Citation
Seroepidemiological studies of herpesvirus-associated diseases of matine turtles: fibropapillomatosis and lung-eye-trachea disease

Material Information

Title:
Seroepidemiological studies of herpesvirus-associated diseases of matine turtles: fibropapillomatosis and lung-eye-trachea disease
Series Title:
Florida Cooperative Fish and Wildlife Research Unit Research Work Order 213
Alternate Title:
RWO 214
Creator:
Klein, Paul A.
Jacobson, Elliot
Affiliation:
University of Florida -- Florida Cooperative Fish and Wildlife Research Unit
Place of Publication:
Gainesville, Fla.
Publisher:
Florida Cooperative Fish and Wildlife Research Unit, University of Florida
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
University of Florida. ( LCSH )
Sea turtles ( LCSH )
Herpesvirus diseases in animals ( LCSH )
Biotic communities -- Florida ( LCSH )
Natural history -- Florida ( LCSH )
City of Gainesville ( local )
Central Florida ( local )
Diseases ( jstor )
Turtles ( jstor )
Antibodies ( jstor )
Spatial Coverage:
North America -- United States of America -- Florida

Notes

Funding:
This collection includes items related to Florida’s environments, ecosystems, and species. It includes the subcollections of Florida Cooperative Fish and Wildlife Research Unit project documents, the Sea Grant technical series, the Florida Geological Survey series, the Coastal Engineering Department series, the Howard T. Odum Center for Wetland technical reports, and other entities devoted to the study and preservation of Florida's natural resources.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved, Board of Trustees of the University of Florida

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FLORIDA COOPERATIVE FISH AND WILDLIFE RESEARCH UNIT
PROJECT STATUS REPORT
TITLE: Seroepidemiological Studies of Herpesvirus-associated Diseases of Marine Turtles: Fibropapillomatosis
and Lung-Eye-Trachea Disease

1.PROJECT OFFICER:

2. PRINCIPAL INVESTIGATOR: Paul A. Klein, Ph.D.
Professor
Department of Pathology, Immunology, and
Laboratory Medicine
College of Medicine
University of Florida, Gainesville, FL

3. CO-PRINCIPAL INVESTIGATOR: Elliott Jacobson, D.V.M., Ph.D.
Professor
Department of Small Animal Clinical Sciences
College of Veterinary Medicine
University of Florida, Gainesville, FL


4. RESEARCH WORK ORDER #: 213
5. FUNDING AGENCY: USFWS

6. START DATE: 07 / 01/ 2001 END DATE: 12 / 31 / 2003
7. This Reporting Period: 8-31-02 to 12-31-03 (FINAL REPORT[see full report attached))


15. REPORTING PERIOD FOR DELIVERABLES: Final report due: 12-31-03
9. ABSTRACT OF PROJECT(maximum 4000 characters):
LETV
Antigenic LETV expressed peptides will be used to develop an LETV ELISA following methods previously established for the
ELISA using whole LET herpesvirus. With expressed antigen, there will be fewer limitations on the number of plasma samples
that can be screened. Plasma samples previously tested by the ELISA using whole virus will be used to validate the specificity of
the assay. Specifically, the plasma samples obtained from the captive-reared turtles prior to and after immunization with
inactivate LETV will be used. Plasma samples from captive-reared green turtles with no known exposure to herpesviruses will
serve as additional known-negative samples.

We will utilize the same plasma bank developed collaboratively with the University of Central Florida to identify marine turtles in
Florida exposed to LETV. This will provide some preliminary data on the extent of infections of free-ranging marine turtles with
the LETV and provide preliminary information about the impact LETV may have on marine turtle health. Since the Cayman
Farm releases turtles on a regular basis and has no health monitoring program for herpesvirus infections (or other infections), this
data will prove illuminating. In addition, The immunological data collected will provide critical seroepidemiological information
for determining the relationship between infection with LETV and the subsequent development of LETD. This evidence may be
used to strengthen the argument that LETV is the etiologic agent of LETD until transmission studies can be performed.

FPHV
Antigenic FPHV expressed peptides will be used to develop an FPHV ELISA following methods previously established for the
LETV specific ELISA assay. The expressed FPHV peptides will be used as antigen to coat 96 well ELISA plates. Plasma samples
previously tested by immunohistochemistry for presence or absence of anti-FPHV antibodies will be used to validate the











specificity of the assay. Plasma samples from captive-reared green turtles with no known exposure to herpesviruses (no anti-
FPHV antibodies) will serve as the known-negative samples. Plasma samples from captive reared green turtles with
experimentally induced FP (have anti-FPHV antibodies) will be used as known-positive samples. In addition, there is a set of
plasma samples collected from wild green turtles with and without FP that have been previously shown to have plasma with and
without anti-FPHV antibodies, respectively. These are important to obtain information about antibody levels in a naturally
infected turtle. We will also utilize our unique monoclonal antibody reagents against marine turtle IgM, 7S IgG, and 5.7S IgG
immunoglobulin subclasses as secondary antibodies so that early antibody responses (IgM) can be differentiated from late
antibody responses (5.7S IgG).

Using the serological tests, such as the FPHV-specific ELISA, we will utilize a large plasma bank developed collaboratively with
the University of Central Florida to identify marine turtles in Florida exposed to FPHV. This plasma bank contains samples from
green and loggerhead turtles collect from the three study sites, Indian River Lagoon, Sebastian Inlet, and Trident basin (Indian
River and Brevard Counties, FL), for over a decade. Unique serial (annual) samples from green turtles that were initially tumor
free but developed tumors in subsequent years as well as samples from tumor regressor and progressor turtles are included in this
plasma bank. The immunological data obtained by testing plasma from these various populations will be evaluated and will
provide critical seroepidemiological information for determining the relationship between infection with the FP-associated
herpesvirus and the subsequent development of fibropapillomatosis. This evidence may be used in lieu of or in association with
transmission studies to strengthen the argument that the FP-associated herpesvirus is the etiologic agent of the disease.
Transmission studies, the "gold standard" for fulfilling Koch's postulates must await cultivation of the virus.


