SUMMARY

Pseudorabies virus (PRV) causes significant mortality and morbidity in swine, the natural host. Modified live vaccines with gene deletions are used worldwide to control the spread of PRV in swine. Little is known about the role of mammalian wildlife in the transmission of pseudorabies virus between swine herds, although it is believed that exposure to virulent wildtype PRV usually results in mortality. However, exposure of wildlife to PRV vaccine may result in immunization, enabling them to survive exposure to wildtype PRV, as well as allowing them to serve as hosts for recombination between wildtype and vaccine strains. Prior evidence indicates that raccoons can develop immunity after exposure to gene-deficient strains of PRV and thereafter survive challenge with virulent PRV. These issues were evaluated in a controlled experiment. The subjects were 16 wild-trapped raccoons. Each received one of 2 genetically engineered vaccines, administered intranasally and orally. One vaccine had a gene deletion for thymidine kinase (TK) and glycoprotein G (gG) and the other an additional deletion for glycoprotein E (gE). There were 4 doses levels for each vaccine: 10³, 10⁴, 10⁵, and 10⁶ TCID₅₀. Two raccoons were assigned to each treatment combination. The 21 day survival rate was 37.5% (3/8) for the gG^-TK^- vaccine; all of the survivors developed PRV antibodies. For the gG^-gE^-TK^- vaccine, there was a 100% 21 day survival rate; 75% of the survivors (all except the lowest dose) developed PRV antibodies. The survivors were challenged intranasally with a 3.2 x 10³ TCID₅₀ dose of the virulent wildtype PRV Shope strain. Two of the remaining 3 gG^-TK^- vaccinated raccoons survived the challenge; for the gG^-gE^-TK^- vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody levels were more likely to survive the challenge with the virulent PRV Shope strain; there was a 100% mortality rate for raccoons lacking detectable PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent wildtype PRV. Thus, raccoon populations are potential reservoirs for virulent PRV. Coinfection with vaccine and wildtype PRV also indicates that the potential exists for recombination between these strains.

Key words: Pseudorabies virus, vaccine, raccoons, immunization, genetic recombination

INTRODUCTION

Pseudorabies virus (PRV), also known as Aujeszky's disease virus, is an alphaherpesvirus, from the same subfamily as human Herpes simplex viruses (Nara, 1985). Swine are the primary host and reservoir for the pathogen. Infection of swine herds with PRV has serious economic consequences for pork producers, due to fetal and perinatal mortality, infertility, and reduced weight gain in finishing pigs (Gustafson and Scherba, 1978; Hoblet et al., 1987; Kliebenstein et al., 1988; Parsons et al., 1990; Bech-Nielsen et al., 1992). Infection of swine with PRV is persistent, in that subsequent to the acute phase of infection, PRV establishes a latent infection in the trigeminal ganglia; under conditions of immunosuppression, PRV may be reactivated, causing disease signs and viral shedding (Gustafson, 1986).

The United States Department of Agriculture Animal and Plant Health Inspection Service administers a nationwide program for eradication of PRV from swine herds. Vaccination is an integral part of PRV eradication programs in the United States (Thawley and Morrison, 1989) and worldwide (van Oirschot, 1989). Vaccination of swine with PRV vaccine reduces, but does not eliminate clinical signs and viral shedding (Donaldson et al., 1984). The most commonly used PRV vaccines are attenuated live viruses, genetically modified from wildtype virus strains by specific gene deletions (Table 1). Virulence is reduced in PRV vaccines by deletions of the genes for thymidine kinase [TK] and, in part, for glycoprotein E [gE] (Mettenleiter et al., 1989). The gE and gG deletions are the basis for the differential diagnostic assays, which specifically detect serum antibodies to gE or gG (Cook et al., 1990; van Oirschot et al., 1990; Schmitt et al., 1991; Weigel et al., 1992; Swenson et al., 1993). Use of diagnostics which test for antibodies to the missing glycoproteins allows differentiation of vaccinated swine from those which are infected with wildtype virus, aiding in the selective removal of animals infected with wildtype virus in eradication programs and in the certification of infection free herd status.

