lisandra Garcia, Dr.. Romulo Albuquerque, Ms. Meeheh and
Mr. Joshua Perry, Ms. Vera and Mr. Goran Radulovic for sharing their wonderful
families with me and making me feel I was part of it. It has been truly a blessing to see all
their kids grow.
I am appreciative of the friendship of Mrs. Jordan Jones, Steffen Wo, Mrs.
Mallory Hick, Mr. Lori Nelson, Mr. Chistine Allen, Dr. Liz Andrews, Mr. Gabriel Falcao
Alencar, Mrs. Brittney Edwards, Mr. Justin Morgan, Mrs. Irina Goloborodko, Mr. Mario
Khoury, Mr. Willi Andrade, Dr. Constanza Valdes, Mr. Joseph Clay and Mr. James Clay.
Their company provided me joy, strength and made my time in Lexington more pleasant.
I am grateful for the support and patience of distant friends during my time away.
Last but not least, I am thankful to the animals that were involved in this work.
v
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