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Worldwide, failure to eradicate a disease in livestock has sometimes been related to wildlife reservoirs of infection. We describe the effects of Eurasian wild boar (Sus scrofa) abundance reduction through increased culling on the prevalence of two chronic infectious diseases, tuberculosis (TB) and Aujeszky's disease (AD), in a region of South-central Spain (SCS). The two infections studied responded differently to an approximately 50% reduction of wild boar abundance. Wild boar TB prevalence remained stable in control sites, whereas it decreased by 21-48% in treatment sites. In one treatment site, the annual wild boar abundance was positively correlated with the annual percentage of skin test reactor cattle. In another treatment site, red deer (Cervus elaphus) M. bovis infection prevalence decreased after culling wild boar. No significant effect of wild boar culling on wild boar ADV seroprevalence was found. The reduction in wild boar TB was achieved despite no alternative M. bovis host being included in the culling strategy. We advocate that culling could become a part of integrated control strategies including habitat and game management changes and vaccination, contributing to increase their success likelihood, or reducing the total expenses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The effect of a bacterial infection on interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) production by porcine cells was studied in specific pathogen-free (SPF) pigs, infected intranasally with Actinobacillus pleuropneumoniae serotype 2. Three experimental groups of five pigs were used: infected non-treated pigs, infected pigs that were treated with enrofloxacin at disease onset, and non-infected, non-treated control pigs. Blood samples were collected from all pigs on the day of infection and on days 1, 4, 7, 13 and 17 post-infection. Sera were analysed for presence of antibodies to A. pleuropneumoniae and for the cytokines IL-6 and IFN-alpha. Ability to produce these cytokines was tested in vitro using whole blood cultures stimulated with inactivated virus (Aujeszky's disease virus infected porcine kidney cells (ADV/PK-15)), inactivated bacteria (A. pleuropneumoniae) or bacterial plasmid (pcDNA3). All cytokine inducers were used neat or pre-incubated with the transfectious agent lipofectin. IL-6 appeared in the serum of all infected non-treated animals but no IFN-alpha was found in the serum of any of the experimental pigs. Accordingly, the bacteria induced a substantial IL-6 but hardly any IFN-alpha production when tested in vitro. However, following incubation with lipofectin, the inactivated bacteria as well as pcDNA3 became efficient inducers of IFN-alpha in whole blood cultures. The increased IFN-alpha production, previously recorded in vitro during the acute phase of infection with A. pleuropneumoniae, was confirmed using lipofected plasmid DNA and it was indicated that leukocytes obtained from infected but apparently cured animals also exhibited an increased production of IFN-alpha. Thus, even mild/sub-clinical bacterial infections may affect cytokine production in pigs.

Sixteen 20 day old pigs, devoid of neutralizing antibody to pseudorabies virus (PRV), were divided into two groups of eight, an the animals of each group were housed in a separate unit. In each group 6 pigs were inoculated intranasally with the thymidine kinase (TK-) mutant (Group 1) or the field strain of PRV (Group 2), each pig receiving an inoculum of 4 ml. The remaining 2 pigs in each group served as uninoculated controls. The only clinical sign observed in the pigs of Group 1 was a transient febrile reaction, in the case of six pigs inoculated with the TK- mutant of PRV, whereas no signs of disease were seen in the uninoculated controls. The virus was isolated from the 6 infected pigs of the group only on post infection day (PID) 2, whereas it was never isolated from the controls. By contrast, the pigs of Group 2, had a severe clinical response and one, among those that were inoculated with the field strain of the PRV, died on PID 9. Virus was consistently isolated from all pigs of Group 2, inoculated and control. On PID 30 all pigs, i.e. the 8 of Group 1 and 7 of the Group 2 which survived to the infection, were subjected to dexamethasone (DMS) treatment. After DMS treatment virus was never isolated from the nasal swabbings obtained from the pigs of Group 1, whereas it was consistently isolated from pigs of Group 2. After 30 d from the start of DMS treatment the pigs were killed and several tissues were collected from each pig for virus detection, by isolation in tissue culture and by PCR analysis. At necropsy no lesions were found in pigs of Group 1, whereas acute pneumonia and gliosis in the trigeminal ganglia were observed in pigs of Group 2. Virus was never isolated from any of the tissues taken from pigs of both, Group 1 and Group 2, nevertheless sequences of PRV were detected by PCR analysis in the trigeminal ganglia of the pigs of both Groups.

Herpesvirus envelope glycoproteins play important roles in mediating infection initiation and also represent major immunogens. We recently showed that a pseudorabies virus (PrV) mutant lacking the essential glycoprotein gD (gp50), after phenotypic complementation by propagation on genetically engineered PrV gD-expressing cell lines was able to infect primary target cells and spread exclusively by means of direct cell-to-cell transmission. Virions released from non-complementing cells that lacked gD were not infectious because of a defect in penetration and so free infectious virions did not arise after infection of animals by phenotypically complemented gD-negative PrV. This formed the basis for the development of novel non-spreading live herpes-virus vaccines. However, the gD-negative PrV mutant still retained a residual level of virulence, which prevented its use as vaccine, and the need to propagate the gD-negative PrV mutant on trans-complementing cell lines resulted in the appearance of wild-type revertants, rescued by the resident gene in the cell line. To overcome these problems we isolated a PrV mutant designated PrV(376) that, in addition to gD, also lacked the non-essential glycoproteins gG, gI and gE. PrV(376), because of the lack of gD, was also dependent on gD-expressing cells for productive replication. Non-complementing cells infected by phenotypically gD-complemented PrV(376) produced non-infectious particles lacking glycoproteins gD and gE as shown by immunoelectron microscopy. Owing to the absence of any homologous sequences between the viral genome and the viral genes resident in the complementing cell line, rescue by homologous recombination was impossible. In cell culture, plaques of PrV(376) were significantly smaller than those of either wild-type, or gD- or gE-deleted mutants, indicating an additive or synergistic effect of the combined deletion on viral cell-to-cell spread capability. Intranasal or intramuscular infection of pigs with phenotypically gD-complemented PrV(376) showed a complete attenuation of viral virulence, with an expected lack of shedding of infectious virus. The PrV(376)-vaccinated pigs exhibited a significant level of protection against challenge infection, measured by survival and weight loss. In summary, PrV(376) represents a novel type of herpesvirus vaccine that combines innocuity, efficacy and biological safety.

