Rev. sci. tech. Off. int. Epiz., 1991, 10 (2), 435-451

Viral haemorrhagic disease of rabbits in Mexico: epidemiology and viral characterization

D.A. GREGG, C. HOUSE, R. MEYER and M. BERNINGER *

Summary: A fatal disease of rabbits was first reported in the People's Republic of China in 1984. Since 1986, the disease has been reported in most countries of Europe and in the Republic of Korea. In 1989 a similar disease, presumably linked to the importation of rabbit meat from the People's Republic of China, spread rapidly through ten states in Mexico; it was eradicated during the same year by "stamping-out" measures. In Mexico, as was the case in other outbreaks, morbidity and mortality reached 80-90% with few clinical signs. In pathogenesis studies, the primary sites of replication were in the small intestinal crypt and villous epithelium, hepatocytes and splenic lymphocytes. Many organs, including the lung and kidney, contained acutely infarcted tissue and haemorrhages resulting from a terminal disseminated intravascular coagulopathy. The disease and the characteristics of the virus isolated in Mexico are similar to isolates from Europe and the Republic of Korea. The comparative morphologic, immunologic, and in situ nucleic acid hybridization evidence for a parvovirus aetiology are summarized.

KEYWORDS: Biotin - Calicivirus - Diagnosis - Electron microscopy -Immunohistochemistry - Nucleic acid hybridization - Parvovirus -Pathogenesis - Rabbits - Viral haemorrhagic disease of rabbits.

I N T R O D U C T I O N

A disease of domestic rabbits causing high morbidity and mortality was first reported in the Nanjing region of the People 's Republic of China in 1984 (10). The disease spread rapidly throughout the country, killing 470,000 Angora rabbits in the first six months (10). The disease was reported in the Republic of Korea in 1985 and had spread throughout the country by 1987 (1). In 1986, an identical disease syndrome was prevalent in Italy, and also in Spain the following year. By 1988, many countries of Central Europe had experienced outbreaks in domestic and wild rabbits (both Oryctolagus cuniculus). A similar disease, called the European brown hare syndrome, was also reported in hares (Lepus sp.) in Sweden (5).

There are nearly as many names for this disease as outbreaks. They include: rabbit viral haemorrhagic disease (10, 20), rabbit plague (4), rabbit viral haemorrhagic

* United States Department of Agriculture, Animal and Plant Health Inspection Service, Science & Technology, National Veterinary Services Laboratories, Foreign Animal Disease Diagnostic Laboratory, Greenport, NY 11944, United States of America.

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pneumonia (17), rabbit viral septicaemia (19), rabbit viral sudden death (9), Picornavirus haemorrhagic fever (1), X disease of rabbits (2), infectious necrotic hepatitis of rabbits (11) and necrotic hepatitis of rabbits (7).

A fatal disease of rabbits was first seen in the area of Mexico City in December 1988 (12). The disease was characterized by sudden death following a short period of depression and fever. Terminal respiratory distress or convulsions were sometimes reported; in some cases, a sero-sanguinous nasal discharge was seen at death. In mid-January, the severity of the disease prompted investigation of the possible viral aetiology of the disease. Diagnostic samples were submitted to the Mexico-United States Commission for the Prevention of Foot and Mouth Disease and other Exotic Diseases of Animals (CPA) and, subsequently, to the Foreign Animal Disease Diagnostic Laboratory (FADDL) on Plum Island, New York.

The source of the Mexican outbreak was traced to the importat ion, through the United States, of 18 metric tons of rabbit meat from the People 's Republic of China to a supermarket chain outside Mexico City (13). The first outbreak was traced to a colony of rabbits belonging to a commercial rabbit breeder and dealer who had been in contact with these frozen rabbit carcasses six days before the first case (13). The disease spread rapidly to other rabbit colonies through contact with individuals who had been on the dealer's premises. Within two months , the disease was reported in ten states close to Mexico City; it was spreading at an a larming rate and causing high mortality. An important subsistence rabbit-raising industry was being devastated by a disease which threatened to eliminate a significant source of meat for a large populat ion.