10. OBJECTIVES OF PROJECT (maximum 4000 characters):

To express LETV antigens, evaluate wild marine turtle populations for exposure to LETV, and conduct seroepidemiological studies as
follows:

a) PCR amplify open reading frames from genes that encode antigenic peptides of LETV identified from the genetic library,
express the peptides, and screen for antigenicity.

b) PCR amplify regions of LETV genome not included in the shotgun library based on sequence information of neighboring
genes to test additional gene products for antigenicity.

a) Identify peptides in LETV infected cells recognized by antibodies in LETV immunized plasma and wild green turtle plasma
in the Western format.

a) Develop serological tests using expressed LETV peptides, and validate the assay with plasma samples previously screened
for presence and absence of anti-LETV antibodies.

a) Screen various populations for exposure to LETV and provide evidence for exposure of wild marine turtles as well as critical
seroepidemiological information for determining the relationship between infection with LETV and the subsequent
development of LETD.

To express FPHV antigens and conduct seroepidemiological studies as follows:

a) PCR amplify open reading frames from genes that encode antigenic peptides of FPHV identified from the LETV genetic
library, express the peptides, and screen for antigenicity.

b) Develop serological tests (like the ELISA developed for LETV) using expressed FPHV peptides, and validate the assay with
plasma samples previously screened for the presence and absence of anti-FPHV antibodies.

a) Screen various populations for exposure to FPHV and provide critical seroepidemiological information for determining the
relationship between infection with the FP-associated herpesvirus and the subsequent development of fibropapillomatosis.










13. PROGRESS STATEMENT (see complete report attached)(maximum 4000 characters):
Herpesviruses are associated with several diseases of marine turtles including lung-eye-tracheal disease (LETD) and
fibropapillomatosis (FP). Critically needed are diagnostic tests for monitoring exposure of marine turtle populations to these
herpesviruses. Using virus-infected cell lysates, we have developed and applied an ELISA to demonstrate that wild green turtles in
Florida are exposed to the LETD-associated herpesvirus (LETV). In contrast, all attempts to cultivate the FP-associated herpesvirus
(FPHV) have been unsuccessful, limiting diagnostic assay development and seroepidemiological studies. Sea turtle conservation
efforts must include the means to monitor the health status of sea turtle populations for exposure to disease-associated
microorganisms. Critically needed are improvements in diagnostics, including the development of defined pathogen-specific antigens
for use in immunoassays that measure disease exposure. While the LETD-associated herpesvirus (LETV) can be cultivated in the
laboratory, efforts to cultivate the FP-associated herpesvirus (FPHV) have been unsuccessful, limiting diagnostic assay development
and epidemiological studies. In this study, we have extensively studied LETV in order to gain critical information about both LETV
and FPHV. This research has demonstrated that marine turtle herpesviruses can persist for extended periods of time as infectious
agents in the marine environment and that wild green turtles in Florida are exposed to the LETD-associated herpesvirus. This is the
first description of LETV infection in free-ranging marine turtles. In addition, data is presented that supports the hypothesis that LETV
and FPHV infections are independent. Furthermore, prior to this study, genomic sequence information for marine turtle herpesviruses
was limited. The only published genomic sequence information was for herpesviral DNA polymerase genes. To our knowledge, the
antigenic proteins identified in this study are not only the first proteins from a reptilian herpesvirus to be cloned and expressed, but
they represent the first reptilian herpesvirus proteins to be identified as immunogenic in their host species. In addition, these studies
have approached the difficult topic of how marine turtle herpesvirus may be transmitted in a pilot experiment on vertical transmission
in nesting turtles (manuscript in preparation). Finally, we have demonstrated using state-of-the art technology that a field portable
assay for measuring exposure of chelonians to infectious agents is feasible. Taken together, these data reveal new levels of complexity
that must be addressed before reliable serodiagnostic assays for herpesvirus infections of chelonians can be developed for widespread
application. The results reported here also raise new concerns about the potential impact of infections by new herpesviruses on
populations of wild marine turtles, an area which has previously been unexplored by turtle biologists. This work does move us closer
to further understanding of herpesvirus infections in sea turtles and the development of assays to detect exposure of wild sea turtles to
FPHV and other infectious agents that threaten their survival.





12. PROJECT SUMMARY STATEMENT (one or two hardhitting sentences that capture project merits):

We have developed immunological tests that can identify marine turtles in Florida (green and loggerhead) that have been exposed
to the LETV herpesvirus. The seroepidemiological data collected provides critical evidence about the relationship between
infection with the FP-associated herpesvirus and the LETV herpesvirus. The data supports the hypothesis that LETV and FPHV
infections are independent infections of marine turtles. The data shows that wild green turtles in Florida are exposed to the
LETD-associated herpesvirus, which is the first description of LETV infection in free-ranging marine turtles. To our knowledge,
the antigenic proteins identified in this study are not only the first proteins from a reptilian herpesvirus to be cloned and
expressed, but they represent the first reptilian herpesvirus proteins to be identified as immunogenic in their host species.

13.KEYWORDS (at least 1; up to 8): Fibropapillomatosis, marine turtles, lung-eye-trachea disease, herpesviruses,
seroepidemiology, recombinant-viral antigens, ELISA,.


14. PUBLICATIONS (Cite all publications resulting from project including proceedings and technical reports. Use Journal
of Wildlife Management style. Also, submit 5 reprints):

Coberley, S.S., Condit, R.C., Herbst, L.H., and P. A. Klein. Identification and Expression of Immunogenic Proteins of a Disease-
associated Marine Turtle Herpesvirus. J. Virol. 76:10553-10558, 2002.

Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Jacobson, E.R., and P.A. Klein. Survey of Florida Green Turtles
for Exposure to a Disease Associated Herpesvirus, Diseases of Aquatic Organisms 47, 159-167, 2001.











Coberley, S.S., Herbst, L.H, Brown, D.R., Ehrhart, L.M., Bagley, D.A., Schaf, S.A., Moretti, R.H., Jacobson, E.R., and P.A. Klein.
Detection of Antibodies to a Disease
associated Herpesvirus of the Green Turtle, Chelonia mydas. J.Clinical Microbiol., 39, 3572-3577, 2001.

Curry, S. S., D. R. Brown, E. R. Jacobson, and P. A. Klein., Ehrhart, L.M., Gaskin,J.M., Blahak, S., Herbst, L.H. and P. A. Klein.,
Persistent Infectivity of a Chelonian Herpesvirus after Exposure to Seawater. J. Wildlife Diseases 36, 792-797, 2000.

15. PRESENTATION CITATIONS (Use Journal of Wildlife Management style, to include Name, Year, Title, Meeting/Conference
Name, City and State.

