Characteristics of the polyriboinosinic acid:polyribocytidylic acid assay for noncytopathogenic bovine viral diarrhea virus.

The use of polyriboinosinic acid:polyribocytidylic acid (poly I:C) for noncytopathogenic bovine viral diarrhea virus (NC-BVDV) assay (PINBA) was studied. Several viruses were tested for their suitability as test challenge viruses. In addition to vesicular stomatitis virus, which previously was shown to be a suitable challenge virus, bovine enteroviruses also were found to be suitable, whereas infectious bovine rhinotracheitis virus and parainfluenza type 3 virus were only marginally suitable. Bovine embryonic skin (BES) cultures developed resistance to PINBA within a few in vitro passages, whereas bovine embryonic lung (BEL) cultures did not. At certain passages, BES cultures were 500,000 times more resistant than BEL cultures. To determine whether the difference in viral titers on BEL and BES cultures was due to NC-BVDV replication, PINBA and fluorescent antibody assays were compared on the cultures. Resistance of BES cultures to PINBA was not due to an inability of the virus to replicate in the cultures, but was due to an inability of PINBA to detect the virus. Viral titers were comparable by fluorescent antibody assay titers on BES and BEL cultures, but were considerably higher than viral titers on BES cultures with PINBA. Variations in viral titers, using PINBA on BEL cultures, were observed and were considered to be due to cultural conditions, such as the presence of low levels of BVDV antibodies in bovine fetal serum used in the medium. Treatment of BEL cultures with poly I:C or interferon showed that NC-BVDV was sensitive to interferon as determined by virus-yield reduction.

Effect of infectious bovine rhinotracheitis virus immunization on viral shedding in challenge-exposed calves treated with dexamethasone.

Calves not vaccinated with infectious bovine rhinotracheitis virus (IBRV) became latently infected when challenge exposed and treated with dexamethasone (DM). Calves that shed IBRV after DM treatment were considered to be latently infected. Vaccination with a temperature-sensitive intranasal vaccine or with formalinized IBRV in Freund's complete adjuvant (IBRV-FCA) protected some, but not all, calves against latent infection--indicating a role for the immune response in preventing latent infection. That all latently infected calves were not detected after DM treatment was indicated by the fact that after a 2nd DM treatment of 3 calves treated 6 months previously and not found to shed virus, 1 of the calves was latently infected. Latently infected calves were inoculated with successive doses of IBRV-FCA and treated with DM. Nonvaccinated calves shed virus, whereas vaccinated calves similarly treated did not shed virus. Because both groups had a comparable cell-mediated immune response, as determined by blastogenic response to IBRV, but the vaccinated group had significantly higher virus-neutralizing antibody titers, a role for humoral antibody in preventing viral shedding was indicated.

Association between route of inoculation with infectious bovine rhinotracheitis virus and site of recrudescence after dexamethasone treatment.

Four calves latently infected with infectious bovine rhinotracheitis virus (IBRV) were used to compare the ease of isolation of virus from neuronal ganglia and from mucosal surfaces. Two calves were slaughtered, and neuronal ganglia (cranial cervical, trigeminal, and 3rd and 4th sacral) were cocultivated on bovine fetal kidney cells. Virus was not isolated. Two calves given dexamethasone for 4 days were slaughtered on the 5th day. Virus was not isolated from cocultivated or macerated neuronal ganglia, but virus was isolated from nasal secretions taken from both calves on the day of slaughter. Eleven calves were inoculated with IBRV via different routes and were treated with dexamethasone 3 to 4 months after inoculation. virus was isolated from the nasal cavities, but not the vaginas of 6 heifers inoculated intranasally, and was isolated from the vaginas, but not the nasal cavities of 2 heifers inoculated intravaginally. Of 3 calves inoculated IV, virus was isolated from the nasal cavities of 3, from the oropharynxes of 2, and from the prepuce of 1.

Evidence for suppression or incomplete maturation of cell-mediated immunity in neonatal calves as determined by delayed-type hypersensitivity responses.

Fifteen bovine fetuses were inoculated in utero 20 to 123 days before birth with a mixture of killed Mycobacterium bovis, tetanus toxoid, and ferritin in Freund's complete adjuvant. On the day of birth (day 0) and when the calves were 21 days of age, the calves were skin-tested to each of the antigens for delayed-type hypersensitivity. Nine delayed-type hypersensitivity responses to the various antigens were obtained at the 0-day test, whereas 28 responses were obtained at the 21-day test. Of those responses that were positive, the mean differences in the double skin-fold thickness before testing and 48 hours later were 5.4 mm for the 0-day and 21-day test and 9.4 mm for the 21-day test. Six control calves that were not inoculated in utero were skin tested on days 0 and 21 and did not exhibit any positive reactions. There was no indication that the interval between immunization and birth had any effect on the immune response. Cellular characteristics of the reactions at 0 and 21 days were the same.