In February, the Mexican government took action to control and eradicate viral haemorrhagic disease (VHD) from Mexico. Efforts commenced with an order to stop sales and the movement of all rabbits and rabbit by-products in affected areas. An intensive publicity campaign was also initiated to alert rabbit breeders of measures to prevent the disease from being introduced to unaffected colonies (13). First in outlying areas and then in the center of the outbreak in Mexico City, all rabbits were collected and destroyed, then buried or incinerated. Owners were given certificates to claim healthy rabbits at a later date and instructions on cleaning and disinfection procedures were circulated. Over 320 pick-up stations or mobile units were established and intensive publicity programs advised owners to bring in all sick or exposed rabbits. Restocking was delayed for two months in each depopulated area. Following this, sentinel rabbits were placed on premises which had been properly cleaned and disinfected. If, after 30 days, sentinel rabbits were seronegative using the haemagglutination inhibition (HI) test for V H D , repopulation was allowed. Repopulation was carried out area by area as they were cleaned up (13).

The program was funded entirely by the Mexican government and employed, for varying periods of t ime, 2,500 local personnel, including 500 veterinary students on summer vacation. Over 110,000 rabbits died or were destroyed in the 13 affected states and the Federal District. Most of the rabbits in infected foci were eliminated in four to five months and few outbreaks were found in Mexico after that time (13). Surveillance for V H D has continued, and a few isolated cases have been found and eliminated using the same eradication measures. No vaccines were used to control V H D in Mexico as this would have caused seroconversion in rabbits and would have interfered with serologic surveillance efforts.

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E X P E R I M E N T A L C L I N I C A L A N D P O S T - M O R T E M F I N D I N G S

Studies were undertaken on Plum Island when the eradication campaign began in Mexico. Pathogenesis studies and comparative virologic studies were conducted to determine the pathology of the disease and to determine the aetiologic agent through cross-reactive methods. Further clinical, pathologic and serologic studies have compared VHD isolates from Italy, Spain and Korea. No differences have been found among any of these isolates.

The clinical disease caused by the Mexican isolate is similar to that reported from the People 's Republic of China and Europe . Predominantly young and adult rabbits die suddenly, following 6 to 24 hours of fever accompanied by mild clinical signs. Most rabbits appear depressed in the final hours and some show a variety of neurologic signs, including excitement, incoordination, opisthotonos and paddling. Sometimes rabbits emit a terminal squeal. A few rabbits have a terminal sero-sanguinous nasal discharge as a result of acute pulmonary oedema and congestion. Gross pathologic lesions include congestion and haemorrhages in the lungs, a fine lobular pattern of necrosis in the liver, resulting in a pale liver with a distinct lobular pat tern, and splenomegaly. The two latter lesions, though most consistent, are subtle. Haemorrhages and congestion may be seen in many other organs and are due to a terminal intravascular coagulopathy rather than to primary involvement of those organs. This is borne out by the fact that rabbits killed before they are in extremis usually do not have haemorrhagic lesions.

V I R U S P U R I F I C A T I O N A N D C H A R A C T E R I S T I C S

To prepare sufficient virus for diagnostic testing and to collect tissues for immunopathologic examination, rabbits were inoculated by the oral-nasal route with clarified and filtered liver homogenate from field cases. Large quantities of stock virus have been purified from the livers of these infected rabbits by centrifugation. Lesser amounts of virus can be purified from the spleen and blood collected terminally. As reported by other workers, the authors have found a nonenveloped, roughly spherical viral particle with small surface projections in negative stained electron microscopy (EM) preparations. There are differences in the size of the virus reported by different workers; these have ranged from 25 to 40 nm, with most reports closer to 30 nm. The measurements made by the authors, using a Zeiss EM10 electron microscope with 2 % phosphotungstic acid staining at p H 8.5, have been between 25 and 30 nm (6). The smaller particles appear to be empty capsid structures which account for about half the populat ion.

The liver has yielded the highest virus concentration, and most workers have extracted virus from this organ for virologic studies. In the experience of the authors, this procedure yields a semi-purified product often contaminated with liver enzymes, ribosomal RNA and nuclear DNA. Results of virus and nucleic acid extraction from different laboratories vary widely. Whether the virus contains single-stranded RNA or DNA is still debated. Therefore, the classification of the virus is still in question. Korean workers (1) and early reports from the People 's Republic of China (18) have called it a Picornavirus, while German (15), Italian (3) and Spanish (16) workers believe it to be a calicivirus. The work of the authors (6, 7) and recent reports from the People's Republic of China (21) indicate that it is caused by a parvovirus.