Paul A. Klein, Daniel Brown, Elliott Jacobson, Lori Wendland, and Mary Brown. Microbial Pathogens, Immunology and Species
Conservation. Workshop on Desert Tortoise Health and Disease. Soda Springs, California, November 14-17, 2002

Brown,M.B., Klein, P.A., and Wendland, L.. Concepts and Importance of Disinfection. 28th Annual Meeting and Symposium of
the Desert Tortoise Council, Las Vegas, NV, February 21-23, 2003


Coberley, S., Condit, R., Herbst, L., and P. A. Klein. The Development of Recombinant Viral Antigens for Detecting Herpesvirus
Infections in Sea Turtles. In Proceedings in the Twenty-second Annual Symposium on Sea Turtle Biology and Conservation. Miami,
Florida. April 4-7, 2002.

Coberley, S.S., Condit, R.C., Herbst, L.H., and P. A. Klein Identification and Expression of Immunogenic Herpesviral for Detecting
Herpesvirus Infections in Sea Turtles. American Society for Microbiology Annual Meeting, May 19"-23, 2002

Hirschmann, R. J., Klein, P.A., Herbst, L.H., Ehrhart, L.M.,and Parkinson, C.L.
An investigation of vertical transmission in the role of the fibropapillomatosis-associated herpesvirus in marine turtles. Florida Keys
Sea Turtle Symposium, Marathon, FL., December 6-7t, 2002

Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Shaf, S.A., Moretti, R.H., Jacobson, E.R., and P.A. Klein. Serological
Detection of Herpesvirus Infections in Green Turtles. Abstract 267.7, The FASEB Journal, Part I, Abstracts 2.1-537.42, Experimental Biolog
2001, March 31-April 4th, 2001.

Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Shaf, S.A., Moretti, R.H., Jacobson, E.R., and P.A. Klein. Serological
Detection of Herpesvirus Infections in Green Turtles. 21st Annual Symposium on Sea Turtle Biology and Conservation, Philadelphia, PA
February 23rd-February 28th,2001, In press.

Coberley, S., Herbst, L.H., Ehrhart, L., Bagley, D., Hirama, S., Schaf, S., Moretti, R.,Jacobson, E., Condit, R. and P. Klein. Detection of
Antibodies to a Disease-associated Herpesvirus of the Green Turtle, Chelonia mydas. American Society for Virology. Madison, Wisconsin.
July 21-25', 2001.

Origgi, F.C., Jacobson, E., Herbst, L.H., Klein, P.A., and Curry, S.S.. Development of Serological Assays for herpesvirus Infections
in Chelonians. 20th Annual Symposium on Sea Turtle Biology and Conservation, Orlando, Fl, February 29th-March 4th, 2000, In
press.

16. THESES/DISSERTATIONS CITATIONS (Use Journal of Wildlife Management style. Also, submit 2 copies):

17. EMPLOYMENT STATUS OF GRADUATED STUDENTS (Provide Position Title, Agency/Company Name, City and State of
employment of any graduated MS or PhD students who graduated):
18. HONORS/AWARDS:
19.
20. PERSONNEL:
NAME POSITION* GENDER MINORITY** DEGREE PROGRAM***

Sadie Coberley, Graduate Student, F (UF), Ph.D.(2002), Interdisciplinary Program in Biomedical Sciences, COM,
Rachel Hirschman, Graduate Student, F, (UCF), M.S.(2003), Aquatic (Department of Biology, UCF)











Dean Bagley, Graduate Student, F, (UCF), M.S.,(2003), Aquatic (Department of Biology, UCF)




* MS Grad student; PhD grad student; Post-doc; Biologist; Technician;....

** B=Black; H=Hispanic; I=American Indian; O=Other; A=Alien

*** Aquatic, Terrestrial, or Integrated

20. NEWS MEDIA INVOLVEMENT:










Final Report
Research Work Order #213
January 20, 2004

Project Title: Seroepidemiological Studies of Herpesvirus-associated Diseases
of Marine Turtles: Fibropapillomatosis and Lung-Eye-Trachea Disease


Principal Investigator:


Co-principal investigator:


Collaborators:


Paul A. Klein, Ph.D.
Professor, Pathology, Immunology, and
Laboratory Medicine
University of Florida, Gainesville, FL

Elliott Jacobson, D.V.M., Ph.D.
Professor, Small Animal Clinical Sciences
College of Veterinary Medicine
University of Florida, Gainesville, FL

Larry Herbst, D.V.M, Ph.D
Associate Professor
Albert Einstein College of Medicine, Bronx, NY

Richard C. Condit, Ph.D.
Department of Molecular Genetics and Microbiology
University of Florida, Gainesville, FL

Daniel Brown, Ph.D.
Assistant Scientist
Department of Pathobiology
College of Veterinary Medicine
University of Florida, Gainesville, FL
Llewellyn Ehrhart, Ph.D., Professor,
Department of Biology, University of Central Florida
Orlando, Florida


Ritchie Moretti and Sue Schaf
Hidden Harbor Marine Environmental
Turtle Hospital, Marathon, Florida


Graduate Students:


Project, Inc. and The


Sadie S. Coberley (Ph.D. Program)
Interdisciplinary Program in Biomedical Sciences
College of Medicine
University of Florida, Gainesville, FL


Rachel Hirschman (M.S. Program)
Department of Biology
University of Central Florida
Orlando, FL






