1. Pathogenicity and Transmission of Reticuloendotheliosis Virus (REV) Isolated from Endangered Prairie Chickens
AUTHORS: Taylor Barbosa, Guillermo Zavala, Sunny Cheng, and Pedro Villegas
Melissa Hopkins
BIOL 5331
Hernández, F., Brennan, L. A., DeMaso, S. J., Sands, J. P., and D. B. Wester On reversing the northern bobwhite population decline: 20 years later. Wildlife Society Bulletin 37: 177–188.

2. Basic Principles of Epidemiology
A high rate of contact between host and parasite encourages the spread of disease.
Indirect effects of habitat destruction increase susceptibility to disease.
Species in conservation programs may contract diseases from related species, and even humans.1
In unnaturally confined conditions, animals remain in contact with the potential sources of infection & disease transmission increases.
- Prairie chickens at fossil rim kept in open-air pens composed mainly of chicken wire, which renders the animals susceptible to potential insect vectors.
When a host population is crowded into a smaller area because of habitat destruction, there will often be lowered habitat quality and food availability, high contract rates, lowered nutritional status, weaker animal, and less resistance to infection.
REV has been documented in chickens, turkeys, ducks, pheasants, geese, Japanese quail, peafowl, and prairie chickens.
Hernández, F., Brennan, L. A., DeMaso, S. J., Sands, J. P., and D. B. Wester On reversing the northern bobwhite population decline: 20 years later. Wildlife Society Bulletin 37: 177–188.

3. Importance of Studying REV
What is REV?
Retrovirus (ex. HIV)
Infects Gallinaceous species
Ex. Attwater’s and Lesser Prairie Chickens
Originally a mammalian virus2
Recently entered bird genome
Spread anthropogenically
Small Knowledgebase
Uncertain transmission method
Insect vector?
Signs of Infection:
Reproductive suppression3
Lymphosarcomas
Enlargement of visceral organs
Delayed growth
Feather development abnormalities
Retrovirus = any of a group of RNA viruses that insert a DNA copy of their genome into the host cell in order to replicate
Spread by humans = It is thought the virus was spread through injections of a malaria-causing parasite (which, unbeknownst to scientists, harbored a strain of REV), which was being heavily studied at the time and required avian hosts to persist, and also into domestic poultry through injection of contaminated viruses
Hernández, F., Brennan, L. A., DeMaso, S. J., Sands, J. P., and D. B. Wester On reversing the northern bobwhite population decline: 20 years later. Wildlife Society Bulletin 37: 177–188.

4. Importance of Studying REV
Conservation Implications
Northern bobwhite decline4
Habitat destruction & fragmentation
Role of disease?
Attwater’s prairie chicken (APC)
High rate of contact = Spread
Economic Impacts
Hunting
Northern bobwhite = ~200 million/year5
Poultry Industry
High rate of contact
Reproductive effects?
Retrovirus = any of a group of RNA viruses that insert a DNA copy of their genome into the host cell in order to replicate
Spread by humans = It is thought the virus was spread through injections of a malaria-causing parasite (which, unbeknownst to scientists, harbored a strain of REV), which was being heavily studied at the time and required avian hosts to persist
4. Hernández, F., Brennan, L. A., DeMaso, S. J., Sands, J. P., and D. B. Wester On reversing the northern bobwhite population decline: 20 years later. Wildlife Society Bulletin 37: 177–188.
5. Burger, L. W., Miller, D. A., and R. I. Southwick Economic Impact of Northern Bobwhite Hunting in the Southeastern United States. Wildlife Society Bulletin 27: 1010–1018.

5. Introduction Several Strains Propagation Japanese Quail
Similar detrimental effects
Propagation
Culture in chicken/duck embryo fibroblasts or DF-1 cells
Japanese Quail
Good model for REV
Rapid development
Related to domestic species
Attwater’s Prairie Chicken
Strain APC-566 used for study
Endangered species
REV contribution
Transmission Method
Mechanism uncertain
Biological reservoirs?
Insect vector?

