Phagocytosis of splenetic neutrophils of mice enhanced by orally administered peptidoglycan from Bifidobacterium thermophilum.

A preparation of peptidoglycan (PG) of swine Bifidobacterium thermophilum was orally administered to SPF-BALB/C and ICR mice and its effect on phagocytosis splenetic neutrophils from PG administered mice was measured by chemiluminescent response (CL) and fluorometric analysis and the result was compared with that of non-treated mice. PG stimulated phagocytosis of neutrophils in a dose-dependent manner, whereas dosage exceeding the optimum concentration (500 microgram) inhibited phagocytosis. The maximum effect on phagocytosis of neutrophils was observed at 3 days after administration of PG 500 microgram. The result of fluorometric analysis was almost similar to that of CL. These results indicate that orally administered PG enhances the activity of the phagocytosis of splenetic neutrophils from mice.

A serological survey of encephalomyocarditis virus infection in pigs in Japan.

A total of 1502 pig sera collected between 1990 and 1991 at 65 farms in 22 prefectures were subjected to neutralization test to encephalomyocarditis (EMC) virus. Of them, three hundred eighty seven pigs (25.8%) from the 55 farms (84.6%) had antibody against EMC virus. The positive rates were rather high in Ehime (53.1%), Tottori (49.1%), Shimane (44.4%) and Nagasaki (38.7%). The positive rate and geometric mean titer of antibody tended to increase with the age. These serological results suggest that the EMC viral infection have been occurred among pigs in Japan.

Distinction between Aujeszky's disease virus-infected and vaccinated pigs by hemagglutination-inhibition test.

A hemagglutination-inhibition (HI) test was applied to distinguish virulent Aujeszky's disease virus infected pigs from those immunized with a glycoprotein gIII deletion vaccine. The vaccine strain, dlg92/dltk, did not have hemagglutination activity with mouse erythrocytes and the pigs vaccinated five times with the dlg92/dltk strain failed to develop HI antibody, although they developed neutralizing antibody with 128 to 512 titers to Aujeszky's disease virus. On the other hand, these pigs produced HI antibody 1 to 2 weeks after virulent virus inoculation. Thus the animals infected with virulent strain were easily differentiated from the animals immunized with the gIII deletion vaccine.

In vivo replication of porcine reproductive and respiratory syndrome virus in swine alveolar macrophages and change in the cell population in bronchoalveolar lavage fluid after infection.

Replication of porcine reproductive and respiratory syndrome (PRRS) virus in swine alveolar macrophages (AM) and cell population in broncho-alveolar lavage fluid (BALF) obtained from PRRS virus-infected pigs were investigated. BALF samples were periodically collected from 6 pigs infected with PRRS virus and 3 non-inoculated control pigs by means of fiber-optic bronchoscope between post-inoculation day (PID) 0 and 56. The mean ratio of macrophages in BALF collected from infected group was 92.7 +/- 3.2% before inoculation and gradually decreased from PID 14. On the other hand, the ratio of lymphocytes was 4.8 +/- 3.2% before inoculation and increased from PID 21 and indicated 41.8 +/- 9.1% on PID 28. After that, they decreased gradually and that of macrophages correspondingly increased. The ratio of neutrophils maintained between 0.7% and 5.1%. The ratios of macrophages, lymphocytes and neutrophils collected from control group were almost stable through the examination. Intracellular PRRS virus antigens in AM were detected from PID 2 by indirect immunofluorescence assay (IIFA). PRRS virus was first isolated from BALF samples collected from inoculated group between PID 2 and 49. From serum, virus was isolated between PID 2 and 21. Antibodies in sera measured by IIFA to PRRS virus were first detected on PID 14 and the antibody titer rose to 1:640 or 1:1,280. The results suggested that PRRS virus replicates in swine AM for a relatively long period.

Replication of virulent and attenuated strains of Aujeszky's disease virus in swine alveolar macrophages.

In vitro and in vivo replication of Aujeszky's disease virus (ADV) in swine alveolar macrophages (AM) was studied using two virulent strains and a vaccine strain with deletions in the thymidine kinase and gIII genes. In vitro, AM were highly permissive to virulent ADV infection. Virus progeny titers of virulent strains in the cell phase and in the fluid phase were higher than 10(7.3) TCID50/ml at 84 hr post-inoculation (PI). For vaccine strain infection, AM were less permissive, yielding virus titers of 10(2.3) TCID50/ml at 84 hr PI. To study in vivo replication of ADV in AM, virus isolations were made from AM collected at intervals from pigs inoculated intranasally with both the virulent and vaccine strains. Virus was isolated from AM samples collected from all pigs infected with the virulent strain from days 2 to 22 PI. On the contrary, no virus was detected in AM samples collected from pigs infected with the vaccine strain. The results presented suggested that in vivo virulent ADV replicates for a relatively long period in swine alveolar macrophages.

