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SPECIAL REVIEW SERIES
HOG CHOLERA: AN UPDATE OF PRESENT KNOWLEDGE
C . TERPSTRA Central Veterinary Institute, Department of Virology, Houtribweg 39, 8221 RA Lelystad, The Netherlands
INTRODUCTION Hog cholera or classical swine fever is a virus disease of pigs which may run an acute, subacute, chronic or clinically inapparent course . Depending on the virulence of the virus strain, the mortality rate can range from almost nil to virtually 100% . The pig is the sole species in which the disease is known to occur naturally . All breeds and ages are susceptible, although adults generally stand a better chance of surviving the infection . Natural outbreaks of hog cholera may also occur in wild boar (Sus srrofa ferns) . The disease was first recorded in Ohio, United States of America (USA), in the early 1830s ; since that time it has spread worldwide . According to the FAO-WHO-OIE Animal Health Yearbook 1989, the disease is present in 36 countries and suspected to be present in another two . The disease is enzootic in South America and the Far East . In recent decades the USA, Canada, Australia, and most European countries have successfully eradicated hog cholera .
CHARACTERISTICS AND PROPERTIES OF THE VIRUS Hog cholera virus (HCV) is antigenically and structurally related to bovine viral diarrhoea virus (BVDV), which is infectious for cattle, small ruminants and pigs . Congenital infections of BVDV cause border disease in sheep and goats, and isolates from these two animal species are usually referred to as border disease virus (Terpstra, 1985) . HCV cross-reacts with BVDV in immunodiffusion (Darbvshire, 1960) and immunofluorescence (Mengeling et al., 1963), and certain strains of HCV and BVDV even induce to a limited extent neutralizing antibody to the heterologous virus species (Dinter, 1963 ; Snowdon & French, 1968) . Cross-neutralization experiments have shown that strains of HCV form a homogeneous group, whereas
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BVDV strains can be classified into distinct serological subgroups which differ from the HCV group (Wensvoort et aL, 1989) . On the level of the genome a nucleotide sequence homology of about 66% and on the amino acid level a homology of about 85% has been demonstrated between strains of HCV and BVDV (Meyers et aL, 1989 ; Moormann et aL, 1990) . On the basis of their antigenic inter-relatedness the viruses have been included in one genus of pestiviruses (Horzinek, 1973) . The taxonomic position of the genus Pestivirus within the family of Togaviridae (Westaway et al., 1985) has become questionable as recent data on molecular features of the genome and replication strategy of pestiviruses have revealed fundamental differences with togaviruses and striking similarities with members of the Flaviviridae (Horzinek, 1990) . HCV can be propagated in porcine kidney cell cultures of which the established cell lines PK 15 and SK-6 are used most frequently . The virus causes no cytopathic effect and either spreads via the medium, or directly from cell to cell via cytoplasmic bridges or by cell division . By these modes persistently infected cell cultures are readily established . HCV is a spherical and enveloped single-stranded RNA virus with a diameter of 40-50 nm (Westaway et al., 1985) . Electron micrographs of ultrathin sections of HCVinfected cells suggest that virus particles assemble and mature within membranebound intracytoplasmic vesicles and are released via exocytosis (Scherrer et al., 1970) . The physicochemical properties are partly dependent on the physical state of the material containing the virus . For example, HCV in cell culture fluid is inactivated after 60 min at 56°C or 10 min at 60°C (Kubin, 1967), whereas in defibrinated blood infectivity is not destroyed at 64°C for 60 min or at 68°C for 30 min (Torrey & Prather, 1963) . No virus could be recovered from cured and canned hams, prepared from experimentally infected pigs, after pasteurization at core temperatures >67°C (Terpstra & Krol, 1976 ; Stewart et al., 1979) . The virus is stable over a wide pH range, but is rapidly inactivated below pH 4 and above pH 11 (Kubin, 1967) . Because the viral envelope contains lipids, solvents such as ether and chloroform, and detergents such as desoxycholate, Nonidet P40, and saponin rapidly inactivate the virus . The virus possesses at least three structural polypeptides : two glycoproteins with molecular weights of 55 000 (El) and 46 000 daltons (E2) located at the surface, and a non-glycosylated core protein (C) of 36 000 daltons (Enzmann, 1988) . The envelope protein El of HCV contains major antigenic determinants, which are conserved and involved in neutralization (Wensvoort et aL, 1990) . The region on the viral genome encoding for El has recently been established (Moormann el al., 1990) and a genetically' engineered live-attenuated Aujeszky's disease virus recombinant expressing El glycoprotein of HCV has been found to be fully protective against both Aujeszky's disease and hog cholera (Van Zijl et al., 1991) .
EPIDEMIOLOGY Direct contact between pigs is the principal means of viral transmission . Under natural conditions infection by the oral and intranasal routes are probably the most common, although infections via abraded skin and by needle can occur as well . Infected pigs may shed virus during the incubation period .
