The development of Anaplasma marginale was studied in Dermacentor andersoni nymphs after they had fed on a calf with ascending Anaplasma infection. Gut tissues were collected on day 4 of tick feeding, from newly replete (fed) nymphs and on postfeeding days (PFD) 5, 10, 15, 20, and were processed for light and electron microscopy to determine density of A marginale colonies. Homogenates of gut tissues were prepared from nymphs collected on the same days and inoculated into susceptible, splenectomized calves to test for infectivity. Anaplasma colonies were detected in gut cells on PFD 5, 10, 15, and 20. Although colony density appeared to be higher on PFD 10 and 15, differences were not significant. Nymphal type-1 colonies were detected in highest numbers on PFD 5 and 10, transitional colonies were seen in highest numbers at PFD 10 and 15, and nymphal type-2 colonies were observed only on PFD 20. Gut homogenates that were collected from ticks at 4 days of feeding, when newly replete, and on PFD 20 caused anaplasmosis when injected into susceptible calves, but homogenates made from ticks collected on PFD 5, 10, and 15 were not infective. The data indicate that of the colony types of A marginale that develop in replete nymphs, nymphal type-1 and transitional colonies may contain organisms that are not infective for cattle.

Pseudorabies virus (PRV), an alpha-herpesvirus, causes substantial economic losses in the swine industry and is currently the focus of eradication and control programs. Some of these programs rely on the ability of veterinarians to differentiate animals exposed to virulent strains of PRV from animals exposed to avirulent vaccine strains of PRV on the basis of a serologic response to nonessential glycoproteins that are deleted in some vaccine strains of PRV. Genetic recombination resulting in the creation of virulent strains of PRV with the same negative immunologic markers as vaccine strains could disrupt these programs. Two strains of PRV were coinoculated either into tissue culture or into sheep to facilitate recombination. Progeny viruses were selected to detect a specific recombinant phenotype. We were able to detect genetic recombination between vaccine strains of PRV following in vitro or in vivo coinoculation of 2 strains of PRV. The selected recombinants had marker-deleted phenotypes in strains with restored virulence genes. Increased virulence was observed in sheep after coinoculation of 2 avirulent vaccine strains of PRV.

The possible role of the complement-mediated bactericidal system in protection of swine against contagious pleuropneumonia was investigated. Strains of Actinobacillus (Haemophilus) pleuropneumoniae representing serotypes 2, 3 and 5 were found to be fully resistant to the bactericidal action of porcine serum from precolostral, clinically normal adult, and chronically infected pigs. All strains were also resistant to hyperimmune rabbit serum, but 3 of 4 strains were sensitive to normal human serum. This bactericidal effect was lost when human serum was previously absorbed with the homologous bacteria, indicating that antibody was necessary for killing. Addition of human serum to porcine serum or to absorbed human serum did not restore the bactericidal system. Pretreatment of the bacteria with undiluted heat-treated human serum also failed to sensitize the bacteria to the absorbed serum, indicating that a heat-labile, absorbable factor may have been required for killing of A pleuropneumoniae. None of the strains was sensitized to porcine serum by sublethal treatment with polymyxin B, a treatment that is known to disrupt the integrity of the outer membrane and induce serum sensitivity in gram-negative bacteria. The ability of A pleuropneumoniae to resist complement killing in vitro may reflect a virulence mechanism in vivo that assists bacteria in avoiding the pulmonary defenses of swine and promotes bacterial invasion of the lungs.

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 X 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% had positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5, IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of AFSV-infected pigs.

Six monoclonal antibodies (MAB) to virulent Mycobacterium bovis is ATCC 19210 were produced, using a suspension of heat-inactivated whole cells. Immunoglobulin isotype for MAB VMB6, VMB73, and VMB93 was IgG1, and for VMB31, VMB99, and VMB119, it was IgG2a. Monoclonal antibodies were examined for cross-reactivity to M tuberculosis, M kansasii, M fortuitum, M paratuberculosis, M avium serovars 1, 2, 4, 8, and 10, M chelonei, M phlei, M scrofulaceum, M smegmatis, Nocardia asteroides, and Rhodococcus equi. Monoclonal antibodies could be grouped on the basis of binding activity by ELISA and immunoblot analysis, in which MAB VMB6, VMB31, and VMB119 had binding activity to M bovis; MAB VMB93 and VMB99 detected M bovis and M tuberculosis antigens, and MAB VMB73 reacted with other mycobacterial species, as well as with N asteroides and R equi. Apparent molecular mass of antigens was 30 to 25 kilodaltons (kD) for VMB6, VMB31, and VMB119 and 63 kD for VMB93 and VMB99, and ranged from greater than 200 to 31 kD for VMB73, as estimated by immunoblot analysis. Monoclonal antibody binding activity to 18 field isolates of M bovis was evaluated, using ELISA. Each of 18 field isolates was detected, using MAB VMB6, VMB31, or VMB119; 10 isolates were detected, using MAB VMB93/VMB99, and 14 were detected by use of MAB VMB73. Use of MAB in ELISA failed to detect antigens from M bovis strain AN-5.

The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P > 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P > 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

Subunit pseudorabies vaccines that contained only purified glycoproteins of either of 2 strains of pseudorabies virus (PRV) were prepared and subsequently tested for safety and efficacy. The strains of virus used for vaccine production differed in at least 2 properties. One strain (Kojnok) was virulent for pigs and was believed to code for the entire complement of viral glycoproteins. The other (Kaplan) was a deletion mutant that was unable to code for structural viral glycoproteins gI and gp63. Purified glycoproteins were dispersed in an oil-in-water emulsion and were administered IM to pigs. Both vaccines were found to be safe and effective immunogens. Neither caused any local or general reactions, as verified by examination of the injection site (local safety) and by vaccination of pregnant sows in PRV-infected and noninfected herds. Sows vaccinated with the gI+ or gI- vaccine protected their pigs at levels of 93 and 92%, respectively, against a severe challenge exposure that killed 98% of pigs born from nonvaccinated sows. Vaccinated pigs were tested for active immunity by intranasal challenge exposure with the NIA 3 strain. Protection was quantitated by measuring the relative daily weight difference, expressed in percent per day, between vaccinated and control pigs during the first week after challenge exposure (deltaG7); the estimated differences were 2.25 and 2.13% for gI+ and gI- vaccines, respectively. The absence of gI and gp63 did not affect the efficacy of this type of subunit glycoprotein vaccines. In pigs that were challenge-exposed intranasally 3 months after 1 injection with the gI- vaccine, the duration of viral excretion was highly reduced, compared with that in control pigs.

Plasmid profiles were compared between nonpiliated and piliated forms of Moraxella bovis isolates. The piliated form of M bovis isolate IBH64 contained 1 fewer plasmid than did the nonpiliated form. Piliated and nonpiliated cells of IBH64 contained plasmids having molecular size of 45, 32.8, 4.9, and 4.6 kilobases (kb). Single- and double-restriction endonuclease digestion by Ava I and Nde I indicated that the size of the additional plasmid carried by the nonpiliated form of IBH64 was approximately 43.6 kb. The M bovis isolates, Newport and GRS, contained the same number of plasmids in either their piliated or nonpiliated form.