Severe leukopenia and liver biochemistry changes in adult rabbits after calicivirus infection

Severe leukopenia and liver biochemistry changes in adult rabbits after calicivirus infection

Research in Veterinary Science 80 (2006) 218–225 www.elsevier.com/locate/rvsc Severe leukopenia and liver biochemistry changes in adult rabbits after...

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Research in Veterinary Science 80 (2006) 218–225 www.elsevier.com/locate/rvsc

Severe leukopenia and liver biochemistry changes in adult rabbits after calicivirus infection ´ guas P.G. Ferreira *, A. Costa-e-Silva, M.J.R. Oliveira, E. Monteiro, E.M Cunha, A.P. A Department of Anatomy, ICBAS (Abel Salazar Institute for Biomedical Sciences) and UMIB (Unit for Multidisciplinary for Biomedical Research), University of Porto, Largo Professor Abel Salazar, 2, Porto 4099-003, Portugal, European Union Accepted 13 May 2005

Abstract Calicivirus infection is the major cause of the severe decrease in the stocks of wild and farm rabbits that has occurred worldwide during the last two decades. Adult rabbits (10-weeks-old) were experimentally infected with a calicivirus inoculum that killed all animals by causing rabbit haemorrhagic disease (RHD) within 24–62 h of infection. The rabbits were used to evaluate blood cell numbers and serum biochemistry every 6 h, starting 12 h after the inoculation of the caliciviruses. No significant changes in blood parameters were observed in most of the rabbits up to 18 h of infection. Severe leukopenia was seen 6 h before death of the infected rabbits; both heterophils and lymphocytes contributed to the decrease in circulating white blood cells. Platelets were also severely decreased in number. Marked enhancement in liver enzymes was seen 6–12 h before death of the infected rabbits. There was also evidence both for cholestasis, as expressed by the elevated levels of direct (conjugated) bilirubin, and for hypoglycemia, an alteration that it is likely to contribute for the seizures that rabbits show during the late stages of RHD. Liver ultrastructure of rabbits that died from RHD revealed extensive hepatocyte vacuolization, severe changes in mitochondrial structure, and depletion of glycogen granules. We conclude that: (i) severe leukopenia characterizes the final hours of calicivirus-induced RHD; (ii) hypoglycemia and cholestasis precede death of rabbits from RHD; (iii) the kinetics of liver enzymes allows an accurate prediction of the time of death of rabbits from calicivirus-induced RHD. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: RHD; Hepatic transaminases; Hypoglycemia; Cholestasis; Ultrastructure

1. Introduction Rabbit haemorrhagic disease (RHD) is a disorder of the species Oryctolagus cuniculus caused by a member of the Caliciviridae family of viruses (Parra and Prieto, 1990; Ohlinger et al., 1990; Moussa et al., 1992; Thiel and Ko¨nig, 1999; Green et al., 2000). The clinical course of RHD is dramatic: it kills 90% of adult rabbits within 1–3 days of infection; on necropsy, the animals show haemorrhagic diathesis and a necrotizing hepatitis (Marcato et al., 1991; Fuchs and Weissenbo¨ck, 1992; Mitro *

Corresponding author. Tel.: +351 222 062204; fax: +351 222 062232. E-mail address: [email protected] (P.G. Ferreira). 0034-5288/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2005.05.007

and Krauss, 1993; Capucci and Lavazza, 1998). The hepatocyte is the major cell target for the replication of caliciviruses; viral antigens have also been detected in the spleen and lungs of rabbits (Alexandrov et al., 1992; Park and Itakura, 1992; Guittre´ et al., 1996; Prieto et al., 2000; Shien et al., 2000; Kimura et al., 2001). Liver damage plays a key role in the pathogenesis of RHD since the animals die from fulminant hepatitis (Mitro and Krauss, 1993). The disseminated intravascular coagulation observed in RHD may be the result of end-stage liver failure (Plassiart et al., 1992; Ueda et al., 1992). In order to better understand the pathogenesis of RHD, we have characterized here the detailed kinetics of cellular and biochemical parameters of blood, using samples collected every 6 h, and along the whole course

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of RHD. The integration of these new data with the ultrastructural pathology of the rabbit liver contributes to a better definition of the pathogenesis of RHD.