Six HPCD (hysterectomy-produced, colostrum-deprived) pigs were inoculated endobronchially with pseudorabies virus (PRV) in the right caudal lobe by means of a bronchoscope. Two pigs, killed on days 5 and 7, had severe purulent pneumonia in the right caudal lobe, associated with an accidental Haemophilus parasuis serovar 4 infection. The three surviving animals were treated with antibiotics. The pigs infected with PRV had necrotizing bronchiolitis and alveolitis. PRV antigen was closely associated with necrotic foci, and was sometimes surrounded by profuse H. parasuis antigen. PRV antigen and IgG- and IgA-containing cells were also detected in bronchioalveolar lavage fluid. These results suggested that the PRV infection destroyed respiratory epithelial cells and allowed H. parasuis to proliferate in the lungs.

Pseudorabies virus was isolated in cell culture from the brain tissue of a 3.5-year-old male Florida panther (Felis concolor coryi). The virus was not isolated from other tissues collected at necropsy. Based upon a nested polymerase chain reaction (PCR), the virus was determined to have the classical wild-type virulent genotype, glycoprotein I+ (gI+) and thymidine kinase+ (TK+).

A polymerase chain reaction (PCR) amplification assay was developed for the detection of Aujeszky's disease virus (ADV) DNA in cell cultures and clinical samples. Pigs vaccinated with commercial ADV vaccines and challenged with a field isolate of ADV were immunosuppressed by dexamethasone treatment. Nasal swabs collected from the pigs at various times post-immunosuppression showed that ADV was excreted for at least four to six days starting from day 8 or day 10 following dexamethasone treatment, by virus isolation and/or PCR. However, PCR only detected latent ADV in the trigeminal ganglia, mandibular lymph node, spleen and tonsils, but not in the brain stem, pons and olfactory lobe of two pigs following dexamethasone treatment, whereas tissue explanation and cocultivation failed to demonstrate the presence of the virus.

Pigs inoculated endobronchially (EB) with 2 ml of virus suspension containing 10(4) TCID50 per ml of the YS-81 strain of pseudorabies virus (PRV), by means of a bronchoscope, all developed viral pneumonia. No pneumonic lesions were observed in intranasally inoculated pigs. Macroscopical and microscopical lesions were localized to the middle to caudal parts of the right caudal lobe and were closely associated with the site at which the inoculum was deposited. PRV became attached to all types of cells and caused destruction of epithelial cells, and viral antigen persisted in the alveolar macrophages. After PRV infection, the total cell number in broncho-alveolar lavage (BAL) fluid was slightly increased and a high titre of PRV was found in the cells of BAL fluid in EB infected pigs. The findings suggest that PRV infection leads to dysfunction of alveolar macrophages before cell death is produced by virus replication.

The virulence, pathogenicity and immunogenicity of two pseudorabies virus (PRV) variants were investigated in 3-week-old pigs that had been intranasally infected. Variant M303 (delta 125,126) lacked amino acids valine (125) and cysteine(126) in an immunodominant antigenic region of glycoprotein I (gI) containing two discontinuous antigenic domains, whereas M304 (delta 59,60) lacked amino acids glycine(59) and aspartic acid(60) in a continuous antigenic domain. M303 (delta 125,126) was not virulent for pigs, but M304 (delta 59,60) was as virulent as wild-type PRV: all pigs died within 8 days of infection. Both gI mutant viruses replicated in the oropharyngeal mucosa, although M304 (delta 59,60) replicated to higher virus titres than M303 (delta 125,126), and virus was recovered from various tissues. However, in contrast to M304 (delta 59,60), M303 (delta 125,126) was not recovered from any central nervous system (CNS) tissues examined. Thus, the tendency of PRV to locate in the CNS was markedly reduced by deleting amino acids valine(125) and cysteine(126) of gI. Pigs immunized with M303 were completely protected against challenge infection; no clinical signs of disease were detected, no virus was shed, and no secondary antibody response was detected. Thus, deleting amino acids valine(125) and cysteine(126) in gI decreases virulence and neurotropism and does not affect immunogenicity.

Retrograde transneuronal viral labelling and immunocytochemical methods were used for revealing neuronal networks controlling the left ventricle myocardium of the rat heart. After injections of 1 microliter pseudorabies virus solution (3 x 10(6) PFU ml-1) into the left ventricle, infected orthosympathetic preganglionic cells were found in the intermediolateral cell groups of the first 6 thoracic spinal segments. Preganglionic parasympathetic neurones were seen both in the nucleus ambiguus/retro-ambiguus area and the dorsal motor vagus nucleus. Large numbers of infected projecting interneurones were found in the rostral, caudal and medial parts of the ventral medulla oblongata, the Kölliker-Fuse nucleus and catecholaminergic cell group A5 and in the paraventricular hypothalamic nucleus.

The propagation of pseudorabies virus (PrV) mutants deficient in essential glycoproteins gp50 and gII was studied after inoculation of transcomplemented gp50- and gII- PrV into the motor hypoglossal (XII) nerves of mice. In this model, viral spread from the infected XII motoneurons involves specific transneuronal transfer to connected cells and local, nonspecific transfer. For comparison, a PrV mutant lacking the nonessential nonstructural glycoprotein gX was included. Although the efficiencies of first-cycle replication were similar for the three viruses, only gX- and gp50- progeny mutants could spread from XII motoneurons via transneuronal and local transfer. The extents of transfer of gX- and gp50- PrV were comparable. The results show that the absence of gp50 does not alter the pattern of transneuronal or local spread of PrV, whereas gII is essential for both processes.

