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

Marques RM, Teixeira L, Aguas AP, Ribeiro JC, Costa-e-Silva A, Ferreira PG - Vet. Res. (2014)

Bottom Line: Remarkably, young rabbits are resistant to RHD.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, ICBAS (Abel Salazar Institute for Biomedical Science) and UMIB (Unit for Multidisciplinary Biomedical Research), University of Porto, Rua de Jorge Viterbo Ferreira n,° 228, 4050-313 Porto, Portugal. ramarques@icbas.up.pt.

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.

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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.
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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.

Mentions: All immunosuppressed, RHDV-infected young rabbits died within 24–72 h of RHDV infection (24–30 h n = 2; 36–48 h n = 6; 60–72 h n = 1) depicting the hyperacute form of RHD, i.e., they died suddenly, often without conspicuous signs of disease, showing prostration, lethargy and anorexia. As expected, all non-immunosuppressed infected young rabbits showed no clinical symptoms of disease after being infected with RHDV; they were euthanized 1 week after the viral inoculation. Immunosuppressed rabbits (infected (IS + I) or not (IS)) showed atrophic thymuses, indicating that immunosuppression had been achieved; this atrophy was confirmed by the ratio thymus (g)/body weight (g) since we found that these ratios were significantly lower in immunosuppressed groups (IS group: 0.00048 ± 0.000049; IS + I group: 0.00054 ± 0.000050; mean ± standard error of the mean (SEM); p < 0.0001) than in control rabbits (PBS group: 0.0024 ± 0.00024; mean ± SEM). The immunosuppressed, RHDV-infected young rabbits showed the typical lesions of RHD, such as enlarged and pale livers with enhanced lobular patterns, congestion and haemorrhagic lesions of several viscera, and bloody foam in the trachea. A significant difference was seen in the spleen volume, with splenomegaly in immunosuppressed, RHDV-infected rabbits in contrast with spleen atrophy in immunosuppressed animals (ratio - spleen (g)/body weight (g); p < 0.05). Young rabbits that were infected with RHDV did not present any macroscopic lesions suggestive of RHD, 7 days after the viral inoculation, when they were euthanized. Widespread liver cell vacuolization was seen in immunosuppressed, RHDV-infected young rabbits; these hepatocytes often showed pyknotic nuclei and karyorrhexis, classic features of cell death (Figure 1A). The TUNEL assay confirmed extensive cell death of hepatocytes and this was illustrated by brown-nuclear staining (early apoptotic cells) or brown-cytoplasmic staining (late apoptotic or necrotic cells) (Figure 2A). Scattered inflammatory cells (predominantly heterophils) were seen in the liver of the animals. In contrast, hepatic tissue of the immunosuppressed rabbits did not reveal any signs of hepatocellular degeneration suggestive of apoptosis. In RHDV-infected young rabbits, at day 7th after viral inoculation, there was evidence of hepatic regeneration as expressed by rebuilding of focal hepatic lesions that had been produced by the infection (Figure 1C). All immunosuppressed, RHDV-infected young rabbits presented an extensive brown-staining of the liver for the RHDV antigen (Figure 1A). In contrast, RHDV-infected young rabbits, euthanized 7 days after infection, exhibited, in general, no RHDV-positive staining (Figure 1C). ELISA revealed that all immunosuppressed, RHDV-infected young rabbits presented elevated viral titers in the liver (240.0 ± 99.06; mean ± SEM), similarly to those found in RHDV-infected adult rabbits that died spontaneously with RHD (170.7 ± 42.67; mean ± SEM; n = 3; data obtained from previous experiments). In contrast, the virus was not detected in the livers of young rabbits that had been submitted to the RHDV infection. The TUNEL assay confirmed marked apoptosis in the thymus of immunosuppressed, RHDV-infected rabbits and this was less extensive in the spleen (Figures 2E-F); these tissue changes were not observed in infected young rabbits not submitted to immunosuppression (Figures 2G-H). As expected, significant apoptosis of thymus lymphocytes was observed in all immunosuppressed animals (Figure 2D). The values of the proinflammatory cytokines, TNF-α, IL-6, and the anti-inflammatory cytokine, IL-10, were determined in the sera of PBS control rabbits, immunosuppressed rabbits and immunosuppressed, RHDV-infected young rabbits. All cytokines had a marked increase in the serum of immunosuppressed, RHDV-infected young rabbits that died with RHD, TNF-α in particular (Figure 3).