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Identification of this virus would be made easier if it could be cultivated in vitro. The authors have tried many different rabbit primary cells and cell lines from various species under various conditions, without success. With each cell type, three blind passages were made with passage of both cells and fluid. No cytopathology or haemagglutination (HA) was observed (Table I) .

T A B L E I

Cell cultures resistant to viral haemorrhagic disease of rabbits

Name Source Description

RKp NVSL Rabbit kidney, primary ERLp NVSL Embryonic rabbit lung, primary RK NVSL Rabbit kidney cell line DRS ATCC Skin, normal rabbit, cell line Oc4T/cc ATCC Rabbit carcinoma, cell line CTPS ATCC Skin, papilloma, rabbit, cell line EREp ATCC Embryonic epidermis, rabbit, cell line Oct 2 (Vx7) ATCC Papilloma, rabbit, cell line SIRC ATCC Rabbit cornea, cell line SF 1 Ep ATCC Epidermis, cottontail, cell line R 9ab ATCC Lung, New Zealand white rabbit, cell line RAB-9 ATCC Skin fibroblast, adult female rabbit, cell line LLC-RK ATCC Kidney, New Zealand white rabbit, cell line BMK lot 1 NVSL Baby mouse kidney, primary L929 PIADC Mouse tumor cell line McCoy NVSL Mouse cell line (originally thought to be human

synovial; is mouse fibroblast) NCTC clone 1469 NVSL Mouse liver cell line P388D NVSL Mouse monocyte-macrophage cell line BHK 21 FADDL Baby hamster kidney cell line (clone) L2 NVSL Rat lung cell line C Yale Rat glial tumor cell line RASK lot 1 NVSL Rat skin cell line Mv 1 Lu NVSL Mink lung cell line 324 K Yale Human foetal SV-40 transformed cell line CRFK NVSL Crandell feline kidney cell line MDCK NVSL Madin Darby canine kidney cell line VERO FADDL Green monkey kidney cell line SK lot 8705 NVSL Swine kidney, primary MVPK FADDL Mengeling-Vaughn porcine kidney cell line ELKp FADDL Embryonic lamb kidney, primary RK VPI Rabbit kidney cell line, originally from G. Siegl ES FADDL Bovine endothelial cell line None FADDL Organ cultures (spleen, liver, lung, testicle, kidney)

from rabbit infected 24 h previously None FADDL Foetal rabbit, primary: lung, intestine, heart, brain,

kidney, liver. Also, these cells were irradiated, inoculated with SV40, or inoculated with adenovirus.

None FADDL Suckling mice (IP, IC, IN) None FADDL Embryonating chicken eggs (IV)

Nota: N o cytopathic effect was observed after three blind passages of cells plus media. N o H A activity in media was recorded after the third passage.

A T C C : American Type Culture Collection, Rockville, Maryland F A D D L : Foreign Animal Disease Diagnostic Laboratory, Greenport, New York NVSL : National Veterinary Services Laboratory, Ames , Iowa PIADC : Plum Island Animal Disease Center, Greenport, New York VPI : Virginia Polytechnic Institute, Blacksburg, Virginia (R. Bates)

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C O M P A R A T I V E P A T H O L O G I C F I N D I N G S

Only rabbits (Oryctolagus cuniculus) have been found to be susceptible to infection. Therefore, investigations conducted by the authors to identify the virus have, of necessity, been done primarily in situ using haematoxylin and eosin (H & E) sections, immunohistochemical staining and transmission electron microscopy (TEM) on various tissues. These results have complemented HI serologic results. Finally, in situ hybridization was used to confirm the identity of this virus.

H I S T O P A T H O L O G Y A N D P A T H O G E N E S I S

The hepatic lesion is most consistently found even in rabbits killed early in the infection. There is severe diffuse portal hepatic necrosis with cytoplasmic and nuclear swelling and margination of the chromatin. Small, multiple intranuclear inclusion bodies can be found in the swollen nuclei. Only the centrolobular hepatocytes are spared, resulting in the characteristic gross reticular pattern of pale necrosis outlining each liver lobule.