Pseudorabies virus (PRV) has been used extensively to map synaptic circuits in the CNS and PNS. A fundamental assumption of these studies is that the virus replicates within synaptically linked populations of neurons and does not spread through the extracellular space or by cell-to-cell fusion. In the present analysis we have used electron microscopy to characterize pathways of viral replication and egress that lead to transneuronal infection of neurons, and to document the non-neuronal response to neuronal infection. Three strains of PRV that differ in virulence were used to infect preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). The data demonstrate that viral replication and transneuronal passage occur in a stepwise fashion that utilizes existing cellular processes, and that the non-neuronal response to infection serves to restrict nonspecific spread of virus by isolating severely infected neurons. Specifically, capsids containing viral DNA replicate in the cell nucleus, traverse the endoplasmic reticulum to gain access to the cytoplasm, and acquire a bilaminar membrane envelope from the trans cisternae of the Golgi. The outer leaf of this envelope fuses with the neuron membrane to release virus adjacent to axon terminals that synapse upon the infected cell. A second fusion event involving the viral envelope and the afferent terminal releases the naked capsid into the bouton. Systematic analysis of serial sections demonstrated that release of virus from infected neurons occurs preferentially at sites of afferent contact. Nonspecific diffusion of virus from even the most severely infected cells is restricted by astrocytes and other non-neuronal elements that are mobilized to the site of viral infectivity. The ability of glia and macrophages to restrict spread of virus from necrotic neurons is the product of (1) temporal differences in the mobilization of these cells to the site of infection, (2) differential susceptibility of these cells to PRV infection, and (3) abortive viral replication in cells that are permissive for infection. The findings provide further insight into the intracellular routes of viral assembly and egress and support the contention that transneuronal spread of virus in the brain results from specific passage of virions through synaptically linked neurons rather than through cell fusion or release of virus into the extracellular space.

Latency represents a challenge for any herpesvirus vaccine. Vaccines could differ in their ability to minimize latency of pseudorabies virus (PRV). To study this possibility, a quantitative and differential PRV-specific polymerase chain reaction (PCR) was developed by the authors. Efficiency of amplification in different tubes is measured by co-amplification with the viral targets (either gI or gp50 genes) of the porcine gene nuclear factor 1. The criteria used to select this approach to a quantitative PCR, as well as for the selection of the standard, are discussed. The amplified products are measured by Southern blot or, alternatively, high performance liquid chromatography. Sensitivity and reproducibility of the technique are evaluated. The ability of this technique to discriminate between the level of latency established by two different PRV strains in trigeminal ganglia is also evaluated. Using this technique, the authors are currently studying whether different vaccines could possess differing levels of ability to minimize, by interference, establishment of wild-type latency.

Tissue homogenates were obtained from swine co-infected with 2 vaccine strains of pseudorabies virus (PRV). Viral isolates derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures, were characterized as recombinant and parental PRV genotypes on the basis of thymidine kinase and glycoprotein X gene combinations. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant PRV. The thymidine kinase and glycoprotein X gene sequences were classified as wild-type or deleted, using a battery of polymerase chain reaction assays. Results substantiate the observation that PRV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection.

A retrospective analysis of DNA restriction fragment length polymorphism (RFLP) of Aujeszky's disease (AD) virus isolates from England and Wales over a 22-year period revealed considerable homogeneity of BamHI restriction endonuclease sites, both in number and size. The appearance of a new DNA RFLP in 1981 coincided with a marked increase in the number of new outbreaks of AD. A polymerase chain reaction (PCR) method was developed to discriminate between the established strains and the new type. By relating these results to pig movement records it was possible to trace the spread of the new type virus which was isolated from 39/60 (65%) of the new outbreaks of AD in England and Wales in 1982. The study suggested that airborne spread of up to 17 km was the most likely route of transmission in many of the new type cases. The discriminatory PCR was shown to be a useful tool for the rapid and sensitive detection of latent AD virus in pigs.

Phenotypically complemented pseudorabies virus gp50 null mutants are able to produce plaques on noncomplementing cell lines despite the fact that progeny virions are noninfectious. To determine whether gp50 null mutants and gp50+gp63 null mutants are also able to replicate and spread in animals, mice were infected subcutaneously or intraperitoneally. Surprisingly, both gp50 mutants and gp50+gp63 double mutants proved to be lethal for mice. In comparison with the wild-type virus, gp50 mutants were still highly virulent, whereas the virulence of gp50+gp63 mutants was significantly reduced. Severe signs of neurological disorders, notably pruritus, were apparent in animals infected with the wild-type virus or a gp50 mutant but were much less pronounced in animals infected with a gp50+gp63 or gp63 mutant. Immunohistochemical examination of infected animals showed that all viruses were able to reach, and replicate in, the brain. Examination of visceral organs of intraperitoneally infected animals showed that viral antigen was predominantly present in peripheral nerves, suggesting that the viruses reached the central nervous system by means of retrograde axonal transport. Infectious virus could not be recovered from the brains and organs of animals infected with gp50 or gp50+gp63 mutants, indicating that progeny virions produced in vivo are noninfectious. Virions that lacked gp50 in their envelopes, and a phenotypically complemented pseudorabies virus gII mutant (which is unable to produce plaques in tissue culture cells), proved to be nonvirulent for mice. Together, these results show that gp50 is required for the primary infection but not for subsequent replication and viral spread in vivo. These results furthermore indicate that transsynaptic transport of the virus is independent of gp50. Since progeny virions produced by gp50 mutants are noninfectious, they are unable to spread from one animal to another. Therefore, such mutants may be used for the development of a new generation of safer (carrier) vaccines.

