A. Costa-e-Silva

Liver enzymes and ultrastructure in rabbit haemorrhagic disease (RHD)

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24–72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150–200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.

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

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.

Early inflammatory response of young rabbits attending natural resistance to calicivirus (RHDV) infection.

Young rabbits (i.e. up to 4 weeks of age) are naturally resistant to infection by rabbit haemorrhagic disease virus (RHDV), the same calicivirus that kills more than 90% of adult rabbits in 3 days or less. To characterize this fascinating model of age-related natural resistance to viral infection, we have studied the kinetics (from 6h up to 7 days) of cytokines and of leukocyte subpopulations in the liver (the target organ for calicivirus replication) and spleen (host systemic response) of RHDV infected young rabbits. Infection was associated with early (6h) elevation of proinflammatory cytokines (TNF-α, IL-1, IFN-α, IFN-γ, IL-6, IL-8). We found that all three major leukocyte subpopulations (macrophages, B and T lymphocytes) were increased in the liver 48h after the RHDV inoculation. At 7 days of infection, B and T lymphocytes were still elevated in the liver of the rabbits. In the spleen, both macrophages and B lymphocytes (but not T cells) were also enhanced. At 7 days, anti-RHDV specific antibodies were present in sera of all young rabbits infected by the virus. We conclude that natural resistance of young rabbits to RHDV infection is associated with a rapid and effective inflammatory response by the liver, with few hepatocytes being infected, and also with a sustained elevation in local and systemic B and T cells.

Saturated salt solution: a further step to a formaldehyde‐free embalming method for veterinary gross anatomy

In the field of veterinary anatomy, most of the specimens used in practical sessions are perfused with fixatives. Thus, they can be used for a longer time, reducing the number of animals for educational purposes. Formalin is the most commonly used fixative, consisting of a 37% formaldehyde solution. However, formaldehyde is a powerful irritant of the eyes and airways and is considered carcinogenic, causing nasopharyngeal cancer in exposed workers and professionals. In the present study, we explored an alternative method to avoid the use of formaldehyde in specimens used for gross anatomy practical sessions. We propose an inexpensive, non‐toxic fixative that is available worldwide, such as sea salt. This method consists of a continuous perfusion of saturated salt solution for a period of 6–8 h, enabling drainage of the solution to avoid a weight increase of the specimen, and allowing salt to be retained in the tissue. The method is based on recirculation of the saturated salt solution instead of maceration. Perfused specimens retained their natural consistency and joint mobility, with no blood, resembling a piece of meat from the slaughterhouse. They could be used immediately without a maceration period, or stored in the fridge until use and then kept in a bath of saturated salt solution for future conservation. In the case of the former, no refrigeration was needed. The specimens did not have an irritating or offensive smell, and could be used for long sessions (several hours per day) and stored for long periods. However, the blood vessels used for perfusion determine the results: a less invasive approach (through common carotid arteries) gave good preservation of the musculoskeletal system, whereas more invasive access to cannulate the abdominal aorta and vena cava caudalis was required to achieve better preservation of the viscera. In conclusion, we propose that perfusion followed by immersion in a saturated salt solution is a good alternative method for the preservation of specimens used in the practical teaching of gross veterinary anatomy. It is a very simple and inexpensive technique, and is much healthier for users than traditional formalin. Moreover, specimens can be preserved for prolonged periods, and maintain a similar appearance and consistency to fresh material.

Inflammatory response of young rabbits to calicivirus infection

Caliciviruses cause rabbit haemorrhagic disease (RHD) that kills more than 90% of the infected adult animals within 1 a 3 days of infection. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to RHD [1]. A mystery of the calicivirus infection is that young rabbits (less than 8-weeks old) are resistant to the infection, in spite of undergoing viral replication in the liver and of expressing transient hepatitis [2,3]. Heterophils were the predominant inflammatory cells seen in hepatic tissue of infected adult rabbits, whereas mononuclear cells dominated the inflammatory infiltrates of the infected young rabbits (4-weeks-old) [4]. In order to define the role of inflammation in the pathogenesis of the calicivirus infection, we have studied the cellular inflammatory response in young rabbits experimentally infected by calicivirus. For this, we have used transmission electron microscopy (TEM) and flow cytometry to identify the inflammatory cells that infiltrate the hepatic tissue of young rabbits at 48 hours of calicivirus infection. In same infected rabbits, lymphoid organs (spleen and thymus) were used to quantify by flow cytometry the total number of leukocytes seen inside these organs. TEM showed that the majority of hepatocytes of infected young rabbits did not present ultrastructural changes and the infiltrates were made up mostly of lymphocytes that depicted membrane contacts with the cell surface of undamaged hepatocytes (fig.1). These lymphocytes could correspond to B-lymphocytes, the major populations of leukocytes that were increased at 48 hours of infection, as we have founded by flow cytometry, in hepatic and splenic tissues. Macrophages and T-lymphocytes were also significantly increased in liver of infected young animals (p<0.001 and p<0.05, respectively) (fig. 2 and 3). We did not observe differences in the number of T-lymphocytes population of the thymus between the infected and control rabbits (fig. 4).We conclude that lymphocytes prevailed in mononuclear infiltrates seen in liver tissue of young rabbits at 48 hours of infection. This liver lymphocytic infiltration of young rabbits suggests the expression of viral antigens on the surface of liver cells of the RHD-resistant animals. In fact, we have recently documented a rise in the title of specific antibodies measured by ELISA in sera of infected young rabbits.