Another characteristic lesion, which has not been reported by others, is necrosis of the small intestinal crypts and subsequent villous atrophy (Fig. 1). This may vary in severity and is often a segmental lesion involving only a few areas of the small intestine at a time (7). This lesion is similar to the intestinal lesion seen in canine parvovirus or feline panleukopenia virus infections. The spleen is variably affected with necrosis in the red pulp and marginal zone, and sometimes in the white pulp.

FIG. 1

Mexican VHD-infected rabbit je junum (36 h post inoculation)

Note severe diffuse acute crypt epithelium necrosis with margination of chromatin. H & E stain

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The complicating pathologic change, which seems to be the cause of sudden death in V H D , is a severe disseminated intravascular coagulopathy (DIC). It is rapidly progressive, resulting in acute thrombosis of major vessels as well as capillaries. Any organ may be affected and the severity of vascular infarction varies. Pulmonary vein thrombosis is the underlying lesion which frequently results in pulmonary congestion and haemorrhaging (Fig. 2). Similarly, the terminal neurologic signs can be attributed to microthrombi in the brain. There is often massive infarction of the kidneys, with both renal vein thrombi and diffuse glomerular thrombosis. Renal tubular degeneration is also present, but may be ischemic in origin. In most fatal cases, DIC results in fibrin deposition in the entire red pulp of the spleen, sometimes causing massive splenic necrosis due to infarction. The pulmonary, splenic and renal vascular thrombi are most remarkable, but may be seen in other septicaemic diseases of rabbits. They are more likely to be attributable to DIC than to viral infection of these organs. It is the hepatic necrosis which is so characteristic and consistent in this disease and, therefore, implies that more emphasis should be placed on the hepatic necrosis than on the haemorrhagic aspects of V H D , which are not pathognomonic .

FIG. 2

Mexican VHD-infected rabbit lung (rabbit died 48 h post inoculation)

Note large fibrin thrombus (T) in pulmonary vein due to a terminal intravascular coagulopathy. Such thrombi can cause pulmonary congestion and a sero-sanguinous nasal discharge. H & E stain

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The liver, small intestine and spleen seem to be the primary target tissues of this virus, and viral replication in the nucleus of affected cells has been confirmed by EM and immunohistochemical staining. Linear and paracrystalline arrays of 25 nm empty capsid structures have been found adjacent to the intranuclear, electron-dense globular inclusions (6) (Fig. 3). Sometimes intranuclear inclusion bodies are found closely apposed to nucleoli. Virions can be found inside the nuclear membrane and free in the cytoplasm of these cells. This nuclear assembly and cytoplasmic distribution of virus represent the typical morphogenesis of a parvovirus.

S T U D I E S

Immunohistochemical studies

Immunostaining of frozen and paraffin-embedded tissue sections with biotin-conjugated Mexican convalescent rabbit antiserum (1:1000 dilution) using the avidin-biotin complex alkaline phosphatase (ABC-AP) method (8) demonstrated the V H D antigen in tissues. Antigen was localized in hepatocytes in periportal areas, in scattered cells of the red pulp and marginal zone of the spleen, as well as within lymphocytes of the periarteriolar lymphoid sheaths and follicles. Immunostaining of liver and spleen cells demonstrate distinct intranuclear inclusion bodies and diffuse staining of the cytoplasm of the same cells (Fig. 4). Positive staining in most other organs can be found intravascularly on red blood cells and in microthrombi. Virus has recently been found by ABC-AP immunostaining and by T E M on the surface of red blood cells (RBCs). Macrophages in the mesenteric lymph node and other organs may have positive immunostaining in the cytoplasm, particularly if they have phagocytized erythrocytes carrying virus. The presence of V H D virus on RBCs may trigger the sudden and massive intravascular coagulation which is undoubtedly responsible for sudden death in V H D .