2

INTRODUCTION
In 1992, an interdisciplinary research team headquartered at the University of Florida began
studies in key targeted areas of fibropapillomatosis (FP) etiology and pathogenesis. At that
time, little was known about FP outside of field studies documenting its prevalence in
different areas of the world and studies of tumor histopathology- Our primary objective was
to develop a broad-based scientific understanding of FP by applying principles of tumor
biology, immunology, pathology, virology, molecular biology, and epidemiology to FP in
the green turtle, Chelonia mydas. Long-term goals included determining the etiology of FP,
the development of assays for FP, and study of any role of environmental co-factors in the
disease. This report is a continuation of that effort and the results reported here bring us
closer to understanding the role of a tumor-associated herpesvirus in marine turtle
fibropapillomatosis. These new findings also raise new concerns about the potential impact
of infections by new herpesviruses, such as lung-eye-trachea-disease virus on populations
of wild marine turtles, an area which has previously been unexplored by turtle biologists.
OVERALL SUMMARY
Sea turtle conservation efforts must include the means to monitor the health status of sea
turtle populations for exposure to disease-associated microorganisms. Critically needed are
improvements in diagnostics, including the development of defined pathogen-specific
antigens for use in immunoassays that measure disease exposure. Herpesviruses are
associated with several diseases of marine turtles including lung-eye-trachea disease
(LETD) and fibropapillomatosis (FP). While the LETD-associated herpesvirus (LETV) can
be cultivated in the laboratory, efforts to cultivate the FP-associated herpesvirus (FPHV)
have been unsuccessful, limiting diagnostic assay development and epidemiological
studies. In this study, we have extensively studied LETV in order to gain critical information
about both LETV and FPHV. We have developed the first ELISA that can detect
exposure of marine turtles to a specific herpesvirus infection (LETV). The specificity of this
ELISA for LETV (ability to distinguish LETV from FPHV) makes it valuable for detecting
exposure to this specific herpesvirus and enhances our ability to conduct
seroepidemiological studies of these disease-associated agents in marine turtles.
This research has demonstrated that marine turtle herpesviruses can persist for extended
periods of time as infectious agents in the marine environment and that wild green turtles in
Florida are exposed to the LETD-associated herpesvirus. This is the first description of
LETV infection in free-ranging marine turtles. The data clearly shows that that LETV and
FPHV infections are independent infections of marine turtles. Furthermore, prior to this
study, genomic sequence information for marine turtle herpesviruses was limited. The only
published genomic sequence information was for herpesviral DNA polymerase genes.
The antigenic proteins identified in this study are not only the first proteins from a reptilian
herpesvirus to be cloned and expressed, but they represent the first reptilian herpesvirus
proteins to be identified as immunogenic in their host species. In addition, these studies
have approached the difficult topic of how marine turtle herpesvirus may be transmitted in a
pilot experiment on vertical transmission in nesting turtles (manuscript in preparation).
Finally, we have demonstrated using state-of-the art technology that a field portable assay
for measuring exposure of chelonians to infectious agents is feasible. Taken together, these
data reveal new levels of complexity that must be addressed before reliable
serodiagnostic assays for herpesvirus infections of chelonians can be developed for
widespread application.
The results reported here also raise new concerns about the potential impact of infections
by new herpesviruses on populations of wild marine turtles, an area which has previously
been unexplored by turtle biologists. This work does move us closer to further






2

INTRODUCTION
In 1992, an interdisciplinary research team headquartered at the University of Florida began
studies in key targeted areas of fibropapillomatosis (FP) etiology and pathogenesis. At that
time, little was known about FP outside of field studies documenting its prevalence in
different areas of the world and studies of tumor histopathology- Our primary objective was
to develop a broad-based scientific understanding of FP by applying principles of tumor
biology, immunology, pathology, virology, molecular biology, and epidemiology to FP in
the green turtle, Chelonia mydas. Long-term goals included determining the etiology of FP,
the development of assays for FP, and study of any role of environmental co-factors in the
disease. This report is a continuation of that effort and the results reported here bring us
closer to understanding the role of a tumor-associated herpesvirus in marine turtle
fibropapillomatosis. These new findings also raise new concerns about the potential impact
of infections by new herpesviruses, such as lung-eye-trachea-disease virus on populations
of wild marine turtles, an area which has previously been unexplored by turtle biologists.
OVERALL SUMMARY
Sea turtle conservation efforts must include the means to monitor the health status of sea
turtle populations for exposure to disease-associated microorganisms. Critically needed are
improvements in diagnostics, including the development of defined pathogen-specific
antigens for use in immunoassays that measure disease exposure. Herpesviruses are
associated with several diseases of marine turtles including lung-eye-trachea disease
(LETD) and fibropapillomatosis (FP). While the LETD-associated herpesvirus (LETV) can
be cultivated in the laboratory, efforts to cultivate the FP-associated herpesvirus (FPHV)
have been unsuccessful, limiting diagnostic assay development and epidemiological
studies. In this study, we have extensively studied LETV in order to gain critical information
about both LETV and FPHV. We have developed the first ELISA that can detect
exposure of marine turtles to a specific herpesvirus infection (LETV). The specificity of this
ELISA for LETV (ability to distinguish LETV from FPHV) makes it valuable for detecting
exposure to this specific herpesvirus and enhances our ability to conduct
seroepidemiological studies of these disease-associated agents in marine turtles.
This research has demonstrated that marine turtle herpesviruses can persist for extended
periods of time as infectious agents in the marine environment and that wild green turtles in
Florida are exposed to the LETD-associated herpesvirus. This is the first description of
LETV infection in free-ranging marine turtles. The data clearly shows that that LETV and
FPHV infections are independent infections of marine turtles. Furthermore, prior to this
study, genomic sequence information for marine turtle herpesviruses was limited. The only
published genomic sequence information was for herpesviral DNA polymerase genes.
The antigenic proteins identified in this study are not only the first proteins from a reptilian
herpesvirus to be cloned and expressed, but they represent the first reptilian herpesvirus
proteins to be identified as immunogenic in their host species. In addition, these studies
have approached the difficult topic of how marine turtle herpesvirus may be transmitted in a
pilot experiment on vertical transmission in nesting turtles (manuscript in preparation).
Finally, we have demonstrated using state-of-the art technology that a field portable assay
for measuring exposure of chelonians to infectious agents is feasible. Taken together, these
data reveal new levels of complexity that must be addressed before reliable
serodiagnostic assays for herpesvirus infections of chelonians can be developed for
widespread application.
The results reported here also raise new concerns about the potential impact of infections
by new herpesviruses on populations of wild marine turtles, an area which has previously
been unexplored by turtle biologists. This work does move us closer to further








understanding of herpesvirus infections in sea turtles and the development of assays to
detect exposure of wild sea turtles to FPHV and other infectious agents that threaten their
survival.

RESULTS SUMMARIES

Note: Copies of papers published are attached These contain the full
details of results, materials, and methods. Other manuscripts are in
preparation.

