Transneuronal viral labelling of rat heart left ventricle controlling pathways.

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.

Experimental infection of monkeys with Herpesvirus suis (Aujeszky's-disease virus).

Monkeys were infected intranasally with Herpesvirus suis. After an incubation period of 7 to 13 days the animals became acutely ill and rapidly died. Clinical signs included salivation, incoordination, ataxia and epileptiform convulsions, but not pruritus. Histopathological changes were confined to the central nervous system, and consisted of destruction of neurones with the formation of intranuclear inclusion bodies, gliosis and perivascular cuffing. Virus was isolated from the brain and spinal cord in the later stages of the illness but neutralising antibodies were not detected in serum. The distribution of lesions indicated direct spread of virus from the inoculation site along cranial nerves to the brain.

Ultrastructure and function in sympathetic ganglia isolated from rats infected with pseudorabies virus.

(1) After inoculation of the pseudorabies virus in the anterior chamber of the eye of the rat, virions can be found only in the neurons of the superior cervical sympathetic ganglion and in the sensory ganglion of the fifth nerve on the inoculated side. Other nervous structures--central or peripheral--are not infected. (2) It is shown that the retrograde axonal flow carries the virus from the eye to the sympathetic neurons. (3) The ultrastructure of the infected neuron has been studied at various intervals after inoculation and at different stages of the viral replication. (4) Excised infected ganglia in vitro show a spontaneous electrophysiological activity that can be recorded on both the post- and preganglionic nerve. Such an activity has never been seen in normal excised ganglion of rat. (5) The shape and frequency of the electrophysiological discharges recorded on the postganglionic nerve have been analyzed at various intervals after inoculation. (6) Correlations established between the ultrastructure, the effect of various drugs and the electrophysiological activity permit the proposal of various hypothesis about the abnormal activity of the infected neurons.

Aujeszky's disease of sheep: experimental studies on the excretion and horizontal transmission of the virus.

Five 5-month-old merino lambs were nasally inoculated with 10(5.0) TCID50 of Aujeszky's disease virus (ADV). The dynamics of virus excretion in the nasal discharges--in agreement with the histologic findings--indicated that ADV also replicates in extraneural sites, in the upper and lower parts of the respiratory tract. The virus was excreted continuously in the nasal discharges, even during the incubation period. The titres, with certain fluctuations, increased gradually up to the final stage of the fatal disease. Following the onset of the clinical disease, the titre of excreted virus (ranging from 10(4.0) to 10(6.0) TCID50/0.1 ml) was comparable with the ADV content found in the nasal discharge of naturally infected piglets. However, the horizontal transmission of ADV to contact lambs failed.

Detection of latent pseudorabies virus in swine using in situ hybridization.

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.

Relative rates of Aujeszky's disease virus attenuation, as assessed in mice.

Field strains of Aujeszky's disease virus (ADV) were attenuated by heat treatment and serial passage at sub-optimal growth temperatures in chicken embryo fibroblasts (CEF). At chosen passage levels, virus was titrated in cell culture and in mice. For each strain, the pathogenicity was expressed as a mouse lethal index (MLI), defined as the inverse of the log10 (CCID50:LD50). MLIs determined for field strains displayed a wide range of comparatively high values. The attenuation of field strains was accompanied by a rapid fall in MLI values, particularly in the initial stages. Heat-treated ADV attenuated faster than untreated ADV, when passaged at 30 degrees C. Passage at 27 degrees C resulted in considerably accelerated attenuation compared to passage at 30 degrees C, in the case of both untreated and heat-treated ADV. MLIs were determined for attenuated ADV strains that had been tested in 6-day-old piglets. Low MLI values were found to correlate with low virulence in piglets and high MLI values with virulence.

The genomic diversity and stability of field strains of suid herpesvirus 1 (Aujeszky's disease virus).

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.

Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's disease vaccines.

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.

Rapid detection of pseudorabies virus genomic sequences in biological samples from infected pigs using polymerase chain reaction DNA amplification.

The presence of the pseudorabies virus (PRV) genome in infected hosts has previously been studied by standard hybridization techniques, which showed the viral genome to be present at very low levels in infected tissues. The recently introduced polymerase chain reaction (PCR) procedure provides an alternative and rapid means of amplifying small quantities of specific DNA sequences. We applied this technique to a study of pigs infected by PRV. The sequence selected for amplification consisted of 222 base pairs lying in the gene coding for the glycoprotein gp50. We used a pair of 20-mer oligonucleotides flanking this sequence as primer and a cloned Stu-Nde fragment containing the sequence as target DNA. To avoid the tedious DNA extraction procedure we performed PCR directly on disrupted cells and detected specific amplification after 25 cycles of PCR with the thermostable Taq DNA polymerase. Amplified products were detected by gel electrophoresis directly. Nasal samples from experimentally and naturally infected pigs were tested by this PCR technique. When compared with tissue culture and serological tests, detection by gel electrophoresis of PCR amplified fragments provided excellent specificity and sensitivity. We concluded that PCR amplification will be a valuable tool for rapid diagnosis of PRV infection in pigs, taking less than 1 h to complete.

Role of different genes in the virulence and pathogenesis of Aujeszky's disease 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.