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Infection with virulent strains gives rise to high levels of virus in blood and other tissues . Large amounts of virus are then excreted in saliva and smaller quantities in urine, nasal and lachrymal fluids (Ressang, 1973) . Viral excretion continues until death or, in pigs which survive, until antibodies have developed . Strains of moderate or low virulence may induce chronic infections in which virus is shed continuously or intermittently until death (Mengeling & Packer, 1969) . When pregnant sows are exposed to such strains, the initial infection will often be inapparent, but the virus can cross the placental barrier and reach the fetuses . Large quantities of virus can be disseminated by piglets born to these so-called `carrier sows' . Congenital infections in which the piglets are born `healthy' are, from an epidemiological point of view, the most insidious . These piglets may shed large quantities of virus for months without showing signs of disease or developing an antibody response (Van Oirschot & Terpstra, 1977) . Not all virus strains spread equally fast . Whereas pigs infected with virulent strains shed large quantities of virus during the entire course of the disease, postnatal infections with strains of low virulence are characterized by a short period of virus multiplication and excretion followed by an antibody response . Consequently, virulent strains usually spread faster in a herd and induce higher morbidity than lessvirulent strains . Movement of pigs is the most common way in which hog cholera is spread . Most feared is road transportation of weaners collected from different breeding farms, sorted and regrouped at markets, and then reloaded for distribution to various fattening farms . Such transportation, often over long distances, may result in a large number of non-traceable contacts . The introduction of viruses of reduced virulence may go unnoticed for weeks or even months and, upon investigation, few animals may prove to be infected . Movement of pregnant `carrier sows' introduces infection into the recipient herd at the time of farrowing and results in a corresponding or even longer delay before the disease is diagnosed . Hog cholera virus can survive in pork and processed pork products . Survival can be prolonged for months when meat is stored cool or even years when stored frozen . In this way the virus can be introduced into areas or countries hitherto free of the disease . Susceptible pigs may contract the disease when fed with contaminated slaughterhouse offal or kitchen leftovers that have not received proper heat treatment . Mechanical transmission by man is of great significance in areas with a high density of pigs and pig herds (Terpstra, 1987) . Farmers, castrators, inseminators, and veterinarians can transmit the virus by contaminated instruments and drugs for parenteral use . The common practice in veterinary medicine of not changing syringes and needles and of not discarding partly used bottles between herds is reason for concern . Although airborne transmission was difficult to establish tinder experimental conditions (Terpstra, 1987), it is conceivable that this mode could play a role in the spread of virus between mechanically ventilated units at close proximity . Transmission by contaminated clothing, footwear, pets, and rodents is rare, since the amounts of virus thus transferred are usually below the minimum infective dose for pigs . The virus can circulate in wild boar and such an infected population is a potential risk to domestic swine, either through the food chain or by direct contact . The latter applies particularly to areas where domestic pigs are kept on free or semi-free range .
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PATHOGENESIS Under natural conditions the virus enters the host via the oronasal route . Natural infections by highly virulent strains are characterized by a lymphatic, a viraemic, and a visceral phase . After initial replication in the epithelial cells lining the tonsillar crypts the virus invades the underlying lymphoreticular tissue from where it is drained to the regional lymph nodes (Ressang, 1973) . Here it multiplies and gives rise to an initial viraemia. Large amounts of virus are produced in secondary target tissues such as the spleen, visceral lymph nodes, bone marrow, and the digestive tract . This results in high virus levels in the blood and invasion of the parenchymatous organs, the respiratory tract and the central nervous system (Ressang, 1973) . Organ infection appears to be mediated via phagocytosis by reticuloendothelial cells and by the growth of virus through the vascular endothelium . Viral replication in leucocytes and in cells of the reticuloendothelial system precipitates a leucopenia and predisposes the animal to secondary bacterial infections . Highly virulent virus spreads throughout the body within 5-6 days . Infections with moderately virulent virus strains follow the same pattern as for highly virulent strains but proceed more slowly and, moreover, virus concentrations in blood and organs tend to be lower . Infections with strains of low virulence are mainly confined to the lymphatic phase, the viraemic phase being brief . Infection of the pregnant sow can result in transplacental transmission of the virus in all stages of pregnancy . The virus usually spreads haematogeneously and grows at one or more sites across the placenta and subsequently spreads from fetus to fetus (Van Oirschot, 1979) . The fetuses become viraemic and virus distribution in the tissues appears to be similar to that in pigs infected postnatally with a virulent strain . The ultimate outcome of infection in utero depends on various factors, e .g. the time of infection and the virulence of the virus strain . Fetuses infected during the first 45 days after conception are more prone to prenatal death or to developing a condition of persistent infection and immunological tolerance than fetuses infected at 65 days and later . On the other hand, fetuses infected with a strain of moderate virulence in the last 45 days of gestation are more likely to show signs of disease at birth or shortly thereafter, or to eliminate the virus in case of a low-virulent strain (Van Oirschot, 1979 ; Hermanns et al., 1981) .
CLINICAL SIGNS Signs of acute hog cholera appear after an incubation period of 2-6 days . The early stage of disease is characterized by fever, dullness, a reluctance to move, and reduced appetite . The signs progress over the next few days . Temperatures of 42 ° C or above may be reached and fever persists until shortly before death . Leucopenia and thrombocytopenia can already be observed before the onset of fever and persist until death . Relatively early in the disease pigs develop conjunctivitis leading to an ocular discharge and in some cases a nasal discharge occurs as well . Evidence of digestive tract involvement includes constipation which is sometimes followed by diarrhoea . Some pigs vomit a yellowish bile-containing fluid . Affected pigs shiver and huddle together . During the terminal stages of the disease most pigs have a typical weaving or staggering gait, often followed by posterior paresis . Occasionally, convulsions are
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seen . A purplish discoloration extending over the skin of the abdomen, snout, ears, and medial sides of the legs may also occur terminally . The mortality rate from acute hog cholera is very high and most pigs die between 10 and 20 days after infection . In subacute cases caused by less virulent strains pigs develop similar but less severe signs of illness and succumb within 30 days . When pigs survive beyond 30 days, the infection is considered chronic (Mengeling & Packer, 1969) . Such infections are characterized by prolonged and intermittent periods with anorexia, fever, viraemia, and diarrhoea . Alopecia followed by dermatitis may occur . Persistent infection leads to severe growth retardation and the development of runted pigs. Pigs with chronic disease may be ill for months but die eventually . Pregnant sows infected by strains of moderate or low virulence may develop the `carrier sow syndrome' . Depending on the stage of gestation and the virulence of the virus strain, congenital infection can result in abortion, fetal mummification, stillbirth, the birth of weak and trembling pigs, neonatal death or the birth of healthy looking but persistently infected piglets . Stillborn and weak piglets often show alopecia, hydrops and subcutaneous oedema (jelly piglets') or, less frequently, malformations of visceral organs and necrosis of peripheral parts of the body . Most piglets that are infected in utero and born alive die shortly after birth, but strains of low virulence may cause `late onset' hog cholera . This form is characterized by normal development for several weeks or months before signs of disease-such as mild anorexia and depression, conjunctivitis, dermatitis, intermittent diarrhoea and locomotor disturbances-start to develop (Van Oirschot & Terpstra, 1977) . Body temperatures are normal or slightly elevated, leucocyte counts are normal to subnormal with a tendency for leucocytosis to develop in the terminal stage of illness . Strains of low virulence often cause only a mild and transient illness and inapparent infections are common .