1. Persistent Infectivity of a Green Turtle Disease-Associated Herpesvirus after
Exposure to Seawater Note: Certain aspects of this research were fundedin part by
RWO 180/194 to P.A. Klein as well as this RWO.
Manuscript: Curry, S., Brown, D.R., Gaskin, J.M., Jacobson, E.R., Ehrhart, L.M., Blahak,
S., Herbst, L.H., and P.A. Klein. Persistent infectivity of a green turtle disease-associated
herpesvirus after exposure to seawater. J. Wildlife Diseases, 36, 792-797, 2000
Summary: Herpesviruses are associated with several diseases of marine turtles including
Lung-Eye-Trachea disease and Gray Patch Disease (GPD) of green turtles (Chelonia
mydas) and fibropapillomatosis of green, loggerhead (Caretta care), and olive ridley
turtles (Lepidochelys olivacea). Stability of chelonian herpesviruses in the marine
environment has not been previously studied. In these experiments, LETD-associated
herpesvirus (LETV) was used as a model chelonian herpesvirus to test viral infectivity after
exposure to seawater. The LETV was grown in terrapene heart (TH-1) cells and then virus
preparations were dialyzed for 24 to 120 hr against aerated artificial or natural seawater or
Hank's balanced salt solution (HBBS). TH-1 cell cultures were inoculated with seawater-
exposed LETV, and on day 10 post-infection cells were scored for cytopathic effect
(CPE). Virus samples tested up to 120 hr after seawater exposure were positive for the
herpesvirus DNA polymerase gene by polymerase chain reaction. Electron microscopy
revealed intact LETV nucleocapsids after exposure of LETV to artificial seawater or HBSS
for 24 hr at 23 C. LETV preparations were then titered for infectivity and were found to
remain infectious after 120 hr of exposure to natural and artificial seawater at 23 C. Similar
results were obtained with a second cultivable chelonian herpesvirus, HV2245. LETV
infectivity could not be detected after 48 hr exposure to artificial seawater at 30 C. Since
LETV remains infectious for extended periods of time in the marine environment, it is
possible that FP-associated and GPD-associated herpesviruses also may be stable.
These findings are significant both for researchers studying the epidemiological association
of herpesviruses with diseases of marine turtles and for individuals who handle turtles in
marine turtle conservation efforts. They provide a proof of concept that herpesviruses
associated with FP are likely to be infectious in the marine environment.

2. Detection of Antibodies to a Disease-associated Herpesvirus
of the Green Turtle, Chelonia mydas
Manuscript: Coberley, S.S., Herbst, L.H, Brown, D.R., Ehrhart, L.M., Bagley, D.A.,
Schaf, S.A., Moretti, R.H., Jacobson, E.R., and P.A. Klein. Detection of Antibodies to a
Disease-associated Herpesvirus of the Green Turtle, Chelonia mydas. J.Clinical Microbiol.,
39, 2001, 3572-3577.








Summary: Lung-eye-trachea disease-associated herpesvirus-is linked with morbidity and
mortality in mariculture-reared green turtles, but its prevalence among and impact on wild
marine turtle populations is unknown. An enzyme-linked immunosorbent assay (ELISA)
was developed for detection of anti-LETV antibodies and could distinguish LETV exposed
green turtles from those with antibodies to fibropapillomatosis-associated herpesvirus.
Plasma from two captive-reared green turtles immunized with inactivated LETV served as
positive controls. Plasma from 42 healthy captive-reared green turtles, and plasma from 30
captive-reared green turtles with experimentally induced fibropapillomatosis (FP) and anti-
FPHV antibodies had low ELISA values on LETV antigen. A survey of wild green turtles
with (n=19) and without FP (n=27) (with and without anti-FPHV antibodies, respectively)
identified individuals with antibodies to LETV regardless of their FP status. The
seroprevalence of LETV infection was 13%. The presence of antibodies to LETV in
plasma samples was confirmed by Western blot and immunohistochemical analyses.
These results are the first to suggest that wild Florida green turtles are exposed to LETV or
to antigenically closely related herpesvirus(es) other than FPHV and that infection with
FPHV and LETV are most likely independent events. This is the first ELISA developed to
detect antibodies for a specific herpesvirus infection of marine turtles. The specificity of this
ELISA for LETV (ability to distinguish LETV from FPHV) makes it valuable for detecting
exposure to this specific herpesvirus and enhances our ability to conduct
seroepidemiological studies of these disease-associated agents in marine turtles.

3. Survey of Florida Green Turtles for Exposure to a Disease-associated
Herpesvirus

Manuscript: Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama,
S.,Jacobson, E.R., and P.A. Klein. Survey of Florida Green Turtles for Exposure to a
Disease Associated Herpesvirus. Diseases of Aquatic Organisms 47:159-167, 2001
Summary: A recently developed enzyme-linked immunosorbent assay (ELISA) was
used to assess exposure of Florida wild green turtles, Chelonia mydas, to LETV, the
herpesvirus associated with lung-eye-trachea disease. Plasma samples from 329 wild
juvenile green turtles netted in the Indian River lagoon, along the Sebastian reef, or in the
Trident basin (Indian River and Brevard Counties, Florida, USA) were tested by ELISA for
the presence of antibodies to LETV. Plasma samples from 180 wild juvenile green turtles
were tested from these study sites to compare the prevalence of anti-LETV antibodies.
While some plasma samples from each site contained anti-LETV antibodies (confirmed by
Western blot analysis), plasma samples collected from the Indian River lagoon had
statistically higher optical density values measured in the ELISA. No statistical differences
were observed when these same plasma samples were analyzed for changes in the level
of anti-LETV antibodies over three years (1997, 1998, and 1999). To explore the
relationship between anti-LETV antibodies and fibropapillomatosis (FP), plasma from 133
green turtles scored for fibropapilloma tumor severity were tested by ELISA. There was
no correlation between tumor severity and the presence of antibodies against LETV.
Additional plasma samples collected from 16 tagged green turtles captured and sampled
more than once (recaptures) were also tested to monitor antibody levels to LETV relative
to the FP status of individual turtles over time. Again there was no clear relationship
between FP tumor status and the presence of antibodies to LETV. Finally, ELISA tests on
plasma from 13 nesting female turtles (9 green and 4 loggerhead) revealed high levels of
anti-LETV antibodies in 11 individuals, including 2 loggerhead turtles. These results
provide strong evidence that wild Florida green turtle populations at these three study sites
are exposed to LETV or a closely related virus and that loggerhead turtles may be
exposed as well. Based on a cutoff optical density value of 0.310, 71 out of the 329 wild
Florida green turtles tested were seropositive for LETV antibodies (seroprevalence =








21.6%). In addition, no relationship between FP tumor severity or status and the presence
of anti-LETV antibodies was found, further supporting the hypothesis that LETV and the
FP-associated herpesvirus are separate infections of marine turtles.

