In this study the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3). The GST fusion protein was recognized by antibodies in serum positive for A. pleuropneumoniae by Western blot analysis. Purified soluble GST-TonB2 was assessed for its ability to protect BALB/c mice against A. pleuropneumoniae infection. Mice were vaccinated with GST-TonB2 subcutaneously and challenged intraperitoneally with either approximately 4.0 × 10(5) colony-forming units (CFU) or approximately 1.0 × 10(6) CFU of A. pleuropneumoniae 4074. They were examined daily for 7 d after challenge. The survival rate of the TonB2-vaccinated mice was significant higher than that of the mice given recombinant GST or adjuvant alone. These results demonstrate that A. pleuropneumoniae TonB2 is immunogenic in mice and should be further assessed as a potential candidate for a vaccine against A. pleuropneumoniae infection. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) based on the GST-TonB2 recombinant protein was developed. Compared with the ApxIVA ELISA, the TonB2 ELISA provided earlier detection of antibodies in pigs at various times after vaccination with A. pleuropneumoniae live attenuated vaccine. When compared with an indirect hemagglutination test, the sensitivity and specificity of the TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA provides an alternative method for rapid serologic diagnosis of A. pleuropneumoniae infection through antibody screening, which would be especially useful when the infection status or serovar is unknown.

Thirty healthy male horses were allotted to 3 groups and treated blindly during 4 days. Group-1 horses received unfractioned calcium heparin (100 IU/kg of body weight, SC, q 12 h). Group-2 horses received a single dose of a low-molecular-weight heparin (50 anti-Xa IU/kg, SC) every morning, and a similar volume of saline solution every evening. Group-3 horses received the vehicle (saline solution), SC, every 12 hours. Citrated and EDTA-anticoagulated blood samples were collected before starting the medication (T-0) and once daily 3 hours after each morning injection (T-3, T-27, T-51, and T-75). The PCV, hemoglobin concentration, RBC and platelet counts, and clotting times (activated partial thromboplastin time and thrombin time) were determined, and a microscopic examination to detect hemagglutination was performed. Plasma concentration of heparin was measured by use of the antifactor Xa activity assay. Bleeding time was determined on the first and fourth days, using a double-template method. The horses given unfractioned heparin had marked agglutination of erythrocytes after the first injection that became more pronounced as treatment progressed. Also, significant decrease in PCV, hemoglobin concentration, and RBC count was observed during treatment. Platelet count was significantly decreased after the first day, and clotting times were significantly prolonged. In contrast to the horses given unfractioned heparin, those given low-molecular-weight heparin did not have any agglutination of erythrocytes during the 4 days of treatment, and there were no significant changes in PCV, hemoglobin concentration, or RBC and platelet counts. Activated partial thromboplastin time increased slightly in the horses given low-molecular-weight heparin, although the values remained within reference range. Both groups of horses achieved adequate concentrations of heparin in plasma for prophylactic purposes, but those given low-molecular-weight heparin achieved those values after the first injection. Bleeding times were not significantly different between heparin-treated horses and horses given saline solution during treatment. We conclude that low-molecular-weight heparin may be used more safely and conveniently in horses, because it does not affect equine erythrocytes, platelets, or clotting and bleeding times.

Heparin inhibited hemagglutination (HA) by pseudorabies virus (PRV), but not HA by Akabane virus, bovine adenovirus type 7, Fukuoka virus, Getah virus, Japanese encephalitis virus, and parainfluenza virus type 3 belonging to the families Bunyaviridae, Adenoviridae, Rhabdoviridae, Togaviridae, Flaviviidae, and Paramyxoviridae, respectively. The minimal inhibitory concentration of heparin required to inhibit 8 HA U of PRV ranged from 0.005 to 0.01 U/ml. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by PRV. Virus-heparin complex formation could be observed by sedimenting heparin with the virus particles.

