ELISA method for detection of influenza A infection in swine.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to monitor virus shedding associated with experimental infection with a field strain of swine influenza in pigs. The assay consisted of a monoclonal anti-nucleoprotein capture antibody and a biotinylated rabbit anti-influenza A (H1N1) sandwich antibody. The antigen-capture system was capable of detecting as little as 1 ng/ml purified virus. The ELISA system surpassed egg cultivation procedures in the detection of low levels of shedding virus. Egg cultivation procedures indicated that most viral shedding had ceased by day 10 postinfection. In contrast, antigen-capture ELISA still showed an ongoing presence of viral antigen. A virus-capture ELISA, using this capture-sandwich antibody system, is equivalent in sensitivity to conventional egg inoculation procedures for the detection of the early phases of virus shedding. The automative potential of an ELISA-based system coupled with a substantially reduced assay time requirement give this virus-capture ELISA a distinct advantage over other cell culture or egg-based diagnostic techniques.

Host humoral factors in natural resistance to Haemophilus somnus.

Natural serum bactericidal activity against Haemophilus somnus in various age groups of cattle was investigated. The level of antibacterial reactivity correlated with the agglutinating titer and age of the given animal. This activity was complement dependent, but the concentration of bactericidal antibody appeared to be the limiting factor in sera which had reduced killing capacity. Immunized animals developed high serum concentrations of bactericidal activity. The results of this study indicated that bactericidal antibodies may be important in host defense from H somnus infection and that immunization can enhance this resistance by stimulating high serum concentrations of bactericidal activity.

Radiolabeled substrate assay to measure inhibition of growth of Haemophilus somnus by normal bovine serum.

An assay was developed to measure the inhibition of cell division of Haemophilus somnus by normal bovine serum. Dilution plate counts, turbidometric measurements, and release of radiolabeled cell constituents were found to be time-consuming or difficult to perform and reproduce. This assay determined the inhibitory effect of normal serum on population growth, by measuring the relative rate of macromolecular (DNA) synthesis in exponential cultures. This method was simple to perform and gave reliable results in 1 day. When this assay was used, a significant difference (P less than 0.025) in serum susceptibility was demonstrated between a H somnus isolate recovered from the trachea of a healthy calf and an isolate recovered at necropsy from a brain lesion of a cow. The implication of this finding, in relation to the pathogenicity of the organism, it briefly discussed.

Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin.

Timed cultures of Pasteurella haemolytica 12296 strain in RPMI 1640 medium (with L-glutamine, pH 7.4) were used to determine the correlation between cytotoxin production and the age of the culture. Cytotoxic activity was measured by a 51Cr-release assay and trypan blue exclusion test with bovine neutrophils as target cells. Results demonstrated that optimal cytotoxin production occurred during the logarithmic phase (peaked at 6 hours) and decreased during the stationary phase of bacterial growth. The cytotoxin was concentrated by sequential ultrafiltration on Diaflo XM 50, XM 100, and XM 300 membranes. The cytotoxin was retained on an XM 300 membrane. These studies indicated that the molecular weight of cytotoxic substance was 300,000 or more. The cytotoxin was heat labile, oxygen stable, and susceptible to extremes of pH and killed bovine neutrophils and mononuclear leukocytes. It was not hemolytic to bovine or ovine RBC. The cytotoxic activity was inactivated by trypsin and did not contain any detectable endotoxin. Bovine fetal serum and serum collected before immunization from neonatal calves did not neutralize the cytotoxic effects of toxin on neutrophils. However, adult bovine serum from 6 cows and an antiserum (against the cytotoxin) neutralized the cytotoxin, as revealed by both the 51Cr-release assay and the trypan blue exclusion test. This was confirmed by transmission electron microscopy. These results indicated that the cytotoxin may be antigenic in cattle. The significance and implications of these findings to bovine pasteurellosis are discussed.

Cytotoxic effects of Pasteurella haemolytica on bovine neutrophils.