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Germany): alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamiltransferase (GGT), total and direct bilirubin, cholesterol, total protein, albumin, glucose, creatinine, urea, sodium, potassium and chloride.

2. Materials and methods 2.4. Transmission electron microscopy (TEM) 2.1. Rabbits Twelve New Zealand White rabbits (Oryctolagus cuniculus) were used in this study when the animals were 10 weeks old. All of the rabbits were kept under standard conditions of housing, and with unrestricted access to food and water; these conditions followed the European Union Directive no. 86/609/CEE on care of animals used in research. The rabbits were attended by veterinary doctors and inspected at least 4 times daily. 2.2. Calicivirus infection The inoculum of calicivirus was obtained as previously described (Ferreira et al., 2004). Ten rabbits were injected intramuscularly with a PBS suspension of caliciviruses that had a 212 titre in haemagglutination units. Repeated blood samples from these rabbits were collected by venipuncture of the marginal ear vein. They were used to determine the kinetics of several blood parameters during the calicivirus infection that leads to RHD. Blood samples of the infected rabbits were obtained before the viral inoculation and every 6 h, starting 12 h after infection and lasting until death of the animals. Liver tissue samples were collected immediately after death of the calicivirus-infected animals and used for electron microscopy. Two rabbits were inoculated with PBS (the vehicle of the calicivirus); they were sacrificed 24 h later, and used as controls of liver ultrastructure.

Small tissue fragments of rabbit liver were fixed by immersion in an aldehyde mixture made up of 4% formaldehyde, 1.25% glutaraldehyde, and 10 mM CaCl2 in ´ guas et al., 1991). 0.05 M cacodylate buffer, pH 7.2 (A The specimens were washed in buffer, postfixed in a ferricyanide-reduced osmium solution made up of 1% potassium ferricyanide and 1% osmium tetroxide made ´ guas, 1982). This fixative/staining in distilled water (A method enhances the visualization of membranes and of glycogen granules of cells observed by TEM. The samples were dehydrated through a series of graded ethanols, embedded in Epon and sectioned in a LKB ultramicrotome. Thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and viewed in a JEOL 100C electron microscope.

3. Results 3.1. Death from RHD All of the 10 rabbits infected with the calicivirus inoculum died with symptoms of RHD within 24–62 h of the viral injection. A few hours before death, rabbits showed seizures and acute respiratory distress, as described before by others (Marcato et al., 1991; Mitro and Krauss, 1993; Cooke and Fenner, 2002). Half of the animals died within 30–36 h (Fig. 1).

2.3. Blood cells and biochemistry Rabbit blood was obtained from the marginal ear vein, and collected into vials that contained or not EDTA (for hematological and biochemical studies, respectively). Sera were separated from cells within 30 min of blood collection, in order to minimize consumption of glucose by cells. Cell smears were made immediately after collection of blood to avoid artefacts caused by cell exposure to anticoagulant. The preparations were treated with Wright stain and microscopically evaluated for cell morphology and relative percentage of leukocytes. Values of erythrocyte number, hemoglobin concentration, packed cell volume, total leukocyte, and platelets counts were obtained using an automated hematology analyzer (Cell-Dyn 3500, Abbott, CA, USA). The following biochemical parameters were measured in sera by spectophotometric methods (Cobas Integra 800, Roche Diagnostics GmbH, Mannheim,

Fig. 1. Distribution of the 10 calicivirus-infected adult rabbits according to the time of death of the animals from RHD.

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3.3. Blood biochemistry

All rabbits showed leukopenia starting 12–18 h after infection, and lasting until the death of the animals. The decrease in circulating leukocytes was severe: most rabbits had lost around 90% of their blood leukocytes at time of death (Fig. 2(a)). Both lymphocytes and heterophils contributed to this severe leukopenia. The lymphopenia started earlier (12–18 h after infection; Fig. 2(b)) than the decrease in blood heterophils (18–24 h; Fig. 2(c)). A moderate to severe decrease in blood platelets was also observed during the last 6 h of RHD (Fig. 2(d)). Values for red blood cells remained unchanged throughout the course of RHD (data not shown).