We investigated the function of antigenic domains on gI in virulence and immunogenicity. Three PRV gI mutants were constructed by deleting nucleotides coding for the following amino acids: valine-125 and cysteine-126, located in a discontinuous antigenic domain (M 303); glycine-59 and aspartic acid-60 located in a continuous antigenic domain (M304); and arginine-67 and alanine-68, located in a discontinuous antigenic domain (M305). Mismatch primers in the polymerase chain reaction were used to introduce the deletions. Anti-gI monoclonal antibodies were used in an immunoperoxidase monolayer assay to distinguish PRV gI mutants from wild-type PRV. The gI mutant viruses were tested for their growth in vitro and for their virulence in mice. The growth properties of PRV gI mutant virus M303 were comparable to the growth properties of a PRV gI-negative mutant (M301): both mutants produced small plaques in various cells, and when grown on swine kidney cells and chicken embryo fibroblasts, their growth was disadvantaged compared to wild-type PRV. However, in embryonal Balb/c mouse cells expressing gI, gI mutant viruses and wild-type PRV produced plaques of the same size, confirming that the mutations in gI are responsible for the small plaque phenotype. The growth properties of PRV gI mutant viruses M 304 and M 305 were comparable to the growth properties of wild-type PRV. When the mean time to death was used as the criterion, the gI mutant viruses M 301 and M 303 were significantly less virulent in mice than wild-type PRV. Four other, independently obtained, PRV mutants all carrying the valine-125 and cysteine-126 deletion (M 308, M 309, M 310 and M 311 respectively) exhibit the same phenotype. Our results show that deleting valine-125 and cysteine-126 in gI decreases plaque size and reduces virulence in mice to the same degree as deleting the gI protein.

Examples are given of observations made with field isolates of suid herpesvirus 1 (SHV-1) which indicate that intergenomic recombination is a common phenomenon associated with the virus. This was further confirmed by experimental co-infection of a pig with 2 virus strains with different, stable and easily identifiable genomic markers, followed by natural transmission to a group of contact pigs. A variety of recombinants was subsequently isolated, while none of the parental strains were re-isolated from any of the pigs. It is suggested that co-invasion of cells and recombination between viral genomes play a role in the life cycle of the virus.

In this study the role of different genes located in the unique short region of the genome of Aujeszky's disease virus was examined. Inactivation of the genes encoding the protein kinase (PK), gp63, and gI reduced virulence of the virus for pigs, in contrast to inactivation of the genes encoding the 28 kDa protein, and gX. There was no correlation between virulence and virus multiplication in vitro or in the oropharynx in vivo. The morphogenesis of the PK mutant was altered. The gI mutant replicated to normal titres in the oropharynx and could be recovered from the trigeminal ganglia but not from other parts of the central nervous system, suggesting that gI facilitates the spread of the virus from neuron to neuron. All mutants induced neutralizing antibody and complete or partial protection against a challenge infection. PK and gp63 were required for the induction of complete protection, although these proteins are reportedly not targets for neutralizing antibody or cytotoxic T cells.

Various procedures of vaccination for pseudorabies were compared for their effects on shedding, latency, and reactivation of attenuated and virulent pseudorabies virus. The study included 6 groups: group 1 (10 swine neither vaccinated nor challenge-exposed), group 2 (20 swine not vaccinated, but challenge-exposed), and groups 3 through 6 (10 swine/group, all vaccinated and challenge-exposed). Swine were vaccinated with killed virus IM (group 3) or intranasally (group 4), or with live virus IM (group 5) or intranasally (group 6). The chronologic order of treatments was as follows: vaccination (week 0), challenge of immunity by oronasal exposure to virulent virus (week 4), biopsy of tonsillar tissue (week 12), treatment with dexamethasone in an attempt to reactivate latent virus (week 15), and necropsy (week 21). Vaccination IM with killed or live virus and vaccination intranasally with live virus mitigated clinical signs and markedly reduced the magnitude and duration of virus shedding after challenge exposure. Abatement of signs and shedding was most pronounced for swine that had been vaccinated intranasally with live virus. All swine, except 4 from group 2 and 1 from group 4, survived challenge exposure. Only vaccination intranasally with live virus was effective in reducing the magnitude and duration of virus shedding after virus reactivation. Vaccination intranasally with killed virus was without measurable effect on immunity. Of the 55 swine that survived challenge exposure, 54 were shown subsequently to have latent infections by use of dexamethasone-induced virus reactivation, and 53 were shown to have latent infections by use of polymerase chain reaction (PCR) with trigeminal ganglia specimens collected at necropsy. Fewer swine were identified by PCR as having latent infections when other tissues were examined; 20 were identified by testing specimens of olfactory bulbs, 4 by testing tonsil specimens collected at necropsy, and 4 by testing tonsillar biopsy specimens. Eighteen of the 20 specimens of olfactory bulbs and 3 of the 4 tonsil specimens collected at necropsy in which virus was detected by PCR were from swine without detectable virus-neutralizing antibody at the time of challenge exposure. One pig that had been vaccinated intranasally with live virus shed vaccine virus from the nose and virulent virus from the pharynx concurrently after dexamethasone treatment. Evaluation of both viral populations for unique strain characteristics failed to provide evidence of virus recombination.

Pigs exposed to fluctuating temperatures (high, 30 +/- 2 degrees C; low, 4 +/- 1 degrees C) were intranasally inoculated with Aujeszky's disease virus (ADV). ADV-infected pigs, exposed to the fluctuating temperatures, showed severe clinical signs and ADV in the nasal secretions persisted longer than in the ADV-infected control pigs kept at the normal temperature (20 +/- 2 degrees C). High concentrations of ADV were isolated from nasal secretions on the 1st day after inoculation of the virus. Pathologically, all ADV-infected pigs had non-suppurative encephalitis and trigeminal ganglionitis. The lesions were more widely distributed in pigs exposed to fluctuating temperatures than in infected control pigs. Two infected pigs given the stress had severe malacic foci in the frontal lobe and four of them had prominent interstitial pneumonia. In the pigs exposed to fluctuating temperatures, a significant number of immunoglobulin-containing cells, especially IgM-containing cells, did not respond to ADV infection. A significant (P < 0.01) difference in the number of IgG- and IgM-containing cells was observed between the ADV-infected pigs exposed to the fluctuating temperature and ADV-infected control pigs, respectively. These results demonstrated that the stress of fluctuating temperatures enhanced the susceptibility to ADV infection.