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

Marques RM, Teixeira L, Aguas AP, Ribeiro JC, Costa-e-Silva A, Ferreira PG - Vet. Res. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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.
Mentions: All immunosuppressed, RHDV-infected young rabbits died within 24–72 h of RHDV infection (24–30 h n = 2; 36–48 h n = 6; 60–72 h n = 1) depicting the hyperacute form of RHD, i.e., they died suddenly, often without conspicuous signs of disease, showing prostration, lethargy and anorexia. As expected, all non-immunosuppressed infected young rabbits showed no clinical symptoms of disease after being infected with RHDV; they were euthanized 1 week after the viral inoculation. Immunosuppressed rabbits (infected (IS + I) or not (IS)) showed atrophic thymuses, indicating that immunosuppression had been achieved; this atrophy was confirmed by the ratio thymus (g)/body weight (g) since we found that these ratios were significantly lower in immunosuppressed groups (IS group: 0.00048 ± 0.000049; IS + I group: 0.00054 ± 0.000050; mean ± standard error of the mean (SEM); p < 0.0001) than in control rabbits (PBS group: 0.0024 ± 0.00024; mean ± SEM). The immunosuppressed, RHDV-infected young rabbits showed the typical lesions of RHD, such as enlarged and pale livers with enhanced lobular patterns, congestion and haemorrhagic lesions of several viscera, and bloody foam in the trachea. A significant difference was seen in the spleen volume, with splenomegaly in immunosuppressed, RHDV-infected rabbits in contrast with spleen atrophy in immunosuppressed animals (ratio - spleen (g)/body weight (g); p < 0.05). Young rabbits that were infected with RHDV did not present any macroscopic lesions suggestive of RHD, 7 days after the viral inoculation, when they were euthanized. Widespread liver cell vacuolization was seen in immunosuppressed, RHDV-infected young rabbits; these hepatocytes often showed pyknotic nuclei and karyorrhexis, classic features of cell death (Figure 1A). The TUNEL assay confirmed extensive cell death of hepatocytes and this was illustrated by brown-nuclear staining (early apoptotic cells) or brown-cytoplasmic staining (late apoptotic or necrotic cells) (Figure 2A). Scattered inflammatory cells (predominantly heterophils) were seen in the liver of the animals. In contrast, hepatic tissue of the immunosuppressed rabbits did not reveal any signs of hepatocellular degeneration suggestive of apoptosis. In RHDV-infected young rabbits, at day 7th after viral inoculation, there was evidence of hepatic regeneration as expressed by rebuilding of focal hepatic lesions that had been produced by the infection (Figure 1C). All immunosuppressed, RHDV-infected young rabbits presented an extensive brown-staining of the liver for the RHDV antigen (Figure 1A). In contrast, RHDV-infected young rabbits, euthanized 7 days after infection, exhibited, in general, no RHDV-positive staining (Figure 1C). ELISA revealed that all immunosuppressed, RHDV-infected young rabbits presented elevated viral titers in the liver (240.0 ± 99.06; mean ± SEM), similarly to those found in RHDV-infected adult rabbits that died spontaneously with RHD (170.7 ± 42.67; mean ± SEM; n = 3; data obtained from previous experiments). In contrast, the virus was not detected in the livers of young rabbits that had been submitted to the RHDV infection. The TUNEL assay confirmed marked apoptosis in the thymus of immunosuppressed, RHDV-infected rabbits and this was less extensive in the spleen (Figures 2E-F); these tissue changes were not observed in infected young rabbits not submitted to immunosuppression (Figures 2G-H). As expected, significant apoptosis of thymus lymphocytes was observed in all immunosuppressed animals (Figure 2D). The values of the proinflammatory cytokines, TNF-α, IL-6, and the anti-inflammatory cytokine, IL-10, were determined in the sera of PBS control rabbits, immunosuppressed rabbits and immunosuppressed, RHDV-infected young rabbits. All cytokines had a marked increase in the serum of immunosuppressed, RHDV-infected young rabbits that died with RHD, TNF-α in particular (Figure 3).

Bottom Line: Remarkably, young rabbits are resistant to RHD.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, ICBAS (Abel Salazar Institute for Biomedical Science) and UMIB (Unit for Multidisciplinary Biomedical Research), University of Porto, Rua de Jorge Viterbo Ferreira n,° 228, 4050-313 Porto, Portugal. ramarques@icbas.up.pt.

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.

Show MeSH
Related in: MedlinePlus