Purified lipopolysaccharides (LPS) from 16 serotypes of Pasteurella multocida were complexed with Aspergillus fumigatus ribosomes. The complexes were used as inocula to prepare antisera, in chickens, for somatic antigen typing by the gel diffusion precipitin test (GDPT). Antisera made against 15 of 16 LPS reacted with their respective specific heat-stable antigens in the GDPT and homologous LPS in the passive hemagglutination test. Antisera could not be made against serotype 15 LPS. Correlation was not observed between intensity of the precipitin reaction in the GDPT and titer to homologous LPS in the passive hemagglutination test. Most antisera cross-related with other heat-stable antigens of other serotypes in the GDPT. Many of these cross-reactions were eliminated by dilution. Cross-reactions that occurred in the GDPT with antisera made against LPS of serotypes 2, 5, 7 and 8 could not be eliminated by dilution.

The sensitivity and specificity of various serologic tests for antibodies to Toxoplasma gondii were compared in 1,000 naturally exposed sows, using isolation of viable T gondii as the definitive test. Serum samples obtained from heart blood of 1,000 sows from Iowa were examined for T gondii antibodies by use of the modified agglutination test (MAT), latex agglutination test (LAT), indirect hemagglutination test (IHAT), and ELISA. Toxoplasma gondii was isolated from 170 hearts of 1,000 sows by bioassays in mice and cats. The percentage of samples diagnosed as positive for each of the serologic tests was: MAT = 22.2% (titer >greater than or equal to 1:20), IHAT = 6.4% (titer greater than or equal to 1:64), LAT = 10.4% (titer greater than or equal to 1: 64), and ELISA = 24.1% (OD > 0.360). The sensitivity and specificity of these tests were calculated respectively to be: 82.9 and 90.29% for MAT, 29.4 and 98.3% for IHAT, 45.9 and 96.9% for LAT, and 72.9 and 85.9% for ELISA. The dye test was run at 1:20 dilution on only 893 sera because of bacterial contamination and presence of anticomplement substances. Dye test antibodies were found in 17.8% of the sera, and sensitivity and specificity were 54.4 and 90.8%, respectively. Thus, the MAT had the highest sensitivity among all serologic tests used.

Four-week-old chickens were inoculated orally with 1,000 or 100,000 oocysts of the ME-49 or GT-1 strain of Toxoplasma gondii, and their antibody responses were measured, using the direct modified agglutination test, latex agglutination test, indirect hemagglutination test, ELISA, and the Sabin-Feldman dye test. Antibodies against T gondii were detected by use of the modified agglutination test and ELISA within 2 weeks of oocyst inoculation, and antibodies persisted until termination of the study by postinoculation day 68. The latex agglutination test was insensitive in detecting T gondii antibodies, and antibodies were not detected by use of the dye and indirect hemagglutination tests. Of tissues bioassayed in mice for tissue cysts by pepsin digestion of individual organs of chickens on postinoculation day 68, tissue cysts were found in the brain of all 5, heart of 3, and leg muscles of 2, but not in the liver and breast muscles. None of the birds developed clinical toxoplasmosis.

Tonsils of 10 calves were inoculated with Pasteurella haemolytica (PH) and the degree of colonization was followed by collecting sequential tonsil wash specimens. Tonsils were colonized for at least 3 weeks after instillation of PH into the tonsillar sinus. Calves with colonized tonsils responded with serum and nasal secretion antibody responses to PH and to leukotoxin. Pasteurelia haemolytica was detected in nasal mucus specimens of 2 calves during the week after inoculation of the tonsils, but all other specimens were culture-negative. Infectious bovine rhinotracheitis virus-induced respiratory tract disease 25 days later did not elicit a population increase of PH in the tonsils, and did not elicit shedding of PH in nasal mucus.