4. Identification of immunodominant LETV herpesvirus antigens

Manuscript: Coberley, S.S., Condit, R.C., Herbst, L.H., and P. A. Klein. Identification
and Expression of Immunogenic Proteins of a Disease-associated Marine Turtle
Herpesvirus. J. Virol. 76:10553-10558, 2002.
Summary: Herpesviruses are associated with several diseases of marine turtles including
lung-eye-trachea disease (LETD) and fibropapillomatosis (FP). Diagnostic tests are critically
needed for monitoring exposure of marine turtle populations to these herpesviruses. An
ELISA assay was previously developed and applied using virus-infected cell lysates as
antigen to document exposure of wild green turtles in Florida to the LETD-associated
herpesvirus (LETV). In contrast, diagnostic assay development and seroepidemiological
studies of FP have been limited by an inability to cultivate the FP-associated herpesvirus
(FPHV). Antibodies to FPHV cross-react with LETV, suggesting a level of conservation of
viral proteins between these two marine turtle herpesviruses. Expression of recombinant
herpesviral proteins could provide an unlimited supply of antigen for LETV and FPHV
diagnostic assays. However, herpesviral antigens recognized by the green turtle humoral
immune response have not been identified. In this study, two approaches were used to
identify immunodominant viral antigens of LETV. The first approach targeted viral proteins
known to be immunogenic and neutralizing in other species and included glycoprotein B.
The second strategy was to identify those immunodominant proteins recognized on
Western blots by plasma antibodies from immunized or naturally infected green turtles. A
38 kDa protein was recognized by LETV and FPHV infected green turtle antibodies and
was resolved by 2D gel electrophoresis. The protein was extracted, digested, and HPLC
purified. The resulting protein fractions were sequenced and the protein was identified as a
scaffolding protein encoded by the overlapping open reading frames of UL26 and
UL26.5. Glycoprotein B and the scaffolding protein were PCR amplified based on the
sequence of a partial LETV genomic library, and products were cloned and expressed in E.
coli. The expressed proteins were recognized on Western blots by antibodies in immune
green turtle plasma. These recombinant herpesviral proteins were evaluated extensively in
ELISA assays as a source of antigen for screening marine turtle populations for exposure
to herpesviruses. They were compared to the intact LETV herpesvirus which was used in
all previous ELISA assays (see above). Unfortunately, these antigens could not
reproducibly replace intact virions in the ELISA plate assays. The ELISA testing was
exhaustive using nickel coated capture ELISA plates and recombinant antigens purified on
nickel affinity columns and a wide variety of different types of ELISA plates, blocking
buffers, and secondary antibodies. This result was not entirely surprising since these
recombinant antigens were denatured during isolation due to the fact that they were not
produced in a soluble form (were in bacterial inclusion bodies) by the bacterial hosts
carrying their respective genes. Additional work is needed to develop new clones which
secrete soluble forms of these immunogenic proteins for use in ELISA plate assays.

5. Investigation of Vertical Transmission in the Spread of Disease-Associated
Herpesviruses in Marine Turtles at the Archie Carr National Wildlife Refuge,
Florida
Manuscript in preparation: Hirschmann, R.J., P.A. Klein, L.M. Ehrhart, L.H. Herbst,and
C.L. Parkinsonl









Summary: Herpesviruses have been associated with several marine turtle diseases
including fibropapillomatosis, lung-eye-trachea disease, and gray patch disease. Diseases
can be transmitted horizontally from one animal to another by contact, or vertically, which is
the spread of a disease from mother to offspring. The current study focused on the role
that vertical transmission may play in the spread of the lung-eye-trachea disease-associated
herpesvirus (LETV) and the fibropapillomatosis-associated herpesvirus (FPHV) in nesting
marine turtles. Previous research has shown evidence of vertical transmission of
herpesviruses in a large number of vertebrates, including humans, birds, fish, and
amphibians. To determine if vertical transmission occurs in marine turtles, DNA was
extracted from eggs laid by the nesting turtles, from the tissues of dead hatchlings, and from
oviductal fluids secreted by the nesting turtles. A PCR (polymerase chain reaction) for the
herpesvirus POL gene was used to indicate the presence of herpesvirus Sequencing of
all products was utilized to identify specific herpesviruses. These studies shed light upon
the possible transmission mechanisms of marine turtle herpesviruses and provide valuable
conservation information.

6. Development of Field Portable Chelonian Serodiagnostics
Manuscript in preparation: Daniel R. Brown, Marianne F. Kramer, Laurie A. Zacher, April
M. Green, and Paul A. Klein Chelonian Serodiagnostics: Development of a Field
Portable
Assay For Detection of Exposure of Tortoises to Mycoplasma agassizi.
[This study was conducted using tortoises and mycoplasma antigen but is currently being
adapted for use in detecting herpesvirus infections of marine turtles.]
Summary: Infectious disease has affected plans for management and conservation of
legally protected chelonians in the United States. Tortoise conservation and recovery plans
now formally include testing for mycoplasmal Upper Respiratory Disease (URTD).
Detection of specific anti-mycoplasma antibodies may be used to diagnose infection and
immune status of chelonians as a tool for disease management. We have evaluated the
feasibility of a field test for specific antibodies against mycoplasma in chelonian plasma,
which would provide nearly instant information for management decision making.
Preliminary trials were conducted of evanescent-wave biosensor technology for detection
of specific anti-Mycoplasma agassizii antibodies in plasma from Gopherus agassizii
tortoises. The evanescent-wave biosensor is a laser-based polystyrene fiber optic sensor
which detects specific G. agassizii anti-M. agassizii antibody bound to M. agassizii whole-
cell lysate antigen. The reporter molecule was Cy5-labeled HL637 monoclonal antibody
against tortoise immunoglobulin. Under various experimental protocols, the signals from
positive control plasma samples from our bank were three to seven times higher than the
signals from negative control plasma samples. A randomized double-blind study was then
conducted to determine the sensitivity, specificity, positive predictive value, and negative
predictive value of the technique. Preliminary analyses of the results indicate a greater than
90% concordance with the traditional ELISA sample categorization, with a 5 minute per
sample, field-portable protocol. Those results suggest that this technology is feasible for
application under field conditions. Understanding the dynamics of disease spread in natural
wildlife populations may also provide valuable new insights into host:pathogen:population
interactions in this era of emerging infectious diseases.
The RAPTORTM (Research International, Woodinville, WA, 98072) is a portable, 4-
channel fluorometric assay system that can be used for high-sensitivity monitoring of
biological agents, toxins, and other analytes. It represents a careful integration of optics,









fluidics, electronics, and software into one compact and rugged system for use in laboratory
settings and field assays. This unit can automatically perform a user-defined, multi-step,
assay protocol while simultaneously tracking fluorescently-tagged chemical reactions
occurring on the surface of each of the system's four disposable optical waveguide sensors.