Viral contamination of bovine fetal lung cultures and bovine fetal serum.

Commercial bovine fetal serum (BFS) and bovine fetal lung (BFL) cells were tested for viruses. The only virus detected in any samples was noncytopathogenic bovine viral diarrhea virus (BVDV). Of 37 BFL cultures initiated, 34 were negative for BVDV, 1 was positive, and 2 were suspicious in that the source of BVDV contamination was not certain. Of 9 lots of irradiated sera tested, 1 (10%) was positive for BVDV; of 21 lots of nonirradiated sera tested, 13 (62%) were positive for BVDV. As judged by intensity of fluorescence in infected cultures, some cell strains were much more susceptible to BVDV than other strains. Heat inactivation of serum at 56 C for 30 minutes was found to be an unreliable method of eliminating BVDV from sera.

Factors affecting the assay of bovine type I interferon on bovine embryonic lung cells.

One preparation of interferon (IF) on 7-day-old bovine embryonic lung cultures free of bovine viral diarrhea virus had titers ranging from 20 to 10,240 in plaque-reduction tests, using bovine vesicular stomatitis virus. Factors believed to contribute to this variation were investigated. Although titers differed on different cell strains, different passages, and on cultures of different ages, variations between the two assays definitely could not be established as a function of these factors. However, IF titers on low passage cultures were usually lower than were titers on subsequent passages of the same cell strain. Calf serum in the diluent for IF reduced the titer one or two twofold dilutions. Over the range of 7 to 24 hours contact of IF with bovine embryonic lung cultures, and titer decreased steadily and was one twofold dilution less at 24 hours than it was at 7 hours. Latent viruses were not found in cultures by electron microscopy. Treatment of cultures with a noncytopathogenic strain of bovine viral diarrhea virus almost eliminated the effect of IF. Naturally occurring production of IF was not a major problem. While IF was on the cells, the pH of IF had a pronounced effect on the titer and may have been responsible for the vatiation observed with different passages, different cell strains, use of cultures of different ages, and use of calf serum in the diluent. Interferon at a low pH had a titer three or four twofold dilutions less than did the IF at a high pH.

Factors affecting the production of bovine type I interferon on bovine embryonic lung cells by polyriboinosinic-polyribocytidylic acid.

Variables believed to affect the amount of interferon (IF) produced in bovine embryonic lung cell cultures by polyriboinosinic-polyribocytidylic acid (poly (rI:rC)) were investigated. A concentration of 100 micron of poly (rI:rC)/ml consistently induced substantial amounts of IF. Bovine viral diarrhea virus infection of cultures did not interfere with induction of IF by poly (rI:rC). Neither the age of cells nor the passage level of the cultures seemed to affect the amount of IF produced. Priming of cultures with large concentrations of IF before treatment with poly (rI:rC) was more effective than priming with small concentrations of IF. Furthermore, priming for 24 hours was more effective than was priming for 12 hours. The use of 2% bovine fetal serum in the medium, subsequent to treatment with poly (rI:rC) markely enhanced IF production. Practically all IF was produced within a few hours after treatment with poly (rI:rC).

Kinetics of detection of blastogenic responses of neonatal calves inoculated in utero with tetanus toxoid, killed Mycobacterium bovis, and killed Brucella abortus.

Nine pregnant cows were laparotomized and their fetuses were immunized with tetanus toxoid, killed Brucella abortus, and killed Mycobacterium bovis. Blastogenesis assays and total leukocyte and differential counts were done when the calves were 1, 2, 3, 7, 14, 21, 28, and 60 days of age. Initial blastogenesis responses to antigens, phytohemagglutinin, and concanavalin A were not positive as frequently as were the responses obtained when the calves were 2 to 3 weeks of age. The probability of obtaining a positive response to an antigen was positively correlated with the magnitude of the response, as determined by delayed-type hypersensitivity skin reactions. Leukocyte and differential WBC counts in immunized calves were similar to those of unimmunized calves. The mean leukocyte count for the immunized calves remained near 16,000 cells/mm3; blood obtained in the first few days after birth contained a greater number of neutrophils than lymphocytes, whereas lymphocyte-to-neutrophil ratio gradually approached those of adult cattle, in which lymphocytes predominate.

Cell-mediated and humoral immune responses of cattle to Brucella abortus, Mycobacterium bovis, and tetanus toxoid: evaluation of immunization and assay techniques.

A concentration of 2.5 X 10(-5) M 2-mercaptoethanol (2-ME) added to the medium in lymphocyte blastogenesis assays increased both the uptake of [3H]thymidine in unstimulated lymphocyte cultures and the probability of detecting antigen-sensitized cattle. The use of 2-ME did not cause lymphocytes from unsensitized cattle to react positively in blastogenesis assays. A crude brucella lysate prepared from Brucella abortus strain 19 was compared with a well-characterized brucella protein allergen prepared from B melitensis and was found to be equally suitable for use in blastogenesis assays. Cell-mediated immunity was produced most effectively in 4-month-old calves by tetanus toxoid, then by Mycobacterium bovis, and least effectively by B abortus.

