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

Ranavirus Outbreak 2011

13 of 27 Eastern box turtles died

Positive for Ranavirus, Herpesvirus, and

Mycoplasma spp.

Severe clinical signs

After Brumation

14 turtles survived the outbreak

Negative oral swabs via PCR

No clinical signs present

Study Purpose

Immunity to reinfection

Viral shedding

Virus persistence in tissues

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

ResultsClinical signs:

4/7 Inoculated

0/4 Controls

Plastron ulcer

Oral ulcers

Swollen sublingual tissue

Results

Only 1 of 7 inoculated turtles died on

Day 22

Petechiae of the tongue

Nasal discharge

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

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

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

Pulmonary Intracytoplasmic

Inclusion Bodies

Necropsy Results

Necropsy and histopathology of 5 Eastern

box turtles

3 inoculated and 2 control

No inclusion bodies

No characteristic Ranavirus lesions

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

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?

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