Microneutralization tests for detection of antibody in bovine serum to three bovine viruses are described. The Madin-Darby bovine kidney cell line was used with parainfluenza 3 virus (PI 3), whereas serially cultivated bovine embryonic kidney cells were used for infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Comparison of micro-hemagglutination-inhibition (HI) with micro-serumneutralization (SN) tests for PI 3 showed the SN test to be more sensitive, more specific, and therefore more useful than the HI test for detecting antibody. Although the effect of trypsin-periodate treatment of serum was to reduce the HI titer of numerous sera by a twofold dilution, sufficient evidence could not be found to indicate that nonspecific HI inhibitors to PI 3 are present in bovine sera.
Six-month-old calves were given one injection or two injections 14 days apart, with a vaccine strain of infectious bovine rhinotracheitis (IBR) virus. Six months ]ater the calves received a similar injection. Serum neutralizing antibody titers to IBI~ virus were determined with and without complement (C'). Nine days after the 1st injection, titers were 16-to 64-fold higher with than without C'. Complement-requiring neutralizing (CRN) antibodies rapidly decreased relative to non-CRN (NCt~N) antibodies. Titers 6 months after the 1st injection, although low, were the same or twofold higher with than without C'. An injection of IBR virus at 6 months stimulated the production of NCRN, but not CRN, antibodies. Differences between titers determined with and without C' of healthy, adult cattle were low. It was concluded that early in the primary response CRN antibodies were preferentially produced compared with NCt~N antibodies and, that if this phenomenon occurs in naturally infected cattle, this observation could be useful from a diagnostic point of view. A possible effect of the injection at 14 days in prolonging the CRN antibody response was also observed.
A serological investigation for neutralizing antibody to bovine respiratory syncytial virus (BRSV) was conducted using an antigenically related human strain of respiratory syncytial virus. Results demonstrated the effectiveness of the procedure. Sixty-seven percent of healthy, adult cattle tested were found to have antibody to BRSV. Calves in several herds were exposed to conditions to encourage development of respiratory tract disease. In three herds in which respiratory tract disease subsequently developed, the incidence of BRSV seroconversions approached 100%. In a herd in which no respiratory tract disease was detected, BRSV seroconversions indicated a high incidence of subclinical infections. It was concluded that, in this country as has been shown in others, BRSV is probably a significant pathogen of the bovine respiratory tract.
The effects of incorporating diethylaminoethyl-dextran (DEAE-D) in the inoculum with bovine respiratory syncytial virus (BRSV) on the infectivity of BRSV was evaluated. A concentration of 40 microgram DEAE-D/ml provided maximal enhancement of infection as determined by the time of onset of cytopathic effect (CPE), the percentage of cells infected by the inoculum, and the amount of virus produced. When DEAE-D was used in the inoculum, the CPE appeared a day earlier, the percentage of cells infected by the inoculum, as determined by the fluorescent antibody test, was increased 11 times, and the viral titer was increased 2 times as compared to results obtained without DEAE-D. Bovine respiratory syncytial virus-infected cultures contained much cell-associated virus which could be liberated by sonication to increase the titer of virus stocks. The use of BRSV-infected cells rather than supernates from BRSV-infected cells increased the rate at which a cytopathic effect developed, although it did not substantially increase the titer of virus which was harvested. The use of DEAE-D in the inoculum and the passage of BRSV-infected cells instead of viral suspensions was found to be the quickest and most effective method of consistently obtaining BRSV with a titer of about 10(5.5) TCID50/ml.
Calves responded to a single intramuscular injection of an attenuated strain of infectious bovine rhinotracheitis virus by producing IgM followed by IgG antibody. Both IgM and IgG antibody produced during the first month were primarily complement-requiring neutralizing antibody (CRNAb), especially IgM antibody. After a month, IgG had replaced IgM as the predominant immunoglobulin, and titers with and without complement (C') decreased in both IgG and IgM fractions. The largest decrease was in the IgM CRNAb fraction. Seven days after a second injection given on day 196, calves responded with an anamnestic IgG response in which CRNAb titers were 1 or 2 two-fold dilutions higher than non-CRNAb titers. One calf developed an IgM response similar to its primary response, whereas inhibition of the IgM response occurred in the other 3 calves which had much lower IgM antibody titers than those attained in the primary response. Twenty-eight days after the second injection the titers of IgG were the same or only a 2-fold dilution less than their 7-day secondary titers, whereas IgM titers generally decreased considerably more than this. Guinea pig and rabbit sera were equally effective as C' sources in potentiating CRNAb, whereas bovine serum was a poor C' source.
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