Twenty four cloned isolates of Aujeszky's disease virus collected from outbreaks of Aujeszky's disease from 1981 through 1989 in Japan were characterized by their restriction endonuclease (RE) cleavage patterns, virulence for mice and thymidine kinase (TK) activity. All of the isolates belonged to Type II of the four types classified by Herrmann et al. (1984). Based on the number and migration rate of the restriction fragments, the isolates were divided into 7 groups with Bam HI, 9 groups with Kpn I, 3 groups with BstE II and 2 groups with Sal I. The results indicate that the RE analysis, especially with Bam HI and Kpn I, provides useful epidemiological information about field isolates of Aujeszky's disease virus. All of the isolates showed virulence for mice ranging from 6.9 to 63.0 (PFU/LD50). It was interesting that the Nagano S87 strain, which had the highest virulence for the mouse, showed unique RE cleavage patterns with four enzymes. On the other hand, ara-T-resistant, TK-negative strain, was avirulent for mice (greater than 10(6.4) PFU/LD50). All of the isolates investigated in this study showed TK activity by the thymidine plaque autoradiography.

The RC/79 strain of the Aujeszky's disease virus was able to induce reproductive failure of pregnant gilts intranasally inoculated at different gestation periods. Four gilts 40-46 days pregnant (group A) and 6 gilts 70-73 days pregnant (group B) were instilled with 0.2 ml x 10(5) tissue culture infectious dose 50 (TCID50/0.2 ml) of the RC/79 strain into each nostril. Two gilts 70-73 days pregnant (group C) were used as non exposed controls. The three groups were kept in separated boxes and they were observed for clinical signs of infections and samples were collected for determination of viral shedding every day. Viral isolation was attempted in Vero cells (figure 1). From the 2nd to 7th day after inoculation, groups A and B showed fever anorexia, sneezing, coughing and depression; and viral isolation from nasal swabs was possible in 7 gilts at days 4 to 11, 9 gilts developed neutralizing antibodies. The virus caused fetal reabsorption in swine during the first period of pregnancy (group A), while infection during late pregnancy resulted in still birth or normal pigs and one mummification (group B). The entire a live litter was composed of no more than 8 suckling pigs in both groups. At necropsy virus from turbinates, ovary , placenta, spleen and lung could be isolated only from 3 gilts (group B, table 1). In 5 of 35 stillbirth and alive fetuses virus could be isolated from spleen (100%), lung (80%), liver (60%) and brain (40%) indicating that the virus has the ability to cross the placental barrier thus producing lesions in porcine fetuses and causing reproductive failure in sows (table 2). Tissue specimens from these 35 fetuses were fixed in 10% formalin, included in paraffin sectioned and stained with hematoxylin and eosin. In 13 fetuses microscopic lesions i.e. necrotic foci were found in lung (60%), liver (40%) and spleen (20%), these alterations were coincident with gross lesions in most of them. Inclusion bodies were absent. The gilts organs did not present gross lesions.

Pregnant sows, immune against pseudorabies after vaccination, were inoculated at 70 days of gestation either with autologous blood mononuclear cells that had been infected in vitro with pseudorabies virus (PRV) or with cell-free PRV. The infected cells or cell-free PRV were inoculated surgically into the arteria uterina. Eight sows (A to H) had been vaccinated with an inactivated vaccine. The titer of seroneutralizing antibodies in their serum varied between 12 and 48. Five sows (A to E) were inoculated with autologous mononuclear cells, infected either with a Belgian PRV field strain or with the Northern Ireland PRV strain NIA3. These 5 sows aborted their fetuses: 2 of them (B and C) 3 days after inoculation, and the other 3 (A, D, and E) 10, 11, and 12 days after inoculation, respectively. Sows F, G, and H were inoculated with a cell-free PRV field strain. They farrowed healthy litters after normal gestation. Neutralizing antibodies were absent against PRV in the sera of the newborn pigs, which were obtained prior to the uptake of colostrum. The 23 fetuses that were aborted in sows B and C 3 days after the inoculation were homogeneous in appearance and size. Foci of necrosis were not detected in the liver. Viral antigens were located by immunofluorescence in individual cells in lungs, liver, and spleen of 15 fetuses. Virus was isolated from the liver, lungs, or body fluids of 12 fetuses. The 39 fetuses that were aborted in sows A, D, and E between 10 and 12 days after inoculation were of 2 types: 17 were mummified and 22 were normal-appearing.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutants of pseudorabies virus defective in either glycoprotein gI or gIII are only slightly less virulent for mice and chickens than is wild-type virus, while mutants defective in both gI and gIII are avirulent. To clarify the reason for the lack of virulence of the gI- gIII- mutants, we have analyzed in some detail the interactions of these mutants with their hosts. The results obtained showed that the gI glycoprotein is an accessory protein that promotes cell fusion. This conclusion is based on the findings that in some cell types, syncytium formation is significantly reduced in mutants deficient in gI. Furthermore, despite efficient replication, gI- mutants form significantly smaller plaques on some cell types. Finally, while wild-type and gI- virus are neutralized similarly by antisera, the size of the plaques formed by gI- mutants, but not by wild-type virus, is reduced by the presence of neutralizing antibodies in the overlay. Passive immunization of mice with neutralizing antipseudorabies virus sera is also considerably more effective in protecting them against challenge with gI- mutants than in protecting them against challenge with wild-type virus. These results show that gI- mutants are deficient in their ability to form syncytia and to spread directly by cell-to-cell transmission and that these mutants spread mainly by adsorption of released virus to uninfected cells. Wild-type virus and gIII- mutants, however, spread mainly via direct cell-to-cell transmission both in vivo and in vitro. We postulate that the lack of virulence of the gIII- gI- virus is attributable to its inability to spread by either mode, the defect in gIII affecting virus spread by adsorption of released virus and the defect in gI affecting cell-to-cell spread. Although a gI- gIII- mutant replicates as well as a gIII- mutant, it will be amplified much less well. Our results with in vitro systems show that this is indeed the case.