PATHOLOGY Acute and subacute hog cholera are septicaemic conditions characterized by multiple haemorrhages of various sizes resulting from hydropic degeneration and necrosis of capillary endothelial cells and from a defect in blood coagulation . Haemorrhages occur throughout the body, most commonly in lymph nodes and the kidneys and less frequently in the heart, serosae, urinary bladder, intestinal mucosae, larynx, epiglottis, muscles, skin, and the subcutis . The lymph nodes are usually swollen and exhibit haemorrhages in the peripheral sinuses which give the nodes a `marbled' appearance . Haemorrhages in the kidney are mainly petechial and situated subcapsularly. The spleen is usually normal in size and haemorrhagic infarcts may be observed, mainly along its edges . In addition, catarrhal, fibrinous, and haemorrhagic inflammatory reactions may occur in the mucosae of digestive and respiratory tracts and in the lungs . Secondary bacterial infections occur frequently . In chronic and `late onset' infections atrophy of the thymus and exostoses at the costochondral junctions of the ribs of young pigs are the commonest lesions (Mengeling & Packer, 1969 ; Van Oirschot & Terpstra, 1977) . Button ulcers are
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occasionally seen in the caecum and proximal part of the large colon, while haemorrhages are usually absent . The most characteristic microscopic lesions are located in the reticulo-endothelial system, particularly in the walls of arterioles, venules and capillaries, where endothelial cells are swollen due to hydropic degeneration . Some terminal vessels are dilated and others are occluded by thrombi which leads to the characteristic gross lesions of hog cholera, e .g . congestion, haemorrhage, infarction, and necrosis . A nonsuppurative meningoencephalitis, characterized by swelling and degeneration of endothelial cells, thrombosis, vascular and perivascular infiltration of lymphocytes, is seen in 70-90% of fatal cases . The vascular lesions are most prevalent in the spleen, lymph nodes, kidneys, and gastrointestinal tract . Proliferation of histiocytes along with depletion of lymphocytes in the germinal centres in lymph nodes, spleen, tonsils, and Peyer's patches occur in chronic and `late onset' hog cholera (Van der Molen & Van Oirschot, 1981) . The interfollicular areas of the lymphoid organs are usually rather well populated .
DIAGNOSIS The clinical signs, macroscopic and microscopic lesions of hog cholera, can vary considerably . An unequivocal diagnosis, therefore, is impossible without laboratory identification of viral antigens or specific antibodies to those antigens, or isolating the virus. Direct immunofluorescence on frozen sections of tonsils, spleen, kidney and distal part of the ileum is the preferred method . Of these organs, tonsils are by far the most important . Ideally, tissues should be collected from several animals and transported to the laboratory without the addition of preservatives and kept as cool as possible but not frozen . Although immunofluorescence is a rapid and reliable technique, a negative result does not entirely exclude hog cholera . Where suspicion remains, more samples should be examined or attempts made at isolating the virus in porcine kidney (PK)-15 cell cultures . These cultures are examined for virus growth by immunofluorescence . Detection of specific antibodies is particularly useful in herds suspected of being infected with strains of low virulence and is imperative when a country desires to become internationally recognized as free from the disease . Since antibodies against BVDV are frequently observed in breeding pigs, only tests that differentiate between bovine virus diarrhoea and hog cholera antibodies, e .g . neutralization tests and an ELISA based on monoclonal antibodies (Wensvoort el al., 1988), are suitable .
DD}ERENTIAL DIAGNOSIS The symptoms and post-mortem lesions of hog cholera are highly variable and resemble those of African swine fever ; difference between these two diseases being more quantitative than qualitative, with the exception of enlargement of the spleen and the haematoma-like visceral lymph nodes, that are characteristic of African swine fever . Oedema of the gall bladder walls and bile ducts and subpleural and interlobular lung oedema are rare in hog cholera, but common in African swine fever .
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Congenital infections of BVDV in pigs, though occurring rarely, can cause clinical signs and post-mortem lesions indistinguishable from those observed in chronic hog cholera (Terpstra & Wensvoort, 1988) . The immunofluorescence test uses conjugates prepared from anti-HCV hyperimmune sera, which do not distinguish between HCV and BVDV antigens . Likewise such conjugates do not differentiate between fluorescence caused by field strains of HCV and the rabbit adapted Chinese (C) vaccine strain, which can be found in the tonsils up to 2 weeks after vaccination . Whenever clinical findings and anamnesis indicate a possible infection with BVDV or vaccination, differentiation can be made by herd serology for HCV and BVDV neutralizing antibodies or rabbit inoculation, respectively . Both methods take several days, which is incompatible with the need for obtaining the earliest possible diagnosis . Conjugates prepared from selected monoclonal antibodies raised against HCV have solved the problem because these allow within hours an unambiguous differentiation between field and vaccine strains of HCV, and between HCV and BVDV (Wensvoort et al., 1986) . Other septicaemic diseases-e .g . babesiosis, eperythrozoonosis, salmonellosis, pasteurellosis, Haemophilus suis, and Erysipelothrix insidiosa infections-can be differentiated from hog cholera either by demonstrating parasites in blood smears or by bacterial cultivation . Generalized haemorrhages unaccompanied by fever may point in young piglets to thrombocytopenia or, in all age groups, to dicumarol or other poisonings . Like chronic hog cholera, malnutrition, enterotoxicosis by Escherichia or Clostridium perfringens and vibrionic dysentery may lead to diarrhoea, growth retardation, or runting . Trembling piglets resulting from intrauterine infection with HCV can be confused with congenital mvoc lon ia .
