Experimental Challenge Study of Ranavirus
Infection in Previously Infected Eastern Box
Turtles (Terrapene carolina carolin...
Ranavirus Outbreak 2011
13 of 27 Eastern box turtles died
Positive for Ranavirus, Herpesvirus, and
Mycoplasma spp.
Seve...
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,...
Results
Clinical 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
or...
Ranavirus qPCR Results
Turtle
Day
0
Day
6
Day
9
Day
13
Day
16
Day
20
Day
22
Day
27
Day
41
Day
55
EBT
R98045
swab 0 137,847...
Necropsy Results
Turtle that died
Intracytoplasmic inclusion bodies:
Kidney, lungs, pancreas, liver, vas deferens
Vasc...
Pulmonary Intracytoplasmic
Inclusion Bodies
Necropsy Results
Necropsy and histopathology of 5 Eastern
box turtles
3 inoculated and 2 control
No inclusion bodies
N...
Tissue Ranavirus qPCR Results
All tissues in the turtle that died positive
Kidney > Liver > Spleen
Other 3 inoculated t...
Discussion and Summary
 All turtles negative after brumation
 Take home points:
1)Can be reinfected
2)Can survive reinfe...
Thank you for listening
A special thank you to all of my co-authors
and the Maryland Zoo in Baltimore’s
wonderful managem...
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Experimental challenge study of ranavirus infection in previously infected eastern box turtles

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2013 International Symposium on Ranaviruses
by Jennifer Hausmann