Pigs (9 [+/- 1] weeks old) were inoculated with Streptococcus suis type 2, pseudorabies virus (PRV), or both. For each pig of groups A, B, and C the inoculum of S suis was 10(9) colony-forming units. For each pig of groups A, B, and D the inoculum of PRV was 5 x 10(3) TCID50 of either PRV strain 4892 (group A, n = 9) or PRV isolate B (group B, n = 9). The PRV strain 4892 is a highly virulent strain; isolate B causes mild clinical signs of infection in inoculated pigs. Group-C pigs (n = 9) were given S suis alone, and group-D pigs (n = 3) were inoculated only with PRV isolate B. Clinical signs of infection and development of lesions were readily seen in pigs of groups A, B, and C. Duration and severity of clinical signs of disease and lesions were reduced in pigs of group C, compared with those of the other 2 groups. Lesions, such as polyarthritis and fibrinous pericarditis, were more abundant and acute in the groups of pigs given mixed challenge exposure, compared with pigs inoculated exclusively with S suis type 2 (group C). The group of pigs inoculated with PRV isolate B alone did not manifest clinical signs of disease or lesions. Average daily gain for group-C pigs was higher, compared with that of other groups; the difference was statistically significant at P less than 0.02 and P less than 0.05 for groups B and D, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus isolation, immunofluorescent staining and DNA probe hybridization, three techniques for the detection of pseudorabies virus (PRV) have been compared in pigs experimentally infected with the Thailand CB-1 strain PRV. The virus isolation and DNA hybridization detection demonstrated a good correlation in detecting infection in live animals by nasal swabbing. In white blood cells an earlier detection was seen with the DNA-hybridization techniques. Consistent results with all the three techniques tested were found in organ materials as nasal mucosa and tonsils as well as in the olfactory bulb.

Pigs (3 and 10 weeks old) were infected intranasally with Aujeszky's disease virus (ADV) mutants that functionally lacked one of the non-essential genes in the unique short region of the genome (except the gene encoding the 11K protein). Virus excretion in oropharyngeal fluid and disease symptoms were monitored. Some pigs were killed to study pathogenesis, whereas others were challenged with virulent ADV 8 weeks after the primary infection. Mutants lacking protein kinase, or glycoproteins gp63 or gI showed reduced virulence, but mutants lacking gX or the 28K protein showed normal virulence. Glycoprotein gI appears to affect the tissue tropism of ADV in pigs, presumably by facilitating the spread of the virus through the central nervous system. In this study, there was no correlation between virulence and virus multiplication in either cultured cells or in the oropharynx in vivo. All mutants induced neutralizing antibody and complete or partial protection against challenge infection. Complete protection was obtained by inoculation with the gI and gX mutants, whereas incomplete protection was obtained using gp63 and protein kinase mutants. Complete clinical and virological protection was associated with the absence of secondary antibody responses in the serum.

Pigs with low levels of maternally derived antibodies were vaccinated twice intramuscularly with 10(5), 10(5.5), or 10(6) plaque forming units (p.f.u.) of the genetically engineered Aujeszky's disease virus (ADV) vaccine strain 783. Strain 783 has deletions in genes encoding glycoprotein gI and thymidine kinase. All vaccinated pigs showed a high level of protection against clinical disease after challenge infection with virulent ADV. Vaccination also reduced virus excretion. The daily mean virus excretion and the mean number of days with virus excretion, fever, mild clinical signs, and growth retardation were higher in pigs vaccinated with 10(5) than in pigs vaccinated with 10(5.5) or 10(6) p.f.u. of strain 783.

Eleven groups of 8 pigs were vaccinated with different vaccinal strains of pseudorabies virus deleted or not for non-essential glycoproteins (gI, gX, gp 63), then were challenged 8-9 weeks later with a virulent strain. Antibodies against the major viral glycoproteins (gII, gIII, gp 50) were titrated at the day of challenge. Excretion of the challenge strain, growth performance of the pigs, and gI antibody responses from pigs vaccinated with a gI minus strain, were monitored after the challenge. The results demonstrated a strong association between gII and gIII antibody titres before the challenge, with the amount of the challenge strain excreted and clinical protection after the challenge. Furthermore, gI antibody titres after the challenge were negatively correlated with the level of gII and gIII antibodies at the day of challenge, the virus excretion and the clinical protection after the challenge. Implications of these results for vaccination of pigs and gI antibody screening for the infection are discussed.

The mouse is a useful laboratory animal for studying various aspects of pseudorabies virus (PRV) virulence. Mice are highly susceptible hosts for PRV infection and are unable to survive acute viral infection. Because of this, mouse models have not been useful for studying PRV latent infections. Here, we report an efficient strategy for establishing latent PRV infections in laboratory mice. Passive transfer of high titered neutralizing antibodies to mice prior to inoculation with highly lethal doses of PRV (Bartha) resulted in survival rates of at least 60% with establishment of latent infections in survivors. Latent PRV infection in mice was demonstrated by: (1) recovery of infectious PRV-Bartha from explants of trigeminal ganglion (TG), and (2) detection of PRV nucleic acids in latently infected TGs by in situ hybridization and polymerase chain reaction (PCR), between 2-8 months post-infection. This PRV latency model indicates that attenuated PRV strains, those currently used extensively in vaccination programs worldwide, can establish a reactivatable latent infection in an experimental host. The mouse model may be particularly useful for examining the molecular bases of PRV latency and reactivation.

Field isolates of suid herpesvirus 1 (Aujeszky's disease virus) from Poland and Hungary were identified by restriction fragment pattern analysis as derivatives of attenuated vaccine strains. The Polish isolates were found to be related to the BUK-TK-900 strain (Suivac A) which is widely used as a live vaccine in Poland, and the Hungarian isolates were related to the Bartha K-61 vaccine strain widely used in Hungary. Pigs experimentally infected with derivatives of BUK-TK-900 or BUK-TK-900 itself were found to develop gI-antibodies, while pigs infected with derivatives of Bartha K-61 showed a gI-negative response.