The RAPTORTM portable, 4-channel fluorometric instrument.


Detection of antibodies to chelonian pathogens using the Raptor. The
waveguide coated with herpesvirus antigen is first exposed to sea turtle
plasma/blood and then to the CY-5 labeled anti-sea turtle IgG antibody. Laser
light excites the bound CY-5 labeled antibody resulting in emitted fluorescence
which is recorded. To run an assay, the user inserts a coupon into the RAPTOR,
introduces the blood sample for testing, and presses the Run Assay key. All of
the fluidics are contained within the instrument and are automated using
computer-controlled miniature valves. The assay is complete in 15 minutes. The
results are stored and can be displayed on the LCD or transferred to a computer.
These assays are under further development.




DISCUSSION
All species of marine turtles have suffered serious population declines from over-
harvesting for their eggs, meat, and shells; entrapment by fishing lines and nets; collisions
with boats; dredging operations; and from destruction of nesting beaches and foraging
habitat, and are currently either threatened or endangered. Permission to study or








conduct experiments with these animals is restricted due their endangered or threatened
status. In addition, access to all life stages of turtles are further limited by their complex life
cycle that takes an individual turtle over thousands of miles of pelagic ocean. Marine
turtles traverse numerous marine habitats during their life history resulting in fragmented
knowledge about marine turtle behavior and ecology at these various sites. Limited
access has made it difficult to determine the impact of infectious diseases on marine turtle
populations, even though it is well-established that infections with pathogens are
capable of causing significant mortality in marine.
Worldwide experience has pointed to the central role of the immunological defense
systems of all animal species in resistance to and recovery from infectious diseases. This
is undoubtedly true in the case of marine turtles. Future improvements in the diagnosis
and control of infectious diseases in marine turtle populations will require an increased
understanding of the immunology of marine turtles, their associated pathogens, and the
environmental factors which may undermine the immune system's ability to cope
effectively with pathogens. Future research in this area will benefit from the networking of
wildlife disease experts, immunologists, and biotechnology researchers to develop and
utilize new tools and assays for management of diseases. Tools which are needed
include up-to-date medical, genetic, and immunological databases, a wider variety of
monoclonal antibodies to immune system components and pathogen-specific antigens,
natural and recombinant pathogen-specific antigens for use in serological assays and as
vaccines, and practical diagnostic immunoassays for measuring disease exposure and
immune system function.
Seroepidemiology, which uses a variety of laboratory-based serological assays, can
determine whether an individual has been infected with the disease-associated
microorganism and how many individuals in a population are infected (seroprevalence of
the disease). Seroepidemiology can track the spread of an infection through a
population or to new populations that have previously been disease free.
Seroepidemiology can help to determine the routes of disease spread and identify
mechanisms of disease transmission (vectors, high risk environmental conditions, etc.).
Seroepidemiology can also be used to provide evidence which links infection with an
infectious agent and the development of the full blown clinical disease. It is especially
useful in cases where the infectious agent cannot be isolated and grown in culture for use
in transmission studies as is currently the case for FP. The seroepidemiological approach
is the cornerstone of Hill's Criteria for Disease Causation. This seroepidemiological
approach has been used successfully in human diseases in which transmission studies
are not feasible, such as Kaposi's sarcoma, a cancer syndrome involving infection with a
newly identified herpesvirus.
The critical factor in using seroepidemiology as a tool to monitor and study infectious
disease in populations is having reliable, sensitive, and specific serological assays that
can detect antibodies against the infectious agent. Such assays require two main
components; antigens from the microorganism and specific secondary antibodies which
can detect the primary anti-infectious agent antibodies in plasma samples from animals.
Previously we have developed unique monoclonal antibody reagents against marine
turtle IgY, IgM, and 5.7s immunoglobulin subclasses as secondary antibodies. These
can detect turtle antibody responses to infectious agents. The research described here
explored the development of viral and pathogen-specific recombinant antigens of both
LETV and FPHV for use in seroepidemiological studies of these two diseases of marine
turtles.








The ability to grow LETV in culture facilitated the development of an ELISA to assess
exposure of populations of wild turtles to this herpesvirus. This has not been the case with
the FP-associated herpesvirus to date. In this study, the LETV-specific ELISA successfully
detected antibodies to LETV in plasma samples collected from 329 wild green turtles at
three study sites on the east coast of Florida (estimated seroprevalence of 21.6%), the first
report of this infection of these populations with this virus. Unfortunately, the developed
recombinant immunodominant LETV antigens could not reproducibly replace intact LETV
virions in ELISA plate assays. This result was not entirely surprising since these
recombinant antigens were denatured during isolation due to the fact that they were not
produced in a soluble form (were in bacterial inclusion bodies) by the bacterial hosts
carrying their respective genes. Additional work is needed to develop new clones which
secrete soluble forms of these immunogenic proteins for use in ELISA plate assays.
The data presented supports the hypothesis that LETV and FPHV infections of green and
loggerhead turtles are independent. Taken together, these data reveal new levels of
complexity that must be addressed before reliable serodiagnostic assays for herpesvirus
infections of chelonians can be developed for widespread application. The results also raise
important new concerns about the potential impact of infections by new herpesviruses on
populations of wild marine turtles, an area which has previously been unexplored by turtle
biologists. This work moves us closer to further understanding of herpesvirus infections in
marine turtles and the development of assays to detect exposure of marine turtles to
FPHV and other infectious agents that threaten their survival.