Psittacine beak and feather disease (PBFD) virus was recovered from the feces and crop washings from various species of psittacine birds diagnosed with PBFD. High concentrations of the virus also could be demonstrated in feather dust collection from a room where 22 birds with active cases of PBFD were being housed. The virions recovered from the feces, crop, and feather dust were confirmed to be PBFD virus by ultrastructural, physical, or antigenic characteristics. Virus recovered from the feather dust and feces hemagglutinated cockatoo erythrocytes. The specificity of the agglutination was confirmed by hemagglutination inhibition, using rabbit antibodies against PBFD virus. During the test period, 26% (8 of 31) of the birds screened were found to be excreting PBFD virus in their feces, and 21% (3 of 14) of crop washings were positive for PBFD virus. Some birds in the sample group had active cases of diarrhea, whereas others had normal-appearing feces. Diarrhea was found to be the only significant indicator of whether a bird was likely to be excreting virus from the digestive tract. These findings suggest that exposure of susceptible birds to PBFD virus may occur from contact with contaminated feather dust, feces, or crop secretions. Viral particles that were morphologically similar to parvovirus (2- to 24 nm-icosahedral nonenveloped virions) also were recovered from feces of some of the birds.

A genomic library to Eperythrozoon suis DNA was constructed in lambda gt11, and from this library, E suis clone KSU-2 was identified as a potential diagnostic probe. In hybridization experiments that used 100-microliter samples of blood collected in chaotropic salt solutions, the KSU-2 probe hybridized strongly with purified E suis organisms and blood samples from splenectomized swine that were parasitized with E suis. However, the probe under stringent conditions did not give radiographic indications of hybridizing with equine blood DNA, bovine blood DNA infected with Anaplasma marginale, canine blood DNA infected with Ehrlichia canis, feline blood DNA infected with Haemobartonella felis, or uninfected swine blood DNA.

Serum samples obtained from feeder calves before and after entry into the market system (days 0 to 7) were assayed for antibodies to Pasteurella haemolytica biotype A, serotype 1 capsular polysaccharide (CPS) and lipopolysaccharide/outer membrane protein (LPSp) by isotype in a kinetic-augmented, antigen-capture ELSIA. These test results, plus indirect hemagglutination (IHA) antibody titers, and hemolysin-in-gel test (HIGT) findings were compared with clinical performance data during the initial 4 weeks in the feedlot (receiving period). High concentrations of HIGT antibody, at the point of initial assembly of feeder calves at weaning and during the subsequent 7-day marketing period, were associated with freedom from bovine respiratory disease (BRD) during the receiving period. High or rapidly increasing concentrations of anti-CPS IgG1 during the marketing period were also associated with less BRD. However, high concentrations of anti-LPSp IgG1 during the marketing period were associated with increased BRD during the receiving period. There was no correlation between the concentrations of antibody determined by IHA tests early in the marketing period and freedom from BRD during the receiving period. High concentrations of antibody determined by this test at entry into the feedlot (day 7) were associated with a high incidence of BRD. Calves vaccinated with a P haemolytica bacterin had significantly (P less than 0.05) higher HIGT values and concentrations of anti-LPPp IgG1 and IHA antibody than did nonvaccinated calves on entry into the feedlot (day 7). Vaccination appeared to have little effect on the amount of anti-CPS IgG1. Of all the tests used to quantitate antibody, the HIGT correlated best with clinical performance. High concentrations of HIGT antibody during the marketing period predicted freedom from BRD during the receiving period. When calves were vaccinated with tissue culture-derived and conventional P haemolytica bacterins in oil adjuvant at 28-day intervals and tested for antibody responses by isotype on days 0, 28, and 35, there were clear and highly significant increases in anti-LPSp IgG1 activity. Also, the amount of anti-LPSp IgM and the HIGT and IHA antibody titers increased significantly. In all tests, the antibody response was highest to the tissue culture-derived bacterin. High concentrations of HIGT antibody, as found in vaccinated animals, were correlated with resistance to transthoracic inoculation of virulent P haemolytica.