Experimental pneumonia of pigs infected with Aujeszky's disease virus and Actinobacillus pleuropneumoniae.

Experimental infections were induced out to examine whether Aujeszky's disease virus (ADV) infection in pigs results in a severe pneumonia by Actinobacillus pleuropneumoniae. Intranasal inoculation of ADV (10(6.9) median tissue culture infective dose/head) in 4-month-old primary specific-pathogen-free pigs was followed by the inoculation of A. pleuropneumoniae type 1 (10(3.1) or 10(5.1) colony-forming-units/head). The pigs inoculated with ADV alone developed clinical signs of Aujeszky's disease but not pneumonia, and those inoculated with A. pleuropneumoniae (10(3.1) CFU/head) alone did not develop clinical symptoms and lung lesions. Whereas all the pigs inoculated dually with ADV and A. pleuropneumoniae (10(3.1) CFU/head) showed severe or very severe clinical symptoms and moderate or severe pneumonia and one of them died. The pigs inoculated with A. pleuropneumoniae (10(5.1) CFU/head) alone had severe clinical symptoms and one of the 2 pigs died acutely. Furthermore, all of the 3 pigs inoculated with ADV and A. pleuropneumoniae (10(5.1) CFU/head) showed clinical symptoms and moderate or severe pneumonic lesions and one pig died of disease. It was concluded that the clinical symptoms of A. pleuropneumoniae became severer by concomitant infection with ADV.

Experimental dual infection of cesarean-derived, colostrum-deprived pigs with Mycoplasma hyopneumoniae and pseudorabies virus.

To determine whether pseudorabies virus (PRV) infection increases the severity of pneumonia by Mycoplasma hyopneumoniae, 18, 10-week-old Cesarean-derived, colostrum-deprived pigs were randomly assigned to 3 groups of 6 pigs each. Pigs in groups A and C were inoculated intranasally with M. hyopneumoniae at 10-week-old. At 11-week-old, pigs in groups B and C were inoculated intranasally with PRV. All pigs were initially seronegative for M. hyopneumoniae and PRV. Three pigs of each group were euthanized at 12-week-old, and remaining pigs at 14-week-old. At necropsy, gross lesions in the lung were observed in the pigs of groups A and C. On post-inoculation-week (PIW) 2 with M. hyopneumoniae (at 12-week-old), lung lesions were recognized in one of the 3 pigs in group A and all the pigs in group C. The mean percentage of the lung lesions were 0.1% in group A and 9.8% in group C. M. hyopneumoniae was isolated from broncho-alveolar lavage fluids (BALF) of pigs in group A with titer of 10(2) to 10(3) CCU/0.2 ml and in group C with titer of 10(5) to 10(6) CCU/0.2 ml. On PIW 4 (at 14-week-old), lung lesions were observed in all the pigs in groups A and C, and the mean percentage of the lung lesions were 8.3% in group A and 17.2% in group C. M. hyopneumoniae was isolated from BALF in group A with titer of 10(4) to 10(7) CCU/0.2 ml and in group C with titer of 10(6) to 10(7) CCU/0.2 ml. PRVs were isolated from nasal swab and tissue samples in groups B and C. After inoculation, antibody against M. hyopneumoniae was detected in groups A and C, and against PRV in groups B and C. Under the present experimental conditions, PRV infection appear to have effect on the severity of experimentally induced acute mycoplasmal pneumonia in young pigs.

Experimental infection of SPF piglets with porcine reproductive and respiratory syndrome (PRRS) viruses isolated from two farms.

Porcine reproductive and respiratory syndrome (PRRS) viruses were isolated from pig samples obtained from two farms characterized by an increased number of stillbirth and high mortality in new-born piglets (farm A), and respiratory distress with high mortality in weaning and growing pigs (farm B) in 1993, respectively. When primary specific pathogen-free piglets, 5-day-old or 13-day-old, were experimentally inoculated with the isolates, they showed clinical signs of depress, anorexia, pyrexia, diarrhea, sitting posture and periocular edema. Rate of the weight gain was reduced in the inoculated piglets compared with the non-inoculated pig. There were no apparent differences in clinical signs between the piglets inoculated with the virus samples derived from farms A and B. Microscopically, the most prominent changes observed in experimentally inoculated piglets were interstitial pneumonia, nonpurulent myocarditis and catarrhal lymphnoditis post inoculation day (PID) 28. Viruses were recovered from tissues collected from the inoculated piglets on PID 7 or 28. Furthermore, the viruses were continuously recovered from the sera from PID 7 to PID 28. Antibodies measured by indirect immunofluorescence assay to PRRS virus were first detected in sera on PID 14, and the antibody titer rose to 1:1280 on PID 28.