PREVENTION AND CONTROL In countries free of hog cholera, measures are aimed at preventing the introduction of the virus . Imports of live pigs and insufficiently heated pork and pork products from infected countries should be prohibited . In addition, kitchen leftovers from aircraft, ships, and international trains should be destroyed as a general precaution against the introduction of exotic diseases of livestock . When, despite these measures, the disease is introduced, zoo-sanitary and veterinary police measures are necessary . Tracing the source of infection and the extent of viral spread is the key to eradication of the disease . Depending on the local situation, control measures may include a ban on the movement of' pigs, close surveillance of farms in the endangered area, and destruction of all pigs on infected premises (stamping out) . Farm buildings should be cleaned and thoroughly disinfected . A solution of I % sodium hydroxide is suitable for disinfecting stables and vehicles . Chlorides and quaternary ammonium compounds supplemented with a non-ionic detergent are effective alternatives . In manure the virus appears to be inactivated within a few days . The buildings are kept uninhabited for the duration of the standstill, which according to internationally accepted regulations may vary from 15 to 30 days . Repopulation of the farm should be closely supervised for suspicious signs of hog cholera . Eradication of hog cholera by means of stamping out, supported by other
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veterinary legislation and zoo-sanitary measures has been achieved in, for example, the Republic of South Africa, Australia, Canada, the USA, the United Kingdom, and the Scandinavian countries . Elsewhere, however, the disease prevails or persisted for many years, despite control measures . The reasons for this are manifold and vary from country to country . Unregistered movement of unidentified pigs often hampers tracing an outbreak to its source . The `carrier sow' syndrome and the presence of strains of low virulence may mask the disease and delay diagnosis . Dissemination of virus in the food chain and lack of diagnostic laboratory facilities may also seriously impede progress towards final eradication . In order to break the uncontrollable spread of field virus, vaccination is or has been used in several countries, either to eliminate the disease, or to reduce the number of outbreaks so that eradication by sanitary measures alone is feasible . Safe and potent modified live vaccines are available, notably the lapinized Chinese strain and the cell-culture-adapted Japanese GPE strain (Sasahara et al., 1969 ; Lin & Lee, 1981) . Experience in Europe has proven beyond doubt that hog cholera can be eradicated from enzootic areas if a systematic and strict regimen of vaccination is pursued for a length of time and that vaccination is supported by veterinary police and zoo-sanitary measures (Terpstra & Robijns, 1977) . To be effective, vaccination should be compulsory and vaccinated animals clearly identified . Due to rapid population turnover vaccinated pigs are replaced quickly by non-immune young stock. To maintain the high degree of immunity required to smother foci of residual virus, supplementary vaccination of 8- to 10-week-old . and newly introduced piglets should be performed at monthly intervals . Of the 450 hog cholera outbreaks diagnosed in the Netherlands during the 1982-1985 epizootic, 63 occurred one month or more after the start of vaccination, i .e . when full protection should have been obtained . Twelve of these 63 were even detected between 9 and 13 months after the start of the vaccination campaign . Analysis of the outbreaks has shown that vaccination is more effective in fattening herds than in breeding and mixed herds, and that in the two latter categories, the efficacy is inversely related to the size of the herd . Apparently, large herds contain sufficient numbers of susceptible piglets for the virus to be perpetuated and to be spread at a low level . The infection lingers on in piglets of about the age of weaning and vaccination prevents the infection from developing into an overt outbreak . The monthly supplementary vaccination programmes therefore should be continued for at least 1 year after the start of a mass vaccination and preferably until 1 year after the last outbreak .
FUTURE DEVELOPMENTS An expansion of intracommunity movements of livestock, meat and meat products is to be expected after abolishing the trade barriers between EC countries in early 1993 . As a consequence, the risk of introducing notifiable diseases like hog cholera and African swine fever, which are present in some member states, will increase . In case of introduction, an early and rapid diagnosis is of the utmost importance in order to minimize losses .
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In recent years, new tools of research have become available for a rapid, sensitive, and specific detection of infectious agents . The advantages of monoclonal antibodies in hog cholera diagnosis have already been described, but other techniques such as the polymerase chain reaction (PCR) and flow cytometry should be mentioned as well . By using reverse transcription followed by the PCR it should be possible to amplify a single RNA fragment of the genome to concentrations either detectable in ethidium bromide stained gels or by probes . The technique is likely to detect the virus in tonsils, blood and other target organs at an earlier stage of infection than immunofluorescence or virus isolation . Flow cytometry is based on the quantitative measurement of fluorescence associated with single cells passing through a flow chamber . The method which has been described for detecting BVDV in blood of persistently viraemic cattle (ovist et al., 1990), would be extremely useful for tracing early HCV infections on suspected premises . If successful, the period of 5 weeks that has to elapse between a suspected contact and a meaningful seroepidemiological investigation could substantially be reduced . According to EC regulations only pigs and pig products from countries or regions with the `officially free' (OF) hog cholera status (i .e . no outbreaks and no vaccination in the last 12 months, and no vaccinated pigs present) have admission to the free trade within the community . The presently available vaccines against hog cholera do not allow differentiation by serological means between vaccinated pigs and pigs that have been exposed to wild strains of HCV . In the event of a hog cholera outbreak, the OF-status will temporarily be suspended, but the country or region will lose its status in case of vaccination . Therefore, eradicating the disease without resorting to vaccination is vital when the pig industry is based on exports . For this reason vaccination was not applied in Belgium during the 1990 hog cholera epizootic (113 outbreaks, one million pigs destroyed, direct losses US$ 270 million ; Westergaard, personal communication) . Vaccines inducing antibodies that can be distinguished from antibodies induced by field strains might remove the objection against vaccination . The way for constructing a serologically distinctive vaccine is open now that the nucleotide sequence and the location of the El gene, which encodes for the protection inducing protein, are known (Moormann et al., 1990) . As a matter of fact, the recombinant Aujeszky's disease virus expressing El protein of HCV (Van Zijl et al,, 1991) is the first product with this property, although its potential in comparison with existing hog cholera vaccines remains to be determined . Analysis of viral nucleic acid by means of PCR and nucleotide sequencing of certain amplified fragments might make it possible to associate genomic differences between field and vaccine strains with various biological properties . Identification of field isolates by these techniques may also prove a fruitful line of research that could shed more light on the epizootiology of the disease .