Cross-reactive serologic studies

Immunostaining of cryosections of infected rabbit spleen using monoclonal and polyclonal antibodies (1:200 dilution) with the ABC-AP indirect method was used to complement HI tests for serologic identification of the suspected parvovirus. These results indicate that the virus cross-reacts most closely with porcine parvovirus (PPV) and minute virus of mice (MVM), a murine parvovirus (6) (Table II) . It is not closely related to the ubiquitous lapine parvovirus or the bovine or carnivore parvovirus groups.

A similar study was conducted to determine if there were any cross-reactions to caliciviruses with isolates from Mexico, Italy, Spain or the Republic of Korea. These results showed that, with HI testing, there was little or no cross-reactivity using group-reactive monoclonal antibodies (MAbs) to the caliciviruses, as well as MAbs and polyclonal antisera to vesicular exanthema of swine and San Miguel sea lion viruses 1 and 2. Similarly, negative results were found with convalescent antisera to feline calicivirus (Table II) .

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FIG. 3

Electron micrograph of VHD-infected hepatocyte nucleus chromatin (C) and nucleolus (N) are marginated

Note several dense viral inclusions bodies (I) (one adjacent to nucleolus) and paracrystalline arrays of empty capsids (arrows) Capsids enlarged in insert. Bar = 1 (jm. Bar in insert - 0.1 /xm

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FIG. 4 A

Mexican VHD-infected spleen immunostained by avidin-biotin method with biotin-conjugated convalescent rabbit serum

made against Italian VHD virus

Note dense staining of vesiculated nuclei (arrows) and intranuclear inclusion bodies (arrowheads).

Counterstained with haematoxylin

FIG. 4 B

Same tissue as 4 A , stained with biotin-conjugated rabbit antiserum made against Korean VHD virus

Same arrow markers and counterstain as 4A

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T A B L E II

Summary of haemagglutination inhibition (HI) and immunostaining with an indirect avidin-biotin complex alkaline phosphatase (ABC-AP) system

on Mexican viral haemorrhagic disease (VHD) using antisera prepared against various parvoviruses and caliciviruses

H I ABC-AP * Antisera or Monoclonal antibody (MAb) titre Spleen-methanol

fixed

Convalescent rabbit (Mexican VHD) ** 1280 + + + Convalescent rabbit (Italian VHD pool) ** 1700 + + + + Convalescent rabbit (Spanish VHD pool) ** 4480 + + + Convalescent rabbit (Korean VHD pool) ** 1440 + + + Lapine parvovirus (in rabbit) 32 + + Normal conventional rabbit 32 + +

Porcine parvovirus MAb 1280 + + + + Normal swine serum pool 640 + +

Minute virus of mice ascitic fluid (84) 1280 + + + + Minute virus of mice ascitic fluid (82) 160 + + Mouse O parvovirus <10 + Rat virus-Yale ascitic fluid 160 + + Rat O parvovirus <10 + + H-l virus ascitic fluid 50 + +

Canine parvovirus MAb 10 +

Convalescent feline parvovirus 10 + Mink enteritis virus (in rabbit) 10 ND Aleutian disease of mink (in rabbit) 10 ND

Calicivirus antisera: Vesicular exanthema of swine (VES) (rabbit) <10 ND San Miguel sea lion virus 1 (SMSV1) (rabbit) <10 ND San Miguel sea lion virus 2 (SMSV2) (rabbit) <10 ND Convalescent feline calicivirus <10 ND Group reactive VES/SMSV MAb 14C5 20 ND Early antigens of VES/SMSV MAb 8F3 20 ND Early antigens of VES/SMSV MAb 8D4 20 ND VES ascitic fluid (in conventional mouse) 40 ND Normal ascitic fluid (in conventional mouse) 40 ND

N D = not done

* All indirect ABC-AP immunostaining done at 1:200 dilution of primary antisera or MAbs. ** Direct ABC-AP staining done with biotin-labelled antisera at 1:1000 dilution.

