The hexon subunit of bovine adenovirus serotype 3 (BAV-3) was purified by use of anion-exchange chromatography. A vaccine composed of the purified hexon in immune-stimulating complexes was administered and induced high titer of virus-neutralizing antibody in rabbits and calves.

Inoculation of swine with a sylvatic isolate of Trichinella spiralis, designated T s nativa, resulted in low numbers of muscle larvae, compared with muscle larvae accumulation in swine inoculated with a pig type of T s spiralis. Despite low infectivity of T s nativa for swine, primary inoculation resulted in high levels of immunity against challenge infection with T s spiralis. This immunity was expressed in accelerated expulsion of challenge adults from the intestine and reduced numbers of muscle larvae. Pigs inoculated with T s nativa developed cellular and humoral responses similar to those in pigs inoculated with T s spiralis. However, in immunoblots, sera from pigs inoculated with T s nativa recognized additional proteins in muscle larvae excretory-secretory (ES) products, compared with sera from pigs inoculated with T s spiralis. Active immunization of pigs with ES products from T s nativa resulted in numerically higher, but not significantly different levels of immunity, compared with pigs immunized with ES from T s spiralis. The highest levels of immunity were obtained in pigs immunized with a T s spiralis newborn larval extract. The combination of ES products and newborn larval extract did not result in additive levels of immunity. These results indicate that the major immune effector response to Trichinella sp in pigs is against the newborn larvae, regardless of the genetic type of Trichinella sp.

An ELISA was developed and tested for its ability to detect antibodies against Salmonella enteritidis in chickens. Various features of the ELISA were evaluated and optimized. The outer membrane protein antigens selected by use of the protein immunoblotting method made the assay specific and sensitive. The assay was evaluated in chickens experimentally infected with S enteritidis. Blood samples collected at weekly intervals after experimental infection with S enteritidis were analyzed by ELISA. Results of the ELISA were compared with those of conventional serum plate and microagglutination tests. The ELISA was more sensitive and specific in the detection of S enteritidis infection than the other 2 conventional tests.

An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (ThFAS) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of ThFAS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of ThFAS were shown to react nonspecifically. Antibodies against the < 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the < 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.

Whole-cell lysates and proteinase K-extracted lipopolysaccharide (LPS) of 19 strains of the group eugonic fermenter-4 (EF-4) were analyzed by electrophoresis and protein immunoblotting. These strains were isolated from dog- and cat-bite abscesses in human beings, ferret and human gastric lesions, and cat-lung infections. These strains represent 2 biovar groupings; EF-4a biovars ferment glucose and possess arginine dihydrolase activity, whereas EF-4b biovars do not. Electrophoresis of whole-cell lysates could distinguish between these biovars groups. Electrophoresis of LPS extracts revealed that all strains of EF-4 possess smooth chemotypes. Two strains of EF-4a reacted weakly in protein immunoblots and revealed distinct LPS profiles. These studies suggests that subgroups of EF-4 biovars may exist.

A dot ELISA was developed for detection of antibodies to Mycobacterium paratuberculosis. The assay was evaluated by testing sera from cattle that were determined, by bacteriologic culturing of feces, to be infected with M paratuberculosis and were suspected of having clinical disease. Further evaluation involved testing sera from cattle in which M paratuberculosis had not been isolated from feces on several attempts. Results of the dot ELISA were positive for sera from 86 of 101 infected cattle, and results were negative for sera from 64 of 64 noninfected cattle. Results of conventional ELISA and agar gel immunodiffusion (AGID) tests were positive for 79 of 99 and for 51 of 101 infected cattle, respectively. The dot ELISA also was evaluated by comparing results of testing 708 sera with results of bacteriologic culturing of matched fecal samples from 262 cattle in 3 central Ohio dairy herds known to include cattle infected with M paratuberculosis. Results of the dot ELISA were positive for 25 of 39 sera from cattle with positive results on culturing of concurrently obtained fecal specimens. The dot ELISA results were negative for 661 of 669 sera from cattle with negative results to culturing of concurrently obtained fecal specimens. The 39 sera from cattle with positive results on bacteriologic culturing of matched fecal specimens had positive results for ELISA and the AGID test 25 and 14 times, respectively. The 669 sera from cattle with concurrently negative results on bacteriologic culturing of feces had negative results to ELISA and the AGID test 559 and 668 times, respectively.

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.

Transstadial and transovarial transmission of Anaplasma marginale by Dermacentor variabilis were attempted with ticks exposed to the organism once by feeding as larvae or nymphs, and twice by feeding as larvae and nymphs. Typical colonies of A marginale were in gut tissues of adults that were infected as larvae, larvae and nymphs, and as nymphs; repeated exposure of ticks did not appear to result in an increase in the number of colonies in the gut of subsequently molted adults nor did it affect severity of the clinical disease that developed in cattle they fed on. In contrast, colonies of A marginale were not found in the midgut epithelium of unfed nymphs exposed as larvae, even though companion nymphs transmitted the parasite, causing severe clinical anaplasmosis in susceptible calves. The organism was not transmitted transovarially by F1 larvae or nymphs from the groups exposed as parent larvae, nymphs, larvae and nymphs, and as adults. Some of the calves fed on by F1 progeny had a few erythrocytic marginale bodies that looked suspiciously like A marginale, as well as postchallenge exposure prepatent periods that were longer than other calves in the transovarial transmission study. Sera from these calves were tested for antibody to A marginale, using a highly sensitive immunoblot technique. Antibodies were not detected in any of the sera.

Seven hybridomas that secreted monoclonal antibodies (MAB) against the peplomer protein and one that secreted MAB against the nucleocapsid protein of Berne virus (proposed family Toroviridae) were isolated. All MAB directed against the peplomer protein neutralized virus infectivity and, with the exception of MAB 6A7, inhibited each other's binding in competition assays. Neutralization of Berne virus infectivity was potentiated when some MAB were used in pairs. The antibodies have been used to localize toroviral proteins in infected cells; use of antipeplomer MAB 6B10 yielded a diffuse intracytoplasmic immunofluorescence, whereas the antinucleocapsid MAB 1F1 detected antigen in the intra- and perinuclear compartments. By use of radioimmune precipitation, protein A of Staphylococcus aureus was found to bind directly to the nucleocapsid polypeptide, without the requirement for specific antibody. Using fluorescein isothiocyanate-conjugated protein A, the intranuclear accumulation of the nucleoprotein of Berne virus was confirmed by results of immunofluorescence.