PRV, similar to other herpesviruses, has a high rate of genetic recombination (Christensen and Lomniczi, 1993). A major concern regarding the use of live gene-deleted PRV vaccines is that recombination of vaccine and wildtype virus may occur, resulting in a recombinant PRV that is virulent but missing the diagnostic marker gene (van Oirschot et al., 1990; Hahn, 1992; Vannier, 1992). Thus, recombination of wildtype and vaccine PRV could result in the spread of virulent PRV strains that are not detectable by standard serological tests used in surveillance and eradication programs. Analysis of the restriction fragment patterns of field isolates of PRV has suggested that natural recombination between PRV strains can occur, including recombination between vaccine and wildtype strains (Christensen and Lomniczi, 1993; Christensen et al., 1992). In addition, several experimental studies involving co-inoculation with wildtype and vaccine PRV have been able to recover recombinant PRV (Katz et al., 1990; Henderson et al., 1990; Henderson et al., 1991; Dangler et al., 1994).

Little is known about the environmental destination and consequences of circulation of PRV wildtype, vaccine, and vaccine-wildtype recombinant strains. Wildtype PRV is believed to spread between swine herds by movements of infected swine (McFerran et al., 1984; Gustafson, 1986), tracking by humans and vehicles (Austin et al., 1992), airborne transmission (Gloster et al. 1984; Christensen et al., 1990; Scheidt et al., 1991), and by wildlife vectors (Kirkpatrick et al., 1980). Initial reports on the role of wildlife in the transmission of PRV documented that disease outbreaks in swine were frequently accompanied by mortality in wild animals such as rats, raccoons and opossums (Maes et al., 1979; Kirkpatrick et al., 1980; Thawley and Wright, 1982; Goyal, 1986). Experimental studies of PRV infection in rats (Maes et al., 1979) and raccoons (Wright and Thawley, 1980) found that these wildlife hosts either died or failed to develop antibodies, suggesting these species were unlikely to be reservoirs for PRV. However, more recent experimental studies with rats (Le Moine et al., 1987) and raccoons (Kirkpatrick et al., 1980) have demonstrated that these species are able to mount an immune response and survive exposure to virulent PRV. Another study demonstrated that raccoons inoculated with an attenuated wildtype strain of PRV were able to develop immunity and survive subsequent challenge with a virulent PRV strain (Platt et al., 1983). In addition, several serological surveys of wildlife have identified the existence of natural reservoirs of PRV infection in raccoons (Platt et al., 1983; Cohen et al., 1989; Mitchell, 1996). None of the raccoons in these serological surveys had clinical signs of pseudorabies.

The experimental studies and epidemiologic surveys cited suggest that raccoons are capable of developing immunity to PRV and thus may serve as a reservoir for PRV infection. Swine vaccinated with live PRV vaccine may excrete vaccine virus, which could be transmitted to raccoons via aerosol droplets. If exposure to PRV vaccine produces an immune response, not only does the probability of establishment of a reservoir of PRV in raccoon populations increase, but coinfection with vaccine and wildtype PRV provides the prerequisite conditions for recombination within this raccoon reservoir.

The experiment described here evaluates the potential for immunization of raccoons after inoculation with genetically engineered live PRV vaccines, and their subsequent ability to survive challenge with virulent wildtype PRV.

MATERIALS AND METHODS

The subjects in this experiment were 16 wild-trapped raccoons. The first part of the experiment evaluated the response to vaccination in a 2 x 4 factorial design, with 2 raccoons in each treatment combination. There were 2 genetically engineered PRV vaccines, one with a gene deletion for thymidine kinase (TK) and glycoprotein G (gG) (PRV/Marker^® Blue; Syntrovet, Inc., Lenexa, KS) and the other including an additional deletion for glycoprotein E (gE) (PRV/Marker^® Gold; Syntrovet, Inc., Lenexa, KS). There were 4 dose levels for each vaccine: 10³, 10⁴, 10⁵, and 10⁶ TCID₅₀, each below the vaccine dose of approximately 10⁷ TCID₅₀ for swine. Vaccine was administered intranasally and orally.

Clinical signs were monitored daily. Animals showing severe respiratory congestion and pruritis characteristic of pseudorabies were euthanized. At days 0, 7, 14, 21, and 28 post-vaccination, blood samples were obtained. On day 28 post-vaccination, the survivors were challenged intranasally with a 3.2 x 10³ TCID₅₀ dose of the virulent wildtype PRV Shope strain. Blood samples were obtained 7 and 14 days after challenge with the virulent wildtype strain. On the 14th day, the animals were euthanized, and the trigeminal ganglia (the site of latent PRV infection) and the tonsils were removed. Antibodies to PRV were determined using the serum neutralization test (Hill et al., 1977).