Evidence for fulminant hepatic failure in RHD was found on analysing the kinetics of liver biochemistry of the infected rabbits (Fig. 3). Values for AST showed the sharpest rise of the all of liver parameters that were studied. These values started to increase 12–6 h before death of the rabbits, and, in just a few hours, AST levels rose from 10 IU/L to 4000–14000 IU/L (Fig. 3(a)). Enhancement of the other liver transaminase (ALT) was also sharp, but it was not as sudden as that of AST (Fig. 3(b)). The timing of elevation of values for both ALP (Fig. 3(c)) and GGT (Fig. 3(d)) resembled the increase in AST values, but their rises were less

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Fig. 3. Individual kinetics of blood values of liver enzymes for each of the 10 adult rabbits that were submitted to calicivirus infection and died of RHD: aspartate aminotransferase (a), alanine aminotransferase (b), alkaline phosphatase (c) and gamma glutamiltransferase (d).

marked (4–6-fold increase of the base values, respectively). Transmission electron microscopy (TEM) of the liver tissue of rabbits that showed these high levels of blood transaminases revealed marked vacuolization of the cytoplasm, and swollen mitochondria with loss of cristae. Some hepatocytes presented pyknotic nuclei, thus indicating that cell death had occurred. It should be noted that there was a large spectrum of ultrastructural alterations among the hepatocytes of these animals. Furthermore, quite different degrees of cell damage could be observed in neighbouring hepatocytes, as it is illustrated in Fig. 4(b) (compare with Fig. 4(a) of a control rabbit).

Interestingly, a mirror image was found on comparing the kinetics of AST with that of blood glucose: the fast decline in blood glucose levels mirrored the equally fast rise in AST values (Fig. 5(a)). Less than one hour before dying, 6 of 10 (6/10) rabbits had less than one third of the blood glucose level that was measured before infection; 2/10 had half of this level, and 2/10 presented two thirds of the normal glucose level. This decrease in blood glucose was accompanied by depletion of glycogen granules in the liver cells studied by TEM (compare Fig. 6(b) with (a) of a control animal). A marked elevation in blood bilirubin values preceded death of the rabbits. This change was derived

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Fig. 4. Transmission electron micrographs of livers from control (a) and calicivirus-infected (b) rabbits. The hepatocytes in b show different degrees of vacuolization. Bar = 2 lm.

from a significant enhancement in direct (conjugated) bilirubin (Fig. 5(b)). The concentrations of urea and creatinine also peaked during the final few hours of RHD (Fig. 5(c) and (d)). Several parameters of blood biochemistry were without change during the whole course of RHD (i.e., cholesterol, total protein, albumin, sodium, potassium and chloride; data not shown).

4. Discussion We contribute here for a better definition of the pathogenesis of RHD by documenting the kinetics of leukocytes and of serum biochemistry of calicivirus-infected adult rabbits. We offer the following conclusions on the pathogenesis of RHD: (i) hypoglycemia and cholestasis precede death of rabbits from the disease; (ii) the kinetics of values of liver enzymes allows an accurate prediction of the time of death of the rabbits; (iii) RHD involves severe leukopenia that is derived from decrease of both heterophils and lymphocytes. 4.1. Hypoglycemia and cholestasis precedes death of rabbits in RHD We found that blood glucose reached low levels during the last 6 h of RHD. This finding was associated with depletion of glycogen granules in liver cells. Hypoglycemia will probably contribute to the seizures that the rabbits show shortly before death by RHD. In fact, marked hypoglycemia is known to damage the central nervous system in similar ways as hypoxia does (Panic-

kar et al., 1998; Tisdall et al., 2000; Yager, 2002). The importance of hypoglycemia in RHD was recently stressed by Tunon et al. (2003), who considered that RHD is an elective animal model to experimentally investigate how to handle fulminant hepatic failure in humans. Cholestasis occurred during the final 6–12 h of RHD, as it was revealed by elevation in the direct variant (conjugated) of bilirubin. This alteration is likely to be the result of severe cell damage caused by caliciviruses in the liver. This was expressed both by the ultrastructural changes of liver cells, and by the high levels of transaminases measured during the final stages of RHD. 4.2. Prediction of time of death of rabbits from RHD A major conclusion from this investigation is that biochemical parameters of the blood can be used to accurately predict the number of hours that rabbits will live until dying from RHD. In fact, blood AST levels over 6000 IU/L were always associated with death of the rabbits within the next 6 h. Therefore, our data show that AST values can be used as an indicator for staging RHD, since rabbits infected with the same calicivirus inoculum will die from 24 and up to 72 h after the infection. The high blood values of AST clearly indicate that fulminant hepatic failure is the primo movens of the calicivirus-induced pathology that leads to death from RHD. The elevated values in transaminases, of AST in particular, can be interpreted as the result of lesion of liver mitochondria, since more than half of AST is known to be associ-