Latent viral DNA was detected by the polymerase chain reaction in trigeminal ganglia of all of 10 pigs that were necropsied 81 or more days after they had been infected intranasally with a thymidine kinase-negative (TK-) vaccine strain of pseudorabies virus (PRV). Failure to reactivate virus from any of the same pigs by earlier treatment with dexamethasone suggested that even though latency can be established with TK- PRV, subsequent reactivation may be a relatively rare event.

In order to investigate the potential involvement of pseudorabies virus (PRV) in swine respiratory disease, nine week old pigs were intranasally inoculated with the PRV strain 4892. Two doses of infection were used: 10(4.5) median tissue culture infectious doses (TCID50)/pig and 10(3.5) TCID50/pig, with ten pigs per group. In the group of pigs inoculated with 10(4.5) TCID50, seven out of ten pigs died within six days after inoculation. The mortality rate in the group of pigs inoculated with the lower dose was only two out of ten and, there were several pigs in this group that showed signs of respiratory distress besides some mild nervous signs. Pseudorabies virus was isolated from various tissues collected postmortem, including alveolar macrophages. Virus localization in tissues was also detected by in situ hybridization. The histopathological examination of the respiratory tract tissues revealed a pathological process that was progressing from mild pneumonia to severe suppurative bronchopneumonia. The isolation of virus from alveolar macrophages provides support to the hypothesis that replication of PRV during the course of infection produces an impairment of the defense mechanisms in the respiratory tract.

Seven deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion. For each vaccine, the levels of clinical protection and viral excretion were compared. Groups of eight pigs were vaccinated twice with attenuated deleted Aujeszky's disease vaccines (which do not express certain glycoproteins: gI, gX or gp63). Pigs were vaccinated at the beginning of the fattening period and challenge took place at the end of it when the pigs were 18-19 weeks old. Live virus vaccines were suspended in water or in an oil-in-water emulsion. The experiment was performed in three successive assays of two groups of eight pigs (except three groups for the first assay). At each assay, a control unvaccinated group of eight pigs was added to compare the effects of challenge between vaccinated and unvaccinated animals. In total, 80 pigs were involved in this experiment. All the vaccinated pigs excreted virus from 3 to 9 d after challenge. However the level of viral excretion and the duration of the period of excretion were reduced after vaccination and especially, when oil-in-water emulsion was used. There were obvious differences between vaccines. With some vaccines, when the level of viral excretion was low, the level of clinical protection was high. However, in other cases, the level of clinical protection could be good despite a higher level of viral excretion. The seroneutralizing titres were significantly and inversely related to a low level of viral excretion but not to the level of clinical protection.

Pseudorabies virus (PRV) latency was investigated, using polymerase chain reaction (PCR). A PCR protocol was developed that specifically amplified a 217-base pair sequence within the gene encoding the essential glycoprotein gp50 of PRV. Using this PCR procedure, the gp50 sequence was amplified from tissues of pigs infected with various doses of PRV (Becker strain). At postinoculation day 64, viral isolation was performed on nasal swab specimens and homogenates of tonsils and trigeminal nerve ganglia obtained from 11 PRV-convalescent, seropositive pigs. Results were negative in all cases. By use of PCR, 11 of 11 pigs had PRV-positive trigeminal nerve ganglia and brain stem, 10 of 11 pigs had PRV-positive tonsils, and 9 of 11 pigs had PRV-positive olfactory bulbs.

The temperature-sensitive (ts), thymidine kinase-deficient (TK-) mutant designated ZHtsTK- strain, of Aujeszky's disease virus (ADV) was isolated from a virulent strain with the treatments using 5-bromodeoxyuridine and arabinosylthymine. The ZHtsTK- strain was easily distinguished from the other virulent ADV strains by plaque size on HmLu-1 and chicken embryo fibroblast cells and by restriction endonuclease analyses using Bam HI, Sal I and Kpn I. The ZHtsTK- strain was avirulent for mice, guinea pigs and rabbits, and produced neutralizing antibodies to ADV in these animals. The rabbits inoculated with the ZHtsTK- strain did not shed detectable amounts of virus after dexamethasone treatment. The ZHtsTK- strain was also avirulent for 5-day-old piglets and did not cause disease. No virus was detected from the piglets inoculated intramuscularly in the nasal swabs or the tissues examined on postinoculation day 9. These findings presented here suggested that there is a significant correlation between pathogenicity and properties such as ts and TK-, and the combination of ts and TK- properties plays a much larger role in reducing virulence for animals.

The spread of Aujeszky's disease virus (ADV) from nasal mucosa via the olfactory pathway was studied in HPCD pigs. ADV antigen was detected in the epithelial cells, nasal gland cells, olfactory nerve cells and peripheral nerve fibres in the nasal cavity and in neuroglial cells in the olfactory bulb. Results indicate that the olfactory pathway is one of the most important neuronal pathways of ADV infection in pigs.

While latent in sensory neurons of infected pigs, pseudorabies virus expresses transcripts from a limited genomic area. These RNAs are transcribed from the strand opposite to that which encodes the pseudorabies immediate-early protein. Using a combination of in situ nucleic acid hybridization performed on latently infected pig trigeminal ganglia and DNA sequencing, 5' and 3' limits of transcription for the pseudorabies LAT transcription unit have been defined. The 5' limit of transcription has been localized to a NarI-BamHI subfragment of the BamHI-6 fragment. Several promoter elements in the correct orientation for the transcript are present including consensus TATA and CAAT boxes and an SP1 site. The 3' limit of transcription has been localized to a HindIII-KpnI subfragment of the BamHI-5 fragment which contains a consensus polyadenylation signal and two termination codones in the correct orientation. From these results we conclude that the region of pseudorabies virus DNA which is active during latency can be no longer than 12.6 kb and completely overlaps the gene encoding the pseudorabies immediate-early protein.