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(Accepted for publication 31 January 1991)
SPECIAL REVIEW SERIES
HOG CHOLERA: AN UPDATE OF PRESENT KNOWLEDGE
C . TERPSTRA Central Veterinary Institute, Department of Virology, Houtribweg 39, 8221 RA Lelystad, The Netherlands
INTRODUCTION Hog cholera or classical swine fever is a virus disease of pigs which may run an acute, subacute, chronic or clinically inapparent course . Depending on the virulence of the virus strain, the mortality rate can range from almost nil to virtually 100% . The pig is the sole species in which the disease is known to occur naturally . All breeds and ages are susceptible, although adults generally stand a better chance of surviving the infection . Natural outbreaks of hog cholera may also occur in wild boar (Sus srrofa ferns) . The disease was first recorded in Ohio, United States of America (USA), in the early 1830s ; since that time it has spread worldwide . According to the FAO-WHO-OIE Animal Health Yearbook 1989, the disease is present in 36 countries and suspected to be present in another two . The disease is enzootic in South America and the Far East . In recent decades the USA, Canada, Australia, and most European countries have successfully eradicated hog cholera .
CHARACTERISTICS AND PROPERTIES OF THE VIRUS Hog cholera virus (HCV) is antigenically and structurally related to bovine viral diarrhoea virus (BVDV), which is infectious for cattle, small ruminants and pigs . Congenital infections of BVDV cause border disease in sheep and goats, and isolates from these two animal species are usually referred to as border disease virus (Terpstra, 1985) . HCV cross-reacts with BVDV in immunodiffusion (Darbvshire, 1960) and immunofluorescence (Mengeling et al., 1963), and certain strains of HCV and BVDV even induce to a limited extent neutralizing antibody to the heterologous virus species (Dinter, 1963 ; Snowdon & French, 1968) . Cross-neutralization experiments have shown that strains of HCV form a homogeneous group, whereas
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BVDV strains can be classified into distinct serological subgroups which differ from the HCV group (Wensvoort et aL, 1989) . On the level of the genome a nucleotide sequence homology of about 66% and on the amino acid level a homology of about 85% has been demonstrated between strains of HCV and BVDV (Meyers et aL, 1989 ; Moormann et aL, 1990) . On the basis of their antigenic inter-relatedness the viruses have been included in one genus of pestiviruses (Horzinek, 1973) . The taxonomic position of the genus Pestivirus within the family of Togaviridae (Westaway et al., 1985) has become questionable as recent data on molecular features of the genome and replication strategy of pestiviruses have revealed fundamental differences with togaviruses and striking similarities with members of the Flaviviridae (Horzinek, 1990) . HCV can be propagated in porcine kidney cell cultures of which the established cell lines PK 15 and SK-6 are used most frequently . The virus causes no cytopathic effect and either spreads via the medium, or directly from cell to cell via cytoplasmic bridges or by cell division . By these modes persistently infected cell cultures are readily established . HCV is a spherical and enveloped single-stranded RNA virus with a diameter of 40-50 nm (Westaway et al., 1985) . Electron micrographs of ultrathin sections of HCVinfected cells suggest that virus particles assemble and mature within membranebound intracytoplasmic vesicles and are released via exocytosis (Scherrer et al., 1970) . The physicochemical properties are partly dependent on the physical state of the material containing the virus . For example, HCV in cell culture fluid is inactivated after 60 min at 56°C or 10 min at 60°C (Kubin, 1967), whereas in defibrinated blood infectivity is not destroyed at 64°C for 60 min or at 68°C for 30 min (Torrey & Prather, 1963) . No virus could be recovered from cured and canned hams, prepared from experimentally infected pigs, after pasteurization at core temperatures >67°C (Terpstra & Krol, 1976 ; Stewart et al., 1979) . The virus is stable over a wide pH range, but is rapidly inactivated below pH 4 and above pH 11 (Kubin, 1967) . Because the viral envelope contains lipids, solvents such as ether and chloroform, and detergents such as desoxycholate, Nonidet P40, and saponin rapidly inactivate the virus . The virus possesses at least three structural polypeptides : two glycoproteins with molecular weights of 55 000 (El) and 46 000 daltons (E2) located at the surface, and a non-glycosylated core protein (C) of 36 000 daltons (Enzmann, 1988) . The envelope protein El of HCV contains major antigenic determinants, which are conserved and involved in neutralization (Wensvoort et aL, 1990) . The region on the viral genome encoding for El has recently been established (Moormann el al., 1990) and a genetically' engineered live-attenuated Aujeszky's disease virus recombinant expressing El glycoprotein of HCV has been found to be fully protective against both Aujeszky's disease and hog cholera (Van Zijl et al., 1991) .
EPIDEMIOLOGY Direct contact between pigs is the principal means of viral transmission . Under natural conditions infection by the oral and intranasal routes are probably the most common, although infections via abraded skin and by needle can occur as well . Infected pigs may shed virus during the incubation period .