In situ nucleic acid hybridization studies

Additional studies were continued at the F A D D L to confirm that the V H D virus is a parvovirus from the porcine and rodent subgroup. Recombinant RNA transcription plasmids containing a portion of the parvoviral genome as template were used to prepare ssRNA-biotin labelled probes to MVM and P P V . These plasmids

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were generously supplied to us by Drs P . Tattersal and T .W. Molitor. Both of these probes represent more than two-thirds of the genome of these viruses. In an RNA-DNA hybridization procedure performed on cryosections of rabbit liver under highly stringent conditions, there was strong hybridization with both probes. The staining was strongest in nuclei of hepatocytes in the periportal zone, correlating to areas of cellular necrosis. The cytoplasm of these cells was also positively stained, but to a lesser extent (Fig. 5). Non-infected liver sections used as negative controls and similarly hybridized were not stained. Under the stringent conditions used for this in situ hybridization, the results indicate that this virus is very closely related to both MVM and P P V .

D I A G N O S I S A N D C O N T R O L

Diagnosis of V H D is based on haemagglutination (HA) of human type O RBCs with extracts of infected liver or spleen. Immunostaining of the liver and spleen can be performed by using the ABC-AP method to confirm diagnosis. Paraffin-embedded tissues can be immunosta ined if care is taken with fixation of tissues. Paraformaldehyde-lysine-periodate (PLP) fixative (14) will preserve tissues for weeks or months , while formalin-fixed tissue may lose reactivity in days to weeks. Negative stained E M preparations of clarified liver homogenate centrifuged at 50,000 g may also aid the diagnosis. T E M of liver tissue has proved even more useful than negative staining. The finding of intranuclear inclusion bodies, capsid arrays and dense viral structures in the nucleus of hepatocytes strongly supports a diagnosis of VHD.

Serology

As many as 20-40% of rabbits in an outbreak may recover; these can be diagnosed by serology using the HI test. All sera are adsorbed with kaolin and human type O RBCs before testing. Titres above 1:80 are considered positive. Titres below 1:80 are common in normal rabbits and are probably due to cross-reaction with other ubiquitous parvoviruses, especially minute virus of mice or, possibly, lapine parvovirus.

Recovered rabbits have been suspected of being one of the most important sources of new VHD outbreaks. Sporadic cases in locations distant from Mexico City, occurring weeks and months after the major outbreak area had been depopulated, suggest that carriers do exist in the field. The authors have shown that the virus is shed in the faeces of recovered rabbits . These studies were conducted under carefully controlled conditions, in separate biologic containment rooms at the FADDL. Drained of urine, faeces from recovered rabbits were collected and transferred to another animal room where susceptible rabbits were housed on the floor, in contact with the faeces. Possible contamination by personnel or fomites was avoided. In sequential experiments in different rooms, contact rabbits died after exposure to two- and four-week post-recovery faeces, but not at a post-recovery period of eight weeks. Such viral shedding in the faeces is to be expected when there is infection of intestinal crypt epithelial cells and sloughing of infected villous epithelial cells into the intestinal lumen, as was demonstrated histologically (Fig. 1).

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FIG. 5 A

Mexican VHD-infected liver section (area of portal necrosis) stained by in situ RNA-DNA hybridization with biotinylated-RNA

probe prepared from a plasmid to porcine parvovirus genome

Note dense nuclear staining (arrows) and fine cytoplasmic stippling

FIG. 5 B

Similar to 5 A but hybridized with plasmid to minute virus of mice parvovirus genome

Note similar nuclear staining as in 5A (arrows)

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FIG. 5 C

Normal rabbit liver section hybridized with probe to porcine parvovirus as a control for 5 A

Cleaning and disinfection The most probable sources of VHD virus seem to be faecal contamination and

any blood or infected tissues. Aerosol transmission is not likely to be an important means of disseminating V H D virus unless faeces or blood are aerosolized during cleaning operations. Farm-to-farm transmission is most likely due to spread of virus on fomites, especially on contaminated shoes or hands, which was probable in the Mexican outbreak. Like other parvoviruses, the VHD virus is very stable. For example, blood from an infected rabbit stored in Alsever's solution at 4°C for nine months still remained infectious when rabbits were inoculated orally. Therefore, thorough cleaning and disinfection of all equipment, cages, houses and vehicles with which infected rabbits have contact is essential to control spread of VHD during an outbreak. Disinfectants which have been used to control V H D include 2 % one-stroke environ, 2 % vanadine, 1% formalin and 0 . 5 % sodium hypochlorite (10% household bleach), and 4 % calcium hydroxide as whitewash. Extensive inactivation studies have not yet been done due to the lack of an in vitro system to assay the virus.

