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Experimental Challenge Study of Ranavirus
Infection in Previously Infected Eastern Box
Turtles (Terrapene carolina carolina) to
Assess Immunity
Hausmann JC, Wack AN, Allender MC,
Cranfield MR, Murphy KJ, Barrett K, Romero
JL, Wellehan JF, Zink C, Bronson E
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Ranavirus Outbreak 2011
13 of 27 Eastern box turtles died
Positive for Ranavirus, Herpesvirus, and
Mycoplasma spp.
Severe clinical signs
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After Brumation
14 turtles survived the outbreak
Negative oral swabs via PCR
No clinical signs present
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Study Purpose
Immunity to reinfection
Viral shedding
Virus persistence in tissues
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Study Design
11 Eastern box turtles
7 Inoculated IM
4 Controls
Monitored for 8 weeks
Blood and oral swabs
Days 0, 6, 9, 13, 16,
20, 27, 41, and 55
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ResultsClinical signs:
4/7 Inoculated
0/4 Controls
Plastron ulcer
Oral ulcers
Swollen sublingual tissue
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Results
Only 1 of 7 inoculated turtles died on
Day 22
Petechiae of the tongue
Nasal discharge
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Ranavirus qPCR Results
All Controls negative on whole blood and
oral swabs
All Inoculated positive on either blood or
oral swabs
Deceased turtle had
overwhelming viremia
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Ranavirus qPCR Results
TurtleDay
0
Day
6
Day
9
Day
13
Day
16Day
20
Day
22
Day
27
Day
41
Day
55
EBT
R98045
swab 0 137,847 1.35E+07 3.21E+09 9.64E+09 1.30E+10 1.32E+10
blood 0 546,000 6.85E+06 9.77E+07 1.55E+08 1.03E+8 1.07E+08
EBT
R98038
swab 0 0 0 6,527 1.20E+06 38,759 5,1491 10,328 47,600
blood 0 0 0 0 0 0 0 0 0
EBT
R98035
swab 0 0 0 2,539 3,223 0 26,132 4,144 0
blood 0 0 0 21,125 0 534 0 0 0
EBT
99R184
swab 0 0 0 0 20,313 58,385 0 19,080 20,862
blood 0 0 0 0 0 619 0 0 0
EBT
R98033
swab 0 0 0 0 65,711 0 0 1,275 3,074
blood 0 0 0 0 0 1,353 0 0 0
EBT
R98049
swab 0 0 0 0 322,948 0 293,427 5,367 1,972
blood 0 0 0 0 0 0 0 0 0
EBT
649
swab 0 0 0 0 0 451,283 0 215,167 1,526
blood 0 0 0 0 0 0 0 0 0
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Necropsy Results
Turtle that died
Intracytoplasmic inclusion bodies:
Kidney, lungs, pancreas, liver, vas deferens
Vasculitis: spleen, pancreas, lungs, and liver
Nephritis, pneumonia, esophagitis, hepatitis,
enteritis
Splenic necrosis
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Pulmonary Intracytoplasmic
Inclusion Bodies
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Necropsy Results
Necropsy and histopathology of 5 Eastern
box turtles
3 inoculated and 2 control
No inclusion bodies
No characteristic Ranavirus lesions
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Tissue Ranavirus qPCR Results
All tissues in the turtle that died positive
Kidney > Liver > Spleen
Other 3 inoculated turtles:
1 positive in spleen
1 positive in liver and bone marrow
1 negative in all tissues
2 Controls:
1 positive in spleen
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Discussion and Summary
All turtles negative after brumation
Take home points:1)Can be reinfected2)Can survive reinfection3)Will shed virus if reinfected
Mortality rate: 14.3% (1/7) vs 48% (13/27) Indicates some level of immunity Risk for rehabilitation Persistence in the tissue?
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Thank you for listening
A special thank you to all of my co-authors
and the Maryland Zoo in Baltimore’s
wonderful management and keeper staff