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Infection with virulent strains gives rise to high levels of virus in blood and other tissues . Large amounts of virus are then excreted in saliva and smaller quantities in urine, nasal and lachrymal fluids (Ressang, 1973) . Viral excretion continues until death or, in pigs which survive, until antibodies have developed . Strains of moderate or low virulence may induce chronic infections in which virus is shed continuously or intermittently until death (Mengeling & Packer, 1969) . When pregnant sows are exposed to such strains, the initial infection will often be inapparent, but the virus can cross the placental barrier and reach the fetuses . Large quantities of virus can be disseminated by piglets born to these so-called `carrier sows' . Congenital infections in which the piglets are born `healthy' are, from an epidemiological point of view, the most insidious . These piglets may shed large quantities of virus for months without showing signs of disease or developing an antibody response (Van Oirschot & Terpstra, 1977) . Not all virus strains spread equally fast . Whereas pigs infected with virulent strains shed large quantities of virus during the entire course of the disease, postnatal infections with strains of low virulence are characterized by a short period of virus multiplication and excretion followed by an antibody response . Consequently, virulent strains usually spread faster in a herd and induce higher morbidity than lessvirulent strains . Movement of pigs is the most common way in which hog cholera is spread . Most feared is road transportation of weaners collected from different breeding farms, sorted and regrouped at markets, and then reloaded for distribution to various fattening farms . Such transportation, often over long distances, may result in a large number of non-traceable contacts . The introduction of viruses of reduced virulence may go unnoticed for weeks or even months and, upon investigation, few animals may prove to be infected . Movement of pregnant `carrier sows' introduces infection into the recipient herd at the time of farrowing and results in a corresponding or even longer delay before the disease is diagnosed . Hog cholera virus can survive in pork and processed pork products . Survival can be prolonged for months when meat is stored cool or even years when stored frozen . In this way the virus can be introduced into areas or countries hitherto free of the disease . Susceptible pigs may contract the disease when fed with contaminated slaughterhouse offal or kitchen leftovers that have not received proper heat treatment . Mechanical transmission by man is of great significance in areas with a high density of pigs and pig herds (Terpstra, 1987) . Farmers, castrators, inseminators, and veterinarians can transmit the virus by contaminated instruments and drugs for parenteral use . The common practice in veterinary medicine of not changing syringes and needles and of not discarding partly used bottles between herds is reason for concern . Although airborne transmission was difficult to establish tinder experimental conditions (Terpstra, 1987), it is conceivable that this mode could play a role in the spread of virus between mechanically ventilated units at close proximity . Transmission by contaminated clothing, footwear, pets, and rodents is rare, since the amounts of virus thus transferred are usually below the minimum infective dose for pigs . The virus can circulate in wild boar and such an infected population is a potential risk to domestic swine, either through the food chain or by direct contact . The latter applies particularly to areas where domestic pigs are kept on free or semi-free range .
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PATHOGENESIS Under natural conditions the virus enters the host via the oronasal route . Natural infections by highly virulent strains are characterized by a lymphatic, a viraemic, and a visceral phase . After initial replication in the epithelial cells lining the tonsillar crypts the virus invades the underlying lymphoreticular tissue from where it is drained to the regional lymph nodes (Ressang, 1973) . Here it multiplies and gives rise to an initial viraemia. Large amounts of virus are produced in secondary target tissues such as the spleen, visceral lymph nodes, bone marrow, and the digestive tract . This results in high virus levels in the blood and invasion of the parenchymatous organs, the respiratory tract and the central nervous system (Ressang, 1973) . Organ infection appears to be mediated via phagocytosis by reticuloendothelial cells and by the growth of virus through the vascular endothelium . Viral replication in leucocytes and in cells of the reticuloendothelial system precipitates a leucopenia and predisposes the animal to secondary bacterial infections . Highly virulent virus spreads throughout the body within 5-6 days . Infections with moderately virulent virus strains follow the same pattern as for highly virulent strains but proceed more slowly and, moreover, virus concentrations in blood and organs tend to be lower . Infections with strains of low virulence are mainly confined to the lymphatic phase, the viraemic phase being brief . Infection of the pregnant sow can result in transplacental transmission of the virus in all stages of pregnancy . The virus usually spreads haematogeneously and grows at one or more sites across the placenta and subsequently spreads from fetus to fetus (Van Oirschot, 1979) . The fetuses become viraemic and virus distribution in the tissues appears to be similar to that in pigs infected postnatally with a virulent strain . The ultimate outcome of infection in utero depends on various factors, e .g. the time of infection and the virulence of the virus strain . Fetuses infected during the first 45 days after conception are more prone to prenatal death or to developing a condition of persistent infection and immunological tolerance than fetuses infected at 65 days and later . On the other hand, fetuses infected with a strain of moderate virulence in the last 45 days of gestation are more likely to show signs of disease at birth or shortly thereafter, or to eliminate the virus in case of a low-virulent strain (Van Oirschot, 1979 ; Hermanns et al., 1981) .
CLINICAL SIGNS Signs of acute hog cholera appear after an incubation period of 2-6 days . The early stage of disease is characterized by fever, dullness, a reluctance to move, and reduced appetite . The signs progress over the next few days . Temperatures of 42 ° C or above may be reached and fever persists until shortly before death . Leucopenia and thrombocytopenia can already be observed before the onset of fever and persist until death . Relatively early in the disease pigs develop conjunctivitis leading to an ocular discharge and in some cases a nasal discharge occurs as well . Evidence of digestive tract involvement includes constipation which is sometimes followed by diarrhoea . Some pigs vomit a yellowish bile-containing fluid . Affected pigs shiver and huddle together . During the terminal stages of the disease most pigs have a typical weaving or staggering gait, often followed by posterior paresis . Occasionally, convulsions are
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seen . A purplish discoloration extending over the skin of the abdomen, snout, ears, and medial sides of the legs may also occur terminally . The mortality rate from acute hog cholera is very high and most pigs die between 10 and 20 days after infection . In subacute cases caused by less virulent strains pigs develop similar but less severe signs of illness and succumb within 30 days . When pigs survive beyond 30 days, the infection is considered chronic (Mengeling & Packer, 1969) . Such infections are characterized by prolonged and intermittent periods with anorexia, fever, viraemia, and diarrhoea . Alopecia followed by dermatitis may occur . Persistent infection leads to severe growth retardation and the development of runted pigs. Pigs with chronic disease may be ill for months but die eventually . Pregnant sows infected by strains of moderate or low virulence may develop the `carrier sow syndrome' . Depending on the stage of gestation and the virulence of the virus strain, congenital infection can result in abortion, fetal mummification, stillbirth, the birth of weak and trembling pigs, neonatal death or the birth of healthy looking but persistently infected piglets . Stillborn and weak piglets often show alopecia, hydrops and subcutaneous oedema (jelly piglets') or, less frequently, malformations of visceral organs and necrosis of peripheral parts of the body . Most piglets that are infected in utero and born alive die shortly after birth, but strains of low virulence may cause `late onset' hog cholera . This form is characterized by normal development for several weeks or months before signs of disease-such as mild anorexia and depression, conjunctivitis, dermatitis, intermittent diarrhoea and locomotor disturbances-start to develop (Van Oirschot & Terpstra, 1977) . Body temperatures are normal or slightly elevated, leucocyte counts are normal to subnormal with a tendency for leucocytosis to develop in the terminal stage of illness . Strains of low virulence often cause only a mild and transient illness and inapparent infections are common .