RESULTS

The Kaplan-Meier survival functions (Lee, 1980) for raccoons receiving each vaccine are shown in Figure 1. Subsequent to vaccination, the 28 day survival rate was 100% (8/8) for the gG^-gE^-TK^- vaccine and 37.5% (3/8) for the gG^-TK^- vaccine; all mortality was within 6-9 days after the vaccine challenge. These differences between vaccines in survival time were statistically significant (2-tailed p < 0.01; Wilcoxon test); there was no apparent effect of vaccine dose on survival time. For the gG^-TK^- vaccine, PRV serum antibodies were not detected prior to death; all of the survivors had developed PRV antibodies. For the gG^-gE^-TK^- vaccine, 75% of the survivors (all except the lowest dose) developed antibodies. Table 2 indicates there was a tendency for higher vaccine dose to be associated with higher antibody titers ( = 0.50; 0.05 < p < 0.1; 2-tailed).

The following results were obtained for the challenge with the virulent wildtype Shope strain of PRV. Two of the remaining 3 raccoons vaccinated with the gG^-TK^- vaccine survived the challenge; for the gG^-gE^-TK^- vaccine, the survival rate was 50% (4/8). For the gG^-gE^-TK^- vaccine, the raccoons with higher vaccine-induced antibody levels were more likely to survive the challenge with the virulent PRV Shope strain (Wilcoxon test: 2 tailed p = 0.096); there was a 100% mortality rate for raccoons lacking detectable PRV antibodies.

DISCUSSION

This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent wildtype PRV. Exposure to low doses of vaccine is unlikely to produce immunity, and high doses may cause mortality. Immunity and mortality are also dependent upon vaccine virulence. For raccoons that survive the initial vaccine challenge, higher vaccine doses are associated with higher levels of antibody produced, and higher antibody levels are associated with a higher survival rate when exposed to virulent wildtype PRV.

With rare exceptions, use of the gG^- PRV vaccines has been discontinued in the US, primarily because of perceived low sensitivity of the gG diagnostic assays (Schmitt et al., 1991; Weigel et al., 1992; Hahn, 1992). Many swine farms previously using gG^- vaccines have switched to using the less virulent gG^-gE^- vaccines. This change may increase the probabilty of survival of wildlife upon exposure to PRV vaccine. However, the lower virulence of the gG^-gE^- vaccine may also decrease the probability of immunization of wildlife populations.

This experiment demonstrates that raccoons can be immunized against PRV, and thus can serve as a reservoir population for PRV infection. Co-infection of raccoons with vaccine and wildtype strains of PRV can occur, and thus the prerequisite conditions for recombination of PRV strains exists in raccoons.

Further analyses planned include attempted isolation of PRV from the tonsils and typing of virus using restriction endonuclease analysis, comparing isolated virus samples to the vaccine and wildtype strains to which the raccoons were exposed in this study.

Field studies have also been initiated for 5 Illinois swine farms infected with PRV that are using modified live gene-deleted PRV vaccines that are adminstered intranasally. Wildlife are being trapped and tested for PRV infection. This study will determine whether reservoirs of wildtype, vaccine, and wildtype-vaccine recombinant PRV can be detected in wildlife populations whose home ranges include commerical swine production facilities.

ACKNOWLEDGEMENT

This research was supported by the Biotechnology Risk Assessment Research Program of the United State Department of Agriculture Cooperative States Research Service.

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Table 1. Gene-deleted pseudorabies virus live vaccines used in the United States (1989-present)

Vaccine Name

Gene Deletions gE gC gG TK

Virulence of Source Strain

PR Vac	X				attenuated
Bioceutic	X			X	virulent
Omnivac	X			X	attenuated
OmniMark	X	X		X	attenuated
Tolvid			X	X	virulent
PRV Marker Blue			X	X	virulent
PRV Marker Gold	X		X	X	virulent

Table 2. Association of vaccine dose with pseudorabies virus antibody titer: gG^-gE^-TK^- vaccine.

Antibody Titer* Vaccine Dose Negative 4 8 16 32 64

103	ÖÖ
104			ÖÖ
105					Ö	Ö
106		Ö		Ö

*Each Ö represents one raccoon.

Figure 1. Kaplan-Meier survival curves for raccoons given each vaccine, not differentiated by dose, after vaccine and wildtype pseudorabies virus challenge