Immunosuppression abrogates resistance of young rabbits to Rabbit Haemorrhagic Disease (RHD)

Abstract

Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naïve adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection.

Figure 1

Immunocytochemistry of RHDV antigen in the liver. A: Light micrograph of a liver paraffin section of an immunosuppressed, RHDV-infected young rabbit that died with RHD showing widespread positive immunodetection of the major RHDV antigen (brown staining). Heterophils are observed surrounding the damaged hepatocytes and inside sinusoids (arrows). The tissue section was labelled with an anti-VP60 mouse antiserum; haematoxylin counterstain; bar = 100 μm. B: A liver section of an immunosuppressed, RHDV-infected young rabbit that died with RHD and that was labeled with control mouse antiserum; haematoxylin counterstain, bar = 100 μm. C: A liver paraffin section of an infected young rabbit that survived the infection and was euthanized 7 days later. Signs of hepatocellular regeneration are visible as expressed by enlarged and often binucleated cells (without the formation of organized hepatic cords); some tissue spaces are occupied by small basophilic cells, with an oval shape, that are suggestive of hepatocyte precursors. The tissue section was labeled with an anti-VP60 mouse antiserum; haematoxylin counterstain, bar = 100 μm.

Figure 2

Immunocytochemistry of apoptosis in the liver, spleen and thymus. Light micrographs of the liver, spleen and thymus paraffin sections where the immunodetection of cellular apoptosis was obtained by TUNEL; counterstaining with haematoxylin. In liver sections, bar = 100 μm; in spleen and thymus sections, bar = 200 μm. A: A liver paraffin section of an immunosuppressed, RHDV-infected young rabbit that died due to RHD. B: A liver paraffin section of an immunosuppressed, RHDV-infected young rabbit that died from RHD; control of the TUNEL assay. C: A spleen paraffin section of an immunosuppressed young rabbit euthanized 7 days after immunosuppression. D: A thymus paraffin section of an immunosuppressed young rabbit euthanized 7 days after immunosuppression. E: A spleen paraffin section of an immunosuppressed, RHDV-infected young rabbit that died from RHD. F: A thymus paraffin section of an immunosuppressed, RHDV-infected young rabbit that died from RHD. G: A spleen paraffin section of an infected young rabbit euthanized 7 days after RHDV infection. H: A thymus paraffin section of an infected young rabbit euthanized 7 days after RHDV infection.

Figure 3

Evaluation of serum cytokines – graphics. A: Comparison of TNF-α levels in the serum of control (PBS) young rabbits (n = 5), immunosuppressed (IS) young rabbits (n = 5), and immunosuppressed RHDV-infected (IS + I) young rabbits (n = 9) that died from RHD; unpaired t test, (***p < 0.0001); the results are shown as a mean ± SEM). B: Comparison of IL-6 levels in the serum of control (PBS) young rabbits (n = 5), immunosuppressed (IS) young rabbits (n = 5) and immunosuppressed RHDV-infected (IS + I) young rabbits (n = 9) that died from RHD; unpaired t test, (*p < 0.05); the results are shown as a mean ± SEM). C: Comparison of IL-10 levels in the serum of control (PBS) young rabbits (n = 5), immunosuppressed (IS) young rabbits (n = 5), and immunosuppressed RHDV-infected (IS + I) young rabbits (n = 9) that died from RHD; unpaired t test, (**p < 0.01); the results are shown as a mean ± SEM).