PATHOLOGY Acute and subacute hog cholera are septicaemic conditions characterized by multiple haemorrhages of various sizes resulting from hydropic degeneration and necrosis of capillary endothelial cells and from a defect in blood coagulation . Haemorrhages occur throughout the body, most commonly in lymph nodes and the kidneys and less frequently in the heart, serosae, urinary bladder, intestinal mucosae, larynx, epiglottis, muscles, skin, and the subcutis . The lymph nodes are usually swollen and exhibit haemorrhages in the peripheral sinuses which give the nodes a `marbled' appearance . Haemorrhages in the kidney are mainly petechial and situated subcapsularly. The spleen is usually normal in size and haemorrhagic infarcts may be observed, mainly along its edges . In addition, catarrhal, fibrinous, and haemorrhagic inflammatory reactions may occur in the mucosae of digestive and respiratory tracts and in the lungs . Secondary bacterial infections occur frequently . In chronic and `late onset' infections atrophy of the thymus and exostoses at the costochondral junctions of the ribs of young pigs are the commonest lesions (Mengeling & Packer, 1969 ; Van Oirschot & Terpstra, 1977) . Button ulcers are
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occasionally seen in the caecum and proximal part of the large colon, while haemorrhages are usually absent . The most characteristic microscopic lesions are located in the reticulo-endothelial system, particularly in the walls of arterioles, venules and capillaries, where endothelial cells are swollen due to hydropic degeneration . Some terminal vessels are dilated and others are occluded by thrombi which leads to the characteristic gross lesions of hog cholera, e .g . congestion, haemorrhage, infarction, and necrosis . A nonsuppurative meningoencephalitis, characterized by swelling and degeneration of endothelial cells, thrombosis, vascular and perivascular infiltration of lymphocytes, is seen in 70-90% of fatal cases . The vascular lesions are most prevalent in the spleen, lymph nodes, kidneys, and gastrointestinal tract . Proliferation of histiocytes along with depletion of lymphocytes in the germinal centres in lymph nodes, spleen, tonsils, and Peyer's patches occur in chronic and `late onset' hog cholera (Van der Molen & Van Oirschot, 1981) . The interfollicular areas of the lymphoid organs are usually rather well populated .
DIAGNOSIS The clinical signs, macroscopic and microscopic lesions of hog cholera, can vary considerably . An unequivocal diagnosis, therefore, is impossible without laboratory identification of viral antigens or specific antibodies to those antigens, or isolating the virus. Direct immunofluorescence on frozen sections of tonsils, spleen, kidney and distal part of the ileum is the preferred method . Of these organs, tonsils are by far the most important . Ideally, tissues should be collected from several animals and transported to the laboratory without the addition of preservatives and kept as cool as possible but not frozen . Although immunofluorescence is a rapid and reliable technique, a negative result does not entirely exclude hog cholera . Where suspicion remains, more samples should be examined or attempts made at isolating the virus in porcine kidney (PK)-15 cell cultures . These cultures are examined for virus growth by immunofluorescence . Detection of specific antibodies is particularly useful in herds suspected of being infected with strains of low virulence and is imperative when a country desires to become internationally recognized as free from the disease . Since antibodies against BVDV are frequently observed in breeding pigs, only tests that differentiate between bovine virus diarrhoea and hog cholera antibodies, e .g . neutralization tests and an ELISA based on monoclonal antibodies (Wensvoort el al., 1988), are suitable .
DD}ERENTIAL DIAGNOSIS The symptoms and post-mortem lesions of hog cholera are highly variable and resemble those of African swine fever ; difference between these two diseases being more quantitative than qualitative, with the exception of enlargement of the spleen and the haematoma-like visceral lymph nodes, that are characteristic of African swine fever . Oedema of the gall bladder walls and bile ducts and subpleural and interlobular lung oedema are rare in hog cholera, but common in African swine fever .
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Congenital infections of BVDV in pigs, though occurring rarely, can cause clinical signs and post-mortem lesions indistinguishable from those observed in chronic hog cholera (Terpstra & Wensvoort, 1988) . The immunofluorescence test uses conjugates prepared from anti-HCV hyperimmune sera, which do not distinguish between HCV and BVDV antigens . Likewise such conjugates do not differentiate between fluorescence caused by field strains of HCV and the rabbit adapted Chinese (C) vaccine strain, which can be found in the tonsils up to 2 weeks after vaccination . Whenever clinical findings and anamnesis indicate a possible infection with BVDV or vaccination, differentiation can be made by herd serology for HCV and BVDV neutralizing antibodies or rabbit inoculation, respectively . Both methods take several days, which is incompatible with the need for obtaining the earliest possible diagnosis . Conjugates prepared from selected monoclonal antibodies raised against HCV have solved the problem because these allow within hours an unambiguous differentiation between field and vaccine strains of HCV, and between HCV and BVDV (Wensvoort et al., 1986) . Other septicaemic diseases-e .g . babesiosis, eperythrozoonosis, salmonellosis, pasteurellosis, Haemophilus suis, and Erysipelothrix insidiosa infections-can be differentiated from hog cholera either by demonstrating parasites in blood smears or by bacterial cultivation . Generalized haemorrhages unaccompanied by fever may point in young piglets to thrombocytopenia or, in all age groups, to dicumarol or other poisonings . Like chronic hog cholera, malnutrition, enterotoxicosis by Escherichia or Clostridium perfringens and vibrionic dysentery may lead to diarrhoea, growth retardation, or runting . Trembling piglets resulting from intrauterine infection with HCV can be confused with congenital mvoc lon ia .