Infection of wild rabbits

The effect of V H D on wild populations of rabbits is still in question. Personal communications indicate that wild European rabbits (O. cuniculus) are dying of VHD in many countries of Europe. Mortality rates in wild European rabbits are high, and the disease may become endemic throughout the range of the European rabbit population. It is not surprising that V H D infects the wild European rabbit , from which the domestic rabbit was derived, as both are the same species. It is probable that a similar, but not identical, virus causes an analogous disease in European brown

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hares (Lepus europaeus) and the varying hare (Lepus timidus) (5). This disease has not been recognized in the Western hemisphere. Oryctolagus sp. rabbits are not indigenous in the wild in Nor th and Central America. There are a number of species of cottontail rabbits (Sylvilagus sp.) and hares (Lepus sp.) in that part of the world, as well as several lesser genera of rabbits. In the preliminary infectivity studies conducted by the authors, Eastern cottontail rabbits (Sylvilagus floridanus) did not show clinical signs of disease, but a domestic rabbit placed in the same room a week later did die. When exposed orally, the cottontail rabbits may possibly shed the virus for a short time. A susceptibility study performed with volcano rabbits (Romerolagus diazzi) in Mexico did not result in clinical disease (13). Further studies were conducted at the F A D D L with black-tailed jackrabbits (Lepus californicus) which were found not to be susceptible and, two weeks post-recovery, did not shed virus. Consequently, it is likely that V H D is genus specific and will not readily become endemic in wild populations of rabbits or hares in North or Central America, Asia and the other areas where Oryctolagus cuniculus is not indigenous in the wild.

Eradication vs. vaccination

Control of V H D in countries where the disease is now endemic has been based on strict sanitation, maintenance of closed rabbit colonies and vaccination of breeding stock. Inactivated vaccines produced from an extract of infected liver offer protection for about six months . Vaccination was not attempted in the Mexican eradication campaign because serologic surveillance for recovered and carrier rabbits would have been made difficult or impossible. Vaccination is also problematic in the face of an outbreak; many rabbits may be only partially protected when exposed to a field virus and could become carriers, serving as a source for future outbreaks. Total eradication of the disease in Mexico was. highly successful and should be the best course for any country without a susceptible wild rabbit population.

C O N C L U S I O N S

Viral haemorrhagic disease of rabbits is a new and devastating disease of rabbits which the authors believe has recently emerged within the family of parvoviruses and is closely related to the minute virus of mice and porcine parvovirus. The Mexican isolate is clinically, pathologically and serologically indistinguishable from isolates from Italy, Spain and the Republic of Korea. The virus primarily causes infection of the liver, small intestine and spleen. Sudden death is common and is usually due to a severe acute coagulopathy, probably instigated by the presence of virus on red blood cells. Congestion, haemorrhages and infarction of many organs are terminal events caused by the coagulopathy, but are not pathognomonic signs of V H D .

The disease may affect only O. cuniculus, the European and domestic rabbit. Since recovered rabbits shed virus in faeces for at least four weeks, all exposed rabbits should be eliminated to help prevent future outbreaks. The Mexican eradication program has shown that V H D can be eliminated from a country by " s tamping-ou t" methods and follow-up surveillance. In countries where a susceptible wild rabbit population is not present and rabbits are an important source of meat, eradication may be the

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best choice for control of V H D . Rabbits are also an important laboratory animal for medical research and, in many countries, are an extensive hobby and fancier industry valued in many millions of dollars. The value of rabbits often extends far beyond the meat and fur value, and this fact should be carefully considered in policy decisions concerning the control of V H D .