Effects of an experimental infection with Actinobacillus pleuropneumoniae on the interferon-alpha and interleukin-6 producing capacity of porcine peripheral blood mononuclear cells stimulated with bacteria, virus or plasmid DNA.

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.

Effect of intratesticular inoculation with Aujeszky's disease virus on genital organs of boars.

Ten 8-10-month-old Belgian Landrace boars were intratesticularly inoculated with 500 TCID50 of a virulent Belgian Aujeszky's disease virus (ADV) isolate (75V19) in 0.1 ml volume. One control boar was similarly inoculated with phosphate-buffered saline solution. The genital organs of six inoculated boars were examined by virus isolation and immunofluorescence. In spite of high virus titers, the fluorescence in the testicles remained limited to a few small foci in the interstitial connective tissue and tunica albuginea at or close to the inoculation site. Neither virus replication, necrosis nor inflammatory lesions could be demonstrated in the epithelium of the seminiferous tubules. However, virus replication was regularly demonstrated in the serosa covering testicles, plexus pampiniformis, ductus deferens and tunica vaginalis. Virus was also isolated from the scrotal fluid. It is suggested that the serosa is the primary target tissue for ADV. The other four boars were inoculated to study the effect of ADV on semen. Severe morphologic alteration and lowered sperm cell concentrations were observed during several weeks after inoculation or until slaughter at 47, 53 and 58 days post inoculation. Virus was isolated from semen of only two out of four boars examined at 9 and 10 days post inoculation.

Horizontal transmission of Aujeszky's disease virus from sheep to pigs.

Eight 2-month-old merino lambs were inoculated intranasally with different (10(2.0)-10(5.0)TCID50) amounts of Aujeszky's disease virus (ADV). Electron microscopic studies indicated that ADV replicated in extra-neural sites, in the epithelial cells of the mucosa of the upper and lower respiratory tract. Although the virus was excreted continuously in nasal discharges, horizontal transmission to contact lambs failed. The surviving exposed and contact lambs had no demonstrable antibodies against ADV and they were susceptible when challenged by ADV. However, the virus was transmitted to susceptible pigs in contact with the exposed lambs. One of the five contact pigs showed characteristic clinical signs of Aujeszky's disease, developed a nonsuppurative meningoencephalomyelitis and ADV was recovered from the brain, nasal discharge and other organs. Restriction enzyme analysis of DNA from this virus confirmed the sheep origin of the isolate. The other 4 pigs seroconverted. ADV infection in sheep is therefore a possible source of infection for pigs, but the lack of horizontal transmission in sheep was confirmed.

The rapid detection of Aujeszky's disease virus in pigs by direct immunoperoxidase labelling.

Direct immunoperoxidase labelling on impression smears of brain and pharynx was compared with virus isolation and direct immunofluorescence for the detection of Aujeszky's disease virus in experimentally-infected pigs. Immunoperoxidase labelling was as sensitive as immunofluorescence and more sensitive than virus isolation for tissue that had been stored at room temperature (approximately 20 degrees C) for up to 144 h.

Experimental infection of pigs with a thymidine kinase negative strain of pseudorabies virus.

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.

Pneumonia in pigs inoculated with pseudorabies virus by means of a bronchoscope.

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.

Immunohistological demonstration of spread of Aujeszky's disease virus via the olfactory pathway in HPCD pigs.

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.

Pathological changes in HPCD pigs with prednisolone induced recrudescence of pseudorabies virus.

In HPCD pigs inoculated with PRV, latent PRV could be reactivated in-vivo by the administration of large doses of prednisolone 3 months after the primary infection. In two pigs, virus shedding was without clinical signs of disease, whereas depression of circulating lymphocytes was prominent. Reactivation of PRV was also demonstrated by cultivation of the brain cortex on the 7th day and the mandibular lymph node on the 9th day after the prednisolone began treatment. Coincident with the virus isolation, characteristic lesions were observed in 2 pigs in the central nervous tissues and mandibular lymph nodes and these were composed of cell necrosis and eosinophilic intranuclear inclusion bodies. Cells containing the intranuclear inclusion bodies had immature and mature PRV particles. Results of the present study with HPCD pigs indicated that the lesions in the brain and lymph node accompanied by eosinophilic intranuclear inclusion bodies were pathogonomonic lesions induced by reactivation of PRV.

Detection of reactivating pseudorabies virus in tissue by immunoperoxidase technique.

Reactivation of pseudorabies virus (PRV) was induced in pigs by prednisolone treatment. The virus was re-isolated from nasal secretions of four, brain cortex of two and mandibular lymph node of three out of 12 pigs, respectively. The characteristic lesion of recurrently infected pigs was focal necrosis in the brain cortex and mandibular lymph node, accompanied by reactivating virus particles in the degenerating cells. Coincident with the pathological lesions, PRV antigen was detected in two of the brain cortex and six of the mandibular lymph node specimens by immunoperoxidase staining. These results suggest that the immunoperoxidase technique was more sensitive than virus isolation for demonstrating the reactivity of PRV in recurrently infected pigs.

Immunopathology in Aujeszky's disease virus-infected pigs exposed to fluctuating temperatures.

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.