PRESENTATIONS AND PUBLICATIONS FROM THIS PROJECT

Presentations.
Paul A. Klein, Daniel Brown, Elliott Jacobson, Lori Wendland, and Mary Brown. Microbial
Pathogens, Immunology and Species Conservation. Workshop on Desert Tortoise Health
and Disease. Soda Springs, California, November 14-17, 2002
Brown,M.B., Klein, P.A., and Wendland, L.. Concepts and Importance of Disinfection.
28th Annual Meeting and Symposium of the Desert Tortoise Council, Las Vegas, NV,
February 21-23, 2003

Coberley, S., Condit, R., Herbst, L., and P. A. Klein. The Development of Recombinant
Viral Antigens for Detecting Herpesvirus Infections in Sea Turtles. In Proceedings in the
Twenty-second Annual Symposium on Sea Turtle Biology and Conservation. Miami,
Florida. April 4-7, 2002.
Coberley, S.S., Condit, R.C., Herbst, L.H., and P. A. Klein. Identification and Expression
of Immunogenic Herpesviral for Detecting Herpesvirus Infections in Sea Turtles. American
Society for Microbiology Annual Meeting, May 19th-23, 2002
Hirschmann, R. J., Klein, P.A., Herbst, L.H., Ehrhart, L.M.,and Parkinson, C.L.
An investigation of vertical transmission in the role of the fibropapillomatosis-associated
herpesvirus in marine turtles. Florida Keys Sea Turtle Symposium, Marathon, FL.,
December 6-7t", 2002








Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Shaf, S.A., Moretti, R.H.,
Jacobson, E.R., and P.A. Klein. Serological Detection of Herpesvirus Infections in Green Turtles.
Abstract 267.7, The FASEB Journal, Part I, Abstracts 2.1-537.42, Experimental Biology 2001,
March 31-April 4t, 2001.
Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Shaf, S.A., Moretti, R.H.,
Jacobson, E.R., and P.A. Klein. Serological Detection of Herpesvirus Infections in Green Turtles.
21st Annual Symposium on Sea Turtle Biology and Conservation, Philadelphia, PA February
23rd-February 28th,2001, In press.
Coberley, S., Herbst, L.H., Ehrhart, L., Bagley, D., Hirama, S., Schaf, S., Moretti, R.,Jacobson,
E., Condit, R. and P. Klein. Detection of Antibodies to a Disease-associated Herpesvirus of the
Green Turtle, Chelonia mydas. American Society for Virology. Madison, Wisconsin. July 21-25",
2001.
Origgi, F.C., Jacobson, E., Herbst, L.H., Klein, P.A., and Curry, S.S.. Development of
Serological Assays for herpesvirus Infections in Chelonians. 20th Annual Symposium on
Sea Turtle Biology and Conservation, Orlando, FI, February 29th-March 4th, 2000, In
press.

Publications.
Coberley, S.S., Condit, R.C., Herbst, L.H., and P. A. Klein. Identification and Expression
of Immunogenic Proteins of a Disease-associated Marine Turtle Herpesvirus. J. Virol.
76:10553-10558, 2002.
Coberley, S.S., Herbst, L.H, Ehrhart, L.M., Bagley, D.A., Hirama, S., Jacobson, E.R., and
P.A. Klein. Survey of Florida Green Turtles for Exposure to a Disease Associated
Herpesvirus, Diseases of Aquatic Organisms 47,159-167, 2001.
Coberley, S.S., Herbst, L.H, Brown, D.R., Ehrhart, L.M., Bagley, D.A., Schaf, S.A.,
Moretti, R.H., Jacobson, E.R., and P.A. Klein. Detection of Antibodies to a Disease
associated Herpesvirus of the Green Turtle, Chelonia mydas. J.Clinical Microbiol., 39,
3572-3577, 2001.
Curry, S. S., D. R. Brown, E. R. Jacobson, and P. A. Klein., Ehrhart, L.M., Gaskin,J.M.,
Blahak, S., Herbst, L.H. and P. A. Klein. Persistent Infectivity of a Chelonian Herpesvirus
after Exposure to Seawater. J. Wildlife Diseases 36, 792-797, 2000.

Related Publications and Presentations (Chelonlan Disease Research by
the P..).

Origgi, F.C, Romero, C.H., Bloom, D.C., Klein, P.A., Gaskin, J.M., Tucker, S.J., Jacobson, E.R.
Experimental Transmission of a Herpesvirus in Greek Tortoises
(Testudo graeca) Vet Pathol 41: 50-61, 2004
Origgi, F.C., Klein, P.A., Tucker, S.J., and E.R. Jacobson. Application of immunoperoxidase-
based techniques to detect herpesvirus infections in tortoises. J. Vet. Diagn. Invest. 15:133-140,
2003
Herbst,LH, Siconolfi-Baez, L, PA Klein, MJ Kerben, IM Schumacher. Induction of
vitellogenin by Estradiol-17beta and development of enzyme linked immunosorbant








assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas) Comp
Biochem Physiol B Biochem Mol Biol, 135:551-63, 2003.
D.R. Brown, I.M. Schumacher, G.S. McLaughlin, L.D. Wendland, M.B. Brown, P.A. Klein, and
E.R. Jacobson. Development and application of diagnostic tests for mycoplasmal infections of
tortoises. Chelonian Conservation and Biology. 4(2): 497-507, 2002.
Origgi, F., Klein, P.A., Mathes, K., Blahak, S., Marschang, R.E., Tucker, S.J., and Jacobson, E.R.,
An Enzyme-linked Immunosorbent Assay (ELISA) for Detecting Herpesvirus Exposure in
Mediterranean Tortoises, Testudo graeca and Testudo Hermanni. J Clin Microbiol. 39:3156-63,
2001
Herbst, L.H., Chahrabarti, R., Klein, P.A., and Achary, M. Differential gene expression
associated with tumorigenicity of cultured green turtle fibropapilloma-derived fibroblasts. Cancer
Genetics and Cytogenetics, 128: 1-5, 2001
F.C. Origgi, C. H. Romero, P.A. Klein, K. H. Berry, and E. R. Jacobson Serological and
Molecular Evidences of Herpesvirus Exposure in Desert Tortoises from the Mojave and
Colorado Deserts of California. Workshop on Desert Tortoise Health and Disease. Soda
Springs, California, November 14-17, 2002
Origgi FC, Romero CH, Klein PA, Berry KH, Johnson A, and Jacobson ER Preliminary
serological and molecular evidences of Tortoise Herpesvirus exposure in Desert Tortoises
(Gopherus agassizii) from the Mojave and the
Colorado Desert of California. ARAV 8th Annual Conference, Reno, Nevada, October 17-
21, 2002.