Effects of cholera toxin on delayed-type hypersensitivity to sheep red blood cells inoculated intranasally into mice.

The effects of cholera toxin (CT) on delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were studied in mice sensitized by intranasal administration of SRBC. CT (1 microgram/mouse), given intranasally together with SRBC (2 x 10(7)/mouse), induced a maximally enhanced DTH response, which reached its peak around 7 days after sensitization, and also induced an accelerated DTH response upon a second administration of SRBC 28 days later. The ability of CT to enhance the DTH to SRBC was lost, either when CT was administered via the intraperitoneal or subcutaneous route, or when CT was introduced into the nasal site from which a large proportion of the SRBC was discharged 2 days after SRBC administration. These results indicate that the cells that are located in the nasal site and participate in the earlier events of DTH response were most affected by CT. The following effects of CT on the earlier events, which occur within 24 hr after the intranasal administration of both CT and SRBC, appeared to be involved in the mechanisms by which CT enhances DTH to SRBC: (i) facilitation of the penetration of the antigen into the nasal tissue; (ii) reinforcement of the migration of immunocompetent cells from the blood to the nasal tissues; (iii) promotion of the ability of Ia-positive macrophages to present the antigenic determinants to T cells; (iv) facilitation of the differentiation of primed T cells to DTH-effector T cells.

Protection against influenza virus infection by vaccine inoculated intranasally with cholera toxin B subunit.

Secretory IgA antibodies in mucosa are known to play an essential role in protection against various infectious agents. To enhance the induction of protective mucosal antibodies, influenza HA vaccine was inoculated intranasally into mice with the B subunit of cholera toxin (CTB), which is known to be an excellent mucosal self-adjuvanting molecule. This combination resulted in high levels of antiviral IgA antibodies in nasal secretions and enhanced serum haemagglutinin-inhibiting (HI) antibodies 4 weeks after inoculation, compared with the inoculation of vaccine alone which induced only a low level of HI serum antibodies and no local IgA antibodies. (Subcutaneous or intraperitoneal inoculation of the vaccine with CTB failed to induce detectable nasal antiviral IgA antibodies). Levels of nasal IgA and serum HI antibodies increased in a dose-dependent fashion with increasing nasal doses of both vaccine and CTB, and correlated with the degree of protection against viral challenge. A greater protective effect was seen with cholera toxin than with its B subunit. Moreover, a second administration of vaccine alone, 4 weeks after the inoculation of the vaccine with CTB, elevated the level of the antiviral IgA nasal antibodies to 10-100 times higher than that of the primary response. These results suggest that either CT or CTB could be used as a potent adjuvant to induce protective secretory antibodies by nasal vaccination against pathogens impinging on respiratory mucosa.

Enhancement of protective antibody responses by cholera toxin B subunit inoculated intranasally with influenza vaccine.

Effects of the B subunit of cholera toxin (CTB) on the primary antibody responses to influenza virus A/PR/8/34 (PR-8) (H1N1) HA vaccine and on protection against viral challenge were investigated in Balb/c mice which were immunized intranasally with both the vaccine and CTB. The dose of CTB (greater than or equal to 1 microgram) inoculated with the vaccine (greater than or equal to 0.15 microgram) induced high responses of both antiviral IgA antibodies in the nasal wash and haemagglutinin-inhibiting (HI) antibody in the serum, enough to provide complete protection against viral challenge four weeks after immunization. High levels of antibody were maintained for more than 16 weeks after inoculation, affording complete protection during this interval. The inoculation of HA vaccine prepared from influenza viruses A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2) together with CTB provided partial protection against PR-8 infection, with production of antiviral IgA antibodies which were cross-reactive to PR-8 antigens whereas immunization with CTB and HA vaccine prepared from a different type of influenza virus (B/Ibaraki/2/85) failed to protect against PR-8 infection. These results indicate that CTB can produce an augmented and persistent antibody response to PR-8 HA vaccine, which is cross-protective to other A-type virus infections. The mechanisms by which CTB enhances the protective antibody responses to the nasally inoculated vaccine were investigated. The ability of CTB to augment antibody responses was lost, either when CTB was inoculated via the intravenous or subcutaneous route, or when CTB was introduced into nasal site one day before or after the vaccine inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)