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ated with mitochondrial membranes (Rej, 1983; Cornelius, 1989; Meyer et al., 1992), and these showed severe ultrastructural damage in our samples. Rabbits and hares are the only species that develop a necrotic hepatitis after infection by calicivirus (Fuchs and Weissenbo¨ck, 1992). The pathogenesis of liver disease caused by calicivirus has been better characterized in the rabbit than in the hare. Reports on the liver disease of the European brown hare syndrome (EBHS) have been limited to the definition of a massive coagulation necrosis that is associated with mitochondrial calcifications (Fuchs and Weissenbo¨ck, 1992; GavierWiden, 1994). Clearly, there is a need for further investigations on the comparative pathogenesis of the RHD and EBHS that plague the two species of lagomorphs.

4.3. Severe leukopenia in RHD We document here that during the last 6 h, before death of the rabbits from RHD, the animals develop severe leukopenia involving both heterophils and lymphocytes. Previous studies have documented that caliciviruses cause a mild necrosis of lymphocytes that are present in lymphoid organs of the infected rabbits, and this phenomenon was reported to be associated with a decrease in lymphocyte counts (Marcato et al., 1991; Plassiart et al., 1992). Our data show that, in addition to lymphocytes, heterophils also decline sharply in RHD, and we illustrate the overall kinetics of the RHD-associated leukopenia. Conceivably, the loss in circulating leukocytes may be derived from different events: (i) direct citotoxicity of the virus to white blood

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Fig. 6. Transmission electron micrographs of a hepatocyte from control (a) and calicivirus-infected (b) rabbits. The control hepatocyte shows numerous small granules of glycogen (black dots scattered in the cytoplasm), whereas the hepatocyte of the rabbits undergoing RHD is vacuolized and depleted of glycogen granules. Bar = 2 lm.

cells; (ii) sequestration of leukocytes out of circulation (e.g., in the infected liver where large numbers of inflammatory cells are observed); (iii) cytopathic action of the virus on the bone-marrow precursors of white blood cells. It remains to be investigated whether any or several of these mechanisms are at play in RHD. Recently, we have documented that the calicivirus infection in young rabbits also causes a decrease in blood heterophils (Ferreira et al., 2004). The herein reported leukopenia of adult rabbits is different from the one of young animals with regards to several features: (i) infected adult rabbits show a decrease in the circulating leukocytes of greater severity than young rabbits do; (ii) both heterophils and lymphocytes participate in the leukopenia of adult rabbits, whereas only heterophils are decreased in infected young rabbits; (iii) the leukopenia of young rabbits depicts a transient nature, whereas the leukopenia in adult animals gets more and more severe until the rabbits die from RHD. Interestingly, both leukopenias are observed with a similar timing after the inoculation of young and adult rabbits with calicivirus. This suggests that the decrease in leukocyte number may be a general effect of calicivirus infection in rabbits, regardless of the animals being resistant (young) or susceptible (adult) to RHD. In addition, the marked severity and the progressive nature of the leukopenia of calicivirus-infected adult rabbits suggest that this pronounced haematological change contributes to the lethal nature of calicivirus infection in the rabbits.

In conclusion, our data contribute for a more detailed definition of the pathogenesis of RHD by offering the timings of alterations of cellular and biochemical parameters of adult rabbits that were experimentally infected with calicivirus and died from RHD. The herein reported discovery of severe leukopenia associated with RHD deserves further investigation.

Acknowledgements We are grateful to Prof. Francisco Parra (University of Oviedo, Spain) for the kind offer of the initial sample of calicivirus suspension that was used to infect our rabbits. We thank Prof. Paulo Ce´lio and Eliane Silva for help regarding the haemagglutination test to quantify the inocula of caliciviruses, and Maria Ju´lia Reis, Dr. Miguel Pereira and Dr. Madalena Costa, for excellent technical assistance. This work was funded by grants from FCT (POCTI/FEDER), Portugal.

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