A C K N O W L E D G M E N T S

The authors are grateful to the following colleagues for the generous supply of diagnostic specimens and reagents: Drs J. Mason, J. Guy, F . Hamdy and J. Lubroth , Mexican-U.S. Foot and Mouth Disease Program, Mexico City, Mexico; Dr R. Bates, Virginia Polytechnic Institute, Blacksburg, Virginia; Dr L. Carmichael, Baker Institute, Cornell University, Ithaca, New York; Dr W. Mengeling, National Animal Disease Center, Ames, Iowa; Dr J. House, FADDL; Dr T. Molitor, University of Minnesota; Drs A. Smith and P . Tattersall, Yale University; Mr S.J. Wessman, National Veterinary Services Laboratories, Ames, Iowa; Dr A. Ferrari , Istituto Zooprofilattico Sperimentali, Turin, Italy; Dr S.H. An, Rural Development Administration, Republic of Korea; Dr J. Plana Duran, Laboratory Sobrino, Spain.

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LA MALADIE HÉMORRAGIQUE VIRALE DU LAPIN AU MEXIQUE : ÉPIDÉMIOLOGIE ET CARACTÉRISATION VIRALE. - D.A. Gregg, C. House, R. Meyer et M. Berninger.

Résumé: Une maladie mortelle des lapins a été signalée en République populaire de Chine dès 1984. A partir de 1986, cette maladie s'est déclarée dans la plupart des pays européens, ainsi qu'en République de Corée. En 1989, une maladie similaire est apparue au Mexique, présumée liée à l'importation de carcasses congelées de lapins en provenance de la République populaire de Chine, et s'est propagée rapidement dans dix états mexicains ; elle a été éradiquée au cours de la même année grâce à la mise en œuvre de l'abattage sanitaire. Au Mexique, comme pour les foyers apparus dans les autres pays, la morbidité et la mortalité ont atteint 80 à 90 %, avec peu de signes cliniques. D'après les études effectuées sur sa pathogénie, les sites primaires de réplication du virus sont l'intestin grêle, l'épithélium villeux, les hépatocytes et les lymphocytes spléniques. De nombreux organes, et parmi eux les poumons et les reins, contiennent des tissus atteints d'infarctus aigus et présentant des hémorragies résultant d'un processus de coagulation intravasculaire disséminée. La maladie et les caractéristiques du virus isolé au Mexique sont semblables à celles observées en Europe et en République de Corée. Les auteurs indiquent, en résumé, que la morphologie, l'immunologie et l'hybridation de l'acide nucléique in situ démontrent que la maladie est due à un parvovirus.

MOTS-CLÉS : Biotine - Calicivirus - Diagnostic - Hybridation de l'acide nucléique - Immuno-histochimie - Lapins - Maladie hémorragique virale du lapin - Microscopie électronique - Parvovirus - Pathogénie.

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LA ENFERMEDAD HEMORRÁGICA VIRAL DEL CONEJO EN MÉXICO: EPIDEMIOLOGÍA Y CARACTERIZACIÓN VIRAL. - D.A. Gregg, C. House, R. Meyer y M. Berninger.

Resumen: Se señaló, por primera vez, una enfermedad mortal de los conejos, en la República Popular de China, en 1984. A partir de 1986, esta enfermedad se había declarado en casi todos los países de Europa así como en la República de Corea. En el año 1989, una enfermedad similar apareció en México y se extiendió rápidamente a diez estados mexicanos; se presume que su aparición está relacionada a la importación de carnes congeladas de conejo en pro venencia de la República Popular de China. Fue erradicada ese mismo año gracias a medidas drásticas de sacrificios sanitarios. En México, como en los demás brotes de esta enfermadad, la morbilidad y mortalidad alcanzaron 80 a 90%, con pocos signos clínicos. Los estudios de patogenia mostraron que las primeras replicaciones del virus se efectúan en la cripta del intestino delgado, en el epitelio villoso, a nivel de los hepatocitos y linfocitos esplénicos. Numerosos órganos, incluyendo los pulmones y riñones, presentan tejidos infartados de manera aguda y hemorragias debidas a un proceso de coagulación intravascular diseminada. La enfermedad, así como las características del virus aislado en México, son las mismas que se determinaron en Europa y República de Corea. Los autores indican en resumen que la morfología, inmunología e hibridización de los ácidos nucléicos in situ evidencian una etiología viral.

PALABRAS CLAVE: Biotina - Calicivirus - Conejos - Diagnóstico -Enfermedad hemorrágica viral del conejo - Hibridización de los ácidos nucléicos - Inmuno-histoquímica - Microscopio electrónico - Parvovirus -Patogenia.

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