PREVENTION AND CONTROL In countries free of hog cholera, measures are aimed at preventing the introduction of the virus . Imports of live pigs and insufficiently heated pork and pork products from infected countries should be prohibited . In addition, kitchen leftovers from aircraft, ships, and international trains should be destroyed as a general precaution against the introduction of exotic diseases of livestock . When, despite these measures, the disease is introduced, zoo-sanitary and veterinary police measures are necessary . Tracing the source of infection and the extent of viral spread is the key to eradication of the disease . Depending on the local situation, control measures may include a ban on the movement of' pigs, close surveillance of farms in the endangered area, and destruction of all pigs on infected premises (stamping out) . Farm buildings should be cleaned and thoroughly disinfected . A solution of I % sodium hydroxide is suitable for disinfecting stables and vehicles . Chlorides and quaternary ammonium compounds supplemented with a non-ionic detergent are effective alternatives . In manure the virus appears to be inactivated within a few days . The buildings are kept uninhabited for the duration of the standstill, which according to internationally accepted regulations may vary from 15 to 30 days . Repopulation of the farm should be closely supervised for suspicious signs of hog cholera . Eradication of hog cholera by means of stamping out, supported by other
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veterinary legislation and zoo-sanitary measures has been achieved in, for example, the Republic of South Africa, Australia, Canada, the USA, the United Kingdom, and the Scandinavian countries . Elsewhere, however, the disease prevails or persisted for many years, despite control measures . The reasons for this are manifold and vary from country to country . Unregistered movement of unidentified pigs often hampers tracing an outbreak to its source . The `carrier sow' syndrome and the presence of strains of low virulence may mask the disease and delay diagnosis . Dissemination of virus in the food chain and lack of diagnostic laboratory facilities may also seriously impede progress towards final eradication . In order to break the uncontrollable spread of field virus, vaccination is or has been used in several countries, either to eliminate the disease, or to reduce the number of outbreaks so that eradication by sanitary measures alone is feasible . Safe and potent modified live vaccines are available, notably the lapinized Chinese strain and the cell-culture-adapted Japanese GPE strain (Sasahara et al., 1969 ; Lin & Lee, 1981) . Experience in Europe has proven beyond doubt that hog cholera can be eradicated from enzootic areas if a systematic and strict regimen of vaccination is pursued for a length of time and that vaccination is supported by veterinary police and zoo-sanitary measures (Terpstra & Robijns, 1977) . To be effective, vaccination should be compulsory and vaccinated animals clearly identified . Due to rapid population turnover vaccinated pigs are replaced quickly by non-immune young stock. To maintain the high degree of immunity required to smother foci of residual virus, supplementary vaccination of 8- to 10-week-old . and newly introduced piglets should be performed at monthly intervals . Of the 450 hog cholera outbreaks diagnosed in the Netherlands during the 1982-1985 epizootic, 63 occurred one month or more after the start of vaccination, i .e . when full protection should have been obtained . Twelve of these 63 were even detected between 9 and 13 months after the start of the vaccination campaign . Analysis of the outbreaks has shown that vaccination is more effective in fattening herds than in breeding and mixed herds, and that in the two latter categories, the efficacy is inversely related to the size of the herd . Apparently, large herds contain sufficient numbers of susceptible piglets for the virus to be perpetuated and to be spread at a low level . The infection lingers on in piglets of about the age of weaning and vaccination prevents the infection from developing into an overt outbreak . The monthly supplementary vaccination programmes therefore should be continued for at least 1 year after the start of a mass vaccination and preferably until 1 year after the last outbreak .
FUTURE DEVELOPMENTS An expansion of intracommunity movements of livestock, meat and meat products is to be expected after abolishing the trade barriers between EC countries in early 1993 . As a consequence, the risk of introducing notifiable diseases like hog cholera and African swine fever, which are present in some member states, will increase . In case of introduction, an early and rapid diagnosis is of the utmost importance in order to minimize losses .
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In recent years, new tools of research have become available for a rapid, sensitive, and specific detection of infectious agents . The advantages of monoclonal antibodies in hog cholera diagnosis have already been described, but other techniques such as the polymerase chain reaction (PCR) and flow cytometry should be mentioned as well . By using reverse transcription followed by the PCR it should be possible to amplify a single RNA fragment of the genome to concentrations either detectable in ethidium bromide stained gels or by probes . The technique is likely to detect the virus in tonsils, blood and other target organs at an earlier stage of infection than immunofluorescence or virus isolation . Flow cytometry is based on the quantitative measurement of fluorescence associated with single cells passing through a flow chamber . The method which has been described for detecting BVDV in blood of persistently viraemic cattle (ovist et al., 1990), would be extremely useful for tracing early HCV infections on suspected premises . If successful, the period of 5 weeks that has to elapse between a suspected contact and a meaningful seroepidemiological investigation could substantially be reduced . According to EC regulations only pigs and pig products from countries or regions with the `officially free' (OF) hog cholera status (i .e . no outbreaks and no vaccination in the last 12 months, and no vaccinated pigs present) have admission to the free trade within the community . The presently available vaccines against hog cholera do not allow differentiation by serological means between vaccinated pigs and pigs that have been exposed to wild strains of HCV . In the event of a hog cholera outbreak, the OF-status will temporarily be suspended, but the country or region will lose its status in case of vaccination . Therefore, eradicating the disease without resorting to vaccination is vital when the pig industry is based on exports . For this reason vaccination was not applied in Belgium during the 1990 hog cholera epizootic (113 outbreaks, one million pigs destroyed, direct losses US$ 270 million ; Westergaard, personal communication) . Vaccines inducing antibodies that can be distinguished from antibodies induced by field strains might remove the objection against vaccination . The way for constructing a serologically distinctive vaccine is open now that the nucleotide sequence and the location of the El gene, which encodes for the protection inducing protein, are known (Moormann et al., 1990) . As a matter of fact, the recombinant Aujeszky's disease virus expressing El protein of HCV (Van Zijl et al,, 1991) is the first product with this property, although its potential in comparison with existing hog cholera vaccines remains to be determined . Analysis of viral nucleic acid by means of PCR and nucleotide sequencing of certain amplified fragments might make it possible to associate genomic differences between field and vaccine strains with various biological properties . Identification of field isolates by these techniques may also prove a fruitful line of research that could shed more light on the epizootiology of the disease .
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(Accepted for publication 31 January 1991)