The interaction of logarithmic- and stationary-phase organisms of Pasteurella haemolytica with bovine neutrophils was evaluated by an opsonocytophagic assay. Only 5% to 8% of the logarithmic-phase P haemolytica 12296 organisms opsonized with normal bovine serum or antiserum were phagocytized. Results from cytotoxicity assays, using the 51Cr-release technique and the trypan blue exclusion test, indicated that the logarithmic-phase organisms liberated a soluble material that was cytotoxic to neutrophils and destroyed their phagocytic capabilities. This hypothesis was verified by transmission electron microscopy studies. Opsonized stationary-phase organisms were completely phagocytized and degraded when exposed to neutrophils at a bacteria/neutrophil ratio of 10:1. However, at a high bacteria/neutrophil ratio of 100:1, only 31% of the bacteria were phagocytized. Prolonged incubation of this mixture resulted in cytotoxic changes in the neutrophils. Seemingly, excess unphagocytized bacteria liberated a soluble substance that was toxic to neutrophils. These findings were confirmed by cytotoxicity assays and transmission electron microscopy studies.

Kinetics of interaction and fate of Pasteurella hemolytica in bovine alveolar macrophages.

To study the role of pulmonary alveolar macrophages (PAMs) in phagocytizing Pasteurella hemolytica, we developed an in vitro cultivation method for preparing them. This procedure provided an adherent monolayer of PAMs which were nonspecific esterase-positive and phagocytized latex beads. The phagocytosis and fate of P. hemolytica (biotype A, serotype 1) by PAMs in suspension were studied. The kinetics of phagocytosis were determined by quantitatively measuring the uptake of 24-h [(3)H]thymidine-labeled bacteria by the PAMs in the presence of opsonins. Results showed that the uptake of P. hemolytica was enhanced in the presence of normal serum or antiserum. A total of 90% of the bacteria were phagocytized in the presence of normal adult bovine serum, and up to 95% were phagocytized in the presence of an antiserum. These studies also showed that normal serum, but not fetal calf serum, contained heat-stable natural antibodies which readily initiated the opsonization of P. hemolytica. The heat-labile complement system was also involved in the opsonization. The fate of P. hemolytica inside the PAMs was investigated by transmission electron microscopy and by the viable plate count method. Approximately 90% of the normal serum- or antiserum-opsonized P. hemolytica were phagocytized by PAMs at a bacteria/PAM ratio of 20:1 and were completely degraded after 60 min of exposure. Prolonged incubation of this mixture of bacteria and PAMs resulted in cytotoxic changes and destruction of PAMs. At a low bacteria/PAM ratio (10:1 or less), there was phagocytosis and killing of bacteria but no cytotoxic changes on the PAMs. The exact mechanism which initiated this phenomenon was not demonstrated. Perhaps toxic substance(s) released by the excess unphagocytized bacteria caused the cytotoxic changes to the PAMs.

Protection of C3H/He mice from experimental Borrelia burgdorferi infection by immunization with a 110-kilodalton fusion protein.

A 110-kDa Borrelia burgdorferi fusion protein, Escherichia coli expressing the fusion protein, transformed E. coli lacking the fusion protein insert, and lyophilized whole B. burgdorferi bacteria were compared for immunogenicity in C3H/He mice. Immunized mice were challenged with a variety of isolates from the United States or the European isolate P/Gau 3 weeks following the last inoculation. An average of 76.7% of the mice immunized with 25 micrograms of lyophilized whole B. burgdorferi cells were protected from infection, while 60% of the mice immunized with the 110-kDa fusion protein were protected. Whole E. coli bacteria expressing the fusion protein protected 57.7% of immunized mice against experimental challenge. Lower levels of protection occurred in mice challenged with the European isolate than in those challenged with isolates originating from the United States. These results demonstrate the potential of the 110-kDa fusion protein for use as a component of a subunit vaccine for prevention of Lyme borreliosis.