6. Introduction Objectives of Study: Pathogenicity Transmission Method
Vertical or Horizontal?
Oncogenicity

7. Materials & Methods PCR Quails, Chickens, & Turkeys
Japanese quail eggs from local hatchery (tested breeders for REV)
Specific pathogen free (SPF) chickens & turkey eggs
History of REV Isolate from APCs
Fossil Rim Wildlife Center ( )
APC-566 strain from blood sample
46 infected APCs
APC-566 genome completely sequenced
Virus Isolation & Titration
Virus Stock
3 passages of DF-1 cells infected w/ REV
Titrated & frozen (-80 C)
Virus Titer
Reed and Muench method
Indirect immunofluorescence
PCR
Diagnostic PCR
Amplify a part of the long terminal repeat of REV

8. Materials & Methods Immunohistochemistry Antibody Assays
Used to detect REV antibodies
ELISA test (IDEXX Laboratories)
Western Blot Assays
Used to confirm ELISA results
Indirect Fluorescent Antibody (IFA) Test
Determine antibody response to REV
Fluorescent dyes
Immunohistochemistry
 Process of detecting antigens in neoplastic cells

9. Materials & Methods Sample Collection Whole blood (plasma & serum)
Heparinized & microcentrifuge tubes
Deceased specimens examined for:
Gross lesions (forced abnormalities)
Small neoplasia (new growth)
Tissues examined by light microscopy

10. Experiment #1 – Japanese Quail
Experimental Design
Experiment #1 – Japanese Quail
First Quail Generation (F1)
Infected as Embryos
6 groups – varying proportion of infected individuals
Control = 120 | Others = 60
0, 5, 10, 25, 50, 100%
6 weeks = separation
Egg production
Eggs incubated & hatched
All breeder birds euthanized & examined (20 wk)
Second Quail Generation (F2)
30 chicks per breeder group
Viremia at hatch (serum)
Kept until 6 weeks
F2 gen. euthanized & examined

11. Experiment #2 – Turkeys & Chickens
Experimental Design
Experiment #2 – Turkeys & Chickens
SPF Chickens
Eggs = Sunrise Farms
Groups of 8 chicks
Including negative control group
Inoculated after hatch
Kept 9 weeks
Euthanized & Examined
SPF Turkeys
Eggs = Southeast Poultry Research Laboratory
Grouped & inoculated similarly to chickens
Kept 14 weeks
Euthanized & Examined

12. Statistical Analysis Difference in Quail Antibody Response
Analysis of Variance (ANOVA) – Duncan Test
SAS Software Package
Differences Between Mortalities
Χ2 Test

13. Results Susceptibility to Infection Viremia
Infected Quail = delayed growth (3 wk)
Mortality Rates (20 wk)
100% inoculated = 61.6%
Negative Control = 10.8%
Mortality rate sig. higher:
At 6 wk in all infected groups
At 20 wk in 25%, 50%, 100%
Viremia
Higher Inoculation = Higher Viremic Birds (20 wk)
Many viremic birds w/ no antibodies (6 wk)
All Nonviremic birds had antibodies
By 20 wk % viremic decreased
All F2 generation (-) for REV
Evidence against vertical transmission

14. Results Oncogenicity & Pathology Antibody Responses
Antibody (+) between 6-20 wk
Many viremia (+)/antibody (-) & vice versa (6 wk)
Similar at 20 wk, except 100% group
100% Inoculated Group (20 wk):
8 of 14 viremia (+)/antibody (+)
5 of 14 viremia (-)/antibody (+)
1 of 12 viremia (+)/antibody (-)
F2 generation (-) for REV antibodies
Oncogenicity & Pathology
Attwatwer’s Prairie Chickens
Specimens from same colony as APC-566
Multiple tumors/organ enlargement
Cutaneous lymphosarcomas*
Kidney & small intestine esp. vulnerable
Chickens
Feather development abnormalities (28 days)
Enlargement of spleen

15. Results Oncogenicity & Pathology Microscopic Pathology Turkeys
Intrafollicular bursal lymphomas*
2 birds experienced:
Enlarged spleen & liver
Renal lymphoma & lymphocytic leukemia
Japanese Quail
Lymphosarcomas = viremia (-)/ antibody (+)
Enlargement of spleen & liver
Microscopic Pathology
Tumors
Solid sheets
Uniform size
Lymphoblastoid* cells in variety of visceral organs

16. Discussion Japanese Quail = Successful Model
REV oncogenic in quail, chickens, & turkeys
Horizontal Transmission
Limitation: young quail
Significant Mortality (Quail)
100% inoculated = 61% mortality
Negative control = 10% mortality
Embryo inoculation*
Low Tumor Detection
6.6.% vs. 92.8% (Witter et al.)
20 wk vs. 90+ wk
Transmission Method
Horizontal = 2 Quail
Mechanism?
Vertical = very low rates*

17. Questions?