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  • As you will hear from Dr. Rich Sim tomorrow, the Maryland Zoo in Baltimore lost 13 of its 27 captive Eastern box turtles to an outbreak of Ranavirus the summer of 2011. There was a 48 % mortality rate.Some of these turtles alsohad co-infections with herpesvirus and mycoplasma, but themorbidity and mortality of this outbreak was attributed to ranavirus. The clinical signs in most of the turtles were very severe as pictured here. Dr. Simwill go into the outbreak and treatment in further detail in his presentation tomorrow, and right now I will be focusing on what occurred after the outbreak.
  • 14 of the box turtles turtles survived the outbreak. All the turtles were negative for Ranavirus on oral swabs after completeinga normal brumation in their enclosure. Post-brumation there were no clinical signs. As far as we are aware, this is the largest known group of chelonians to survive a ranavirus outbreak. These turtles represented a unique population of Eastern box turtles that could shed light on whetherbox turtles that survive a ranavirus infection are potentially immune to the virus or chronic carriers.
  • Therefore, the purpose of this study was be to begin to determine whether 1) previously infected turtles appear to be immune to re-infection with morbidity; 2) if virus shedding occurs during a repeated exposure to ranavirus; and 3) if the virus persists in the tissues. The hope was that this knowledge would help to form recommendations for wildlife rehabilitators and veterinarians dealing with Eastern box turtles and other chelonians.
  • To assess survival, immunity, and shedding post-infection, an experimental challenge study was performed. In this study the surviving eastern box turtles that had confirmed positive ranavirusqPCRtests during the outbreak were re-infected with the outbreak strain of Ranavirus. Seven turtles were inoculated intramuscularly with a predetermined dose of infectious virus and four control turtles were injected with an equal volume of saline. The turtles were kept in separate rooms. The temperature was attempted to be maintained between 70-75 degrees, which is the optimal temperature for the virus to replicate.The turtles were monitored for 8 weeks with blood and oral swabs collected for qPCR.
  • During the 8 weeks of the study, four of the inoculated turtles showed clinical signs and none of the control turtles. The most common clinical signs were lethargy, weight loss, ocular discharge and periocular swelling. Other less common clinical signs included: mild oral ulcers, plastron ulcers, and sublingual swelling.
  • Only one of the seven inoculated turtles and none of the controls died. There was no significant difference in survival between the two groups.The one turtle diedon day 22 after developing ocular-nasal discharge, lethargy, and petechia of the tongue as pictured here.
  • All controls were negative on oral swabs and whole blood by quantitative PCR for Ranavirus. All of the inoculated turtles were positive on either oral swabs or WB samples or both for ranavirus. The turtle that died had an overwhelming viremia as compared to the other turtles sampled.
  • This chart shows the viral loads of the inoculated turtles on blood and oral swabs. Only one turtle was positive on day 6. This was the turtle that died during the study. On day 13 two other turtles became positive. Then, on day 16, 6 of 7 turtles were positive. All 7 turtles had a positive test result by day 20.5/6 were still positive on oral swabs only on day 55, the last day of the study. All turtles were positive by oral swabs. But, 3/7 were never positive on blood.These turtles may have had a very short viremicphase, which was not caught on the samples.
  • The necropsy results of the turtle that died during the study showed gross and histologic lesions similar to the turtles that died during the outbreak. The cause of death for this turtle was considered to be due to a secondary bacterial infection associated with the underlying ranavirus infection. This turtle had intracytoplasmic inclusion bodies in multiple organs. In addition, thehistopathology showed vasculitits and inflammation in multiple organs. All these lesions were characteristic for Ranavirus infections.
  • Five of the surviving ten turtles were euthanized at the end of the study for histopathology and qPCRtesting. 3 inoculated and 2 controls where euthanized. No intracytoplasmic inclusion bodies were seen. And, nocharacteristic lesions for Ranavirus were seen.
  • On quantitative PCR for ranavirus on the tissues of the necropsied turtles revealed variable positive results. The turtles that diedwas positive on all tissues tested: Kidney had the highest level, then liver, and then spleen. The 3 inoculated turtles tested revealed varying tissues to be positive. These tissue were positive forranavirus, even though no intracytoplasmic inclusion bodies were seen on histopathology. The one innoculatedturtle that was negative on all tissues was negative on blood and oral swabs at time of death. Two controls were necropsied. All tissues on the controls were negative, except the spleen of one of the turtles.
  • The remaining five turtles awoke frombrumationandwere negative for Ranavirus on oral swabs and WB. They are currently healthy and showing no clinical signs of the virus. In conclusion this study indicates that:Eastern box turtles can be re-infected with the same strain of ranavirusThey can survive re-infection without supportive care. And, the clinical signs during re-infection are much less severe and can be mild or completely absent. But based on this study, turtles will shed virus from the oral cavity if re-infected with ranavirusThe fact that the morality rate was 14.3% (1/7) in this study versus the 48% (13/27) in the original outbreak despite not receiving any supportive treatment, indicates that some level of immunity to the virus is gained by eastern box turtles after infection. However the turtlecan still become re-infected and shed the virus. Because of this, there is a risk that a rehabilitated and released Eastern Box Turtle could become re-infected and shed ranavirusinto the environment without showing clinical signs, potentially spreading the disease to other turtles and amphibians. In addition, as one of the control turtles had virus still present in the spleen at necropsy, despite not being positive on oral swabs or blood for ranavirus for over a year. This could indicate that there is persistence of the virus in the tissues. The major draw back of this study is the low number of turtles studied, making it difficult to say for certain if persistence in the tissues occurs. This study is an initial exploration into the immune response to reinfection with ranavirus in eastern box turtles. And further studies should be pursued to assess immunity and persistence in other chelonian species.
  • thank you all forlistening.
  • Experimental challenge study of ranavirus infection in previously infected eastern box turtles

    1. 1. 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
    2. 2. Ranavirus Outbreak 2011 13 of 27 Eastern box turtles died Positive for Ranavirus, Herpesvirus, and Mycoplasma spp. Severe clinical signs
    3. 3. After Brumation 14 turtles survived the outbreak Negative oral swabs via PCR No clinical signs present
    4. 4. Study Purpose Immunity to reinfection Viral shedding Virus persistence in tissues
    5. 5. 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
    6. 6. Results Clinical signs: 4/7 Inoculated 0/4 Controls Plastron ulcer Oral ulcers Swollen sublingual tissue
    7. 7. Results Only 1 of 7 inoculated turtles died on Day 22 Petechiae of the tongue Nasal discharge
    8. 8. 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
    9. 9. Ranavirus qPCR Results Turtle Day 0 Day 6 Day 9 Day 13 Day 16 Day 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
    10. 10. 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
    11. 11. Pulmonary Intracytoplasmic Inclusion Bodies
    12. 12. Necropsy Results Necropsy and histopathology of 5 Eastern box turtles 3 inoculated and 2 control No inclusion bodies No characteristic Ranavirus lesions
    13. 13. 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
    14. 14. Discussion and Summary  All turtles negative after brumation  Take home points: 1)Can be reinfected 2)Can survive reinfection 3)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?
    15. 15. 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

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