We report here genetic recombination between 2 USDA-licensed vaccine strains of pseudorabies virus co-inoculated into swine. The vaccine strains, one of which was a conventionally attenuated strain and the other, a genetically engineered deleted strain containing a negative immunologic marker, had complementary genomes. Co-inoculation resulted in the creation of novel strains of pseudorabies virus containing negative immunologic markers with restored virulence genes. Plaque-purified recombinant progeny viruses were found in 2 litters of pigs in which both strains were co-inoculated IM, a litter in which both strains were co-inoculated oronasally, and a litter in which the conventionally attenuated strain was inoculated oronasally and the genetically engineered strain was inoculated IM. Recombinant phenotypes and recombinant restriction fragment patterns were observed. The creation, spread, and potential misdiagnosis of these types of recombinant strains could disrupt control and eradication programs that are based on the serologic identification of swine infected with potentially virulent strains of pseudorabies virus.

Because of the importance of environmental survival of pseudorabies virus to proposals to eradicate the virus from swine in the United States, survival of the virus was studied in various diluents and on combinations of diluents and solid fomites at 25 C. Suspensions of the virus in phosphate-buffered saline and saline G solutions remained infectious for at least 10 days. Infectivity of other virus/diluent suspensions decreased to less than 10 plaque-forming units/ml in 14 days (swine urine), 7 days (well water), 4 days (swine saliva), 2 days (lagoon water and swine nasal washings), and 1 day (swine pit effluent, chlorinated water, and bile). Suspensions of pseudorabies virus in saline G solution and on the solid fomites, whole corn, and steel remained infectious for at least 7 days. Infectivity of other virus/diluent/fomite combinations decreased to less than 10 plaque-forming units/ml in 7 days. The role of the fomites as vehicles for transmission of infection is discussed.

Aujeszky's disease virus was isolated from the brain of a horse which had shown severe neurological signs, including excessive sweating, muscle tremors and periods of mania. Pathological examination revealed a non-suppurative meningoencephalitis. The virus was propagated in cell culture and inoculated into the conjunctiva and nostrils of two ponies. The ponies developed fever seven days after inoculation and subsequently started to behave abnormally, showing severe neurological signs on the ninth day after inoculation. One pony became excited and the other was depressed. One pony died on the ninth day after inoculation and the other was euthanased on the 10th day. Both ponies had a significant increase in serum antibody titre against the virus. The virus was recovered from several parts of the brains and the eyes of the ponies. Aujeszky's disease in horses therefore fulfils Koch's postulates. Although horses do not appear to be very susceptible to the virus, Aujeszky's disease should be included in the differential diagnosis of horses with fatal or transient neurological signs of disease in areas where the virus is endemic.

After intranasal inoculation of pseudorabies virus in 10-week-old pigs, the morphogenesis of three pseudorabies virus strains was studied in nasal mucosa. Virulent NIA-3 virus rapidly invaded the lamina propria of the nasal mucosa. NIA-3 nucleocapsids were enveloped at the nuclear membrane. Invasion of the lamina propria was delayed after intermediate virulent 2.4N3A virus infection or absent after nonvirulent Bartha virus infection. 2.4N3A virus was mainly enveloped by membranes of the smooth endoplasmic reticulum. Bartha virus replication was delayed for at least 24 h as compared with NIA-3 virus, and Bartha virus was also enveloped by membranes of the smooth endoplasmic reticulum. The virulence of 2.4N3A and Bartha virus may be diminished because of altered envelopment and delayed viral replication and release.

The genomic diversity among isolates of suid herpesvirus 1 (SHV-1) collected in the same herd and among clones from the same isolate was studied by restriction fragment pattern (RFP) analysis using BamHI. Tentatively defining a field strain as a transmissible entity, it was concluded that strains of SHV-1 commonly comprise distinguishable genomic variants. Contrary to the hypothesis of genomic lability, it is suggested that the pool of variants is sufficiently stable to specifically characterize a strain. The impact of the results on epizootiological methods based on identification of field isolates is discussed in connection with Aujeszky's disease in Denmark.

Attenuated, gene-deletion mutants of pseudorabies virus (PRV) were tested for their ability to establish a reactivatable latent infection in pigs. The viruses (designated A, B, and C) were from each of three vaccines commercially available in the United States. Viruses A and C were similar in that they had genetically engineered gene deletions for thymidine kinase (TK) and glycoprotein X (gX); however, they had been prepared from genetically different parental strains. Virus B was TK positive, but had a naturally occurring gene deletion for glycoprotein I (gI). Four pigs were exposed oronasally to each of the viruses, and 10 weeks later they were treated with dexamethasone in an attempt to induce virus reactivation. All of the viruses replicated after initial exposure as evidenced by virus isolation from nasal swabs and the pigs' immune responses. Virus reactivation was subsequently induced by dexamethasone treatment in two of four pigs exposed to virus A. Notably, both pigs remained free of serum antibody for gX. Restriction endonuclease analysis and tests for TK activity and the presence of gX indicated that reactivated virus was similar, if not identical, to virus A used to establish latent infection. Virus shedding after dexamethasone treatment was not identified for either of the other pigs exposed to virus A nor for any of the pigs exposed to viruses B or C. The results indicated that attenuated, TK-negative PRV can establish a reactivatable, latent infection in pigs.

We have examined methods for detection of pseudorabies virus (PRV) latency in three groups of swine; naturally infected animals obtained from a field case; animals which have been experimentally infected with Becker or Iowa strains of PRV; and single reactors (single seropositive animals within PRV-free herds). In situ hybridization was shown to be more sensitive than explanation/co-cultivation for the detection of latent virus. Nervous tissues, in particular the trigeminal ganglia, were found to be the most reliable source for detecting latent PRV. The presence of latent PRV was not detected in lymphoid tissues examined.