Bovine respiratory syncytial virus infection of bovine embryonic lung cultures: enhancement of infectivity with diethylaminoethyl-dextran and virus-infected cells.

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.

Bovine respiratory syncytial virus infection of bovine embryonic lung cultures: a kinetic study by the fluorescent antibody technique.

Development of fluorescence in bovine embryonic lung cells infected with bovine respiratory syncytial virus (BRSV) was studied by the fluorescent antibody (FA) test. Similar patterns of fluorescence were seen with the direct FA test, in which the immunoglobulin G fraction of antiserum to BRSV was conjugated with fluorescein isothiocyanate and used; and the indirect test, in which antiserum to the Long strain of respiratory syncytial virus and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G were used. In different trials, fluorescence was first detected between 16 and 18 hours after inoculation with BRSV. Fluorescence always was confined to the cytoplasm. Before 24 hours, fluorescence consisted of fine fibrils, usually parallel to the long axis of the cell, and cytoplasmic granules. After 24 hours, coincident with rounding of the cells, fluorescence slowly moved to the periphery of the cytoplasm. Under the growth conditions used, syncytia did not develop. By the FA test and as determined by the release of BRSV into the supernatant fluid, the minimal time for a single cycle of infection was between 24 and 26 hours.

Susceptibility of bovine macrophage and tracheal-ring cultures to bovine viruses.

Cultures of macrophages initiated from peripheral blood monocytes and organ cultures of tracheal rings were tested for their susceptibility to bovine viruses. With several notable exceptions, viruses cytopathogenic for bovine embryonic lung cultures were cytopathogenic for macrophages. Although cowpox virus replicated in macrophages, pseudocowpox did not, and although pseudorabies virus replicated within macrophages, infectious bovine rhinotracheitis and DN-599 herpesviruses did not. Bluetongue virus established an interesting relationship with macrophages. Whereas bluetongue virus was initially cytopathogenic for macrophages, it lost its cytopathogenicity on repeated passage, although it was capable of continued replication in macrophages. When subsequently passaged onto bovine embryonic lung cultures, it regained its cytopathogenicity. Parainfluenza-3, bovine viral diarrhea, and infectious bovine rhinotracheitis viruses readily destroyed ciliary activity in tracheal-ring cultures, as contrasted with the inability of bovine respiratory syncytial virus to destroy ciliary activity, even though bovine respiratory syncytial virus was able to replicate within ciliated epithelial cells of tracheal rings.

Bovine immunoglobulin G subclass receptor sites on bovine macrophages.

Macrophage cultures were initiated from bovine peripheral blood monocytes. The following antiserums were prepared: rabbit immunoglobulin (Ig) M anti-sheep erythrocyte (RBC) serum, bovine IgG1 anti-rabbit Ig serum, and bovine IgG2 anti-rabbit Ig serum. Erythrocytes treated singly with any of these serums failed to mediate attachment of RBC to macrophages. Erythrocytes treated with rabbit IgM anti-sheep RBC serum followed by either bovine IgG 1 or IgG2 anti-rabbit Ig serum attached to bovine macrophages, as seen microscopically. Results indicate that bovine macrophages contain receptors for both bovine IgG subclasses.

Bovine peripheral blood monocyte cultures: growth characteristics and cellular receptors for immunoglobulin G and complement.

Bovine peripheral blood monocytes were cultured in vitro for as long as 60 days. Although mitotic figures were not observed, the amount of cytoplasm was observed to increase; most cultures developed multinucleated cells, some containing as many as 200 nuclei. In multinucleated cells, the nuclei were peripherally distributed around a cytoplasmic matrix and surrounded by an undulating membrane. Receptors on macrophages were identified with erythrocytes sensitized with various immunoglobulins and complement (C'). A receptor for immunoglobulin G, but not immunoglobulin M, was detected. By the use of freshly prepared equine serum, a C' receptor was detected; however, by the use of freshly prepared bovine serum, C' receptor was not detected.

A paravaccinia virus isolated from cattle.

Isolation of viruses from calves with acute respiratory tract disease were attempted on bovine embryonic lung cell cultures. An isolate obtained from one calf with oral lesions and respiratory disease, designated 44-M-E482, was characterized as a paravaccinia virus on the basis of biological and physical properties. The calf from which the paravaccinia virus 44-M-E482 was isolated did not possess serum neutralizing antibody in its convalescent sera; neither did experimentally inoculated calves possess serum neutralizing antibody to the isolate. However, a low titer of serum neutralizing antibody was produced in one calf after several intravenous injections of the virus. Inoculation of calves with 44-M-E482 into the oral mucosa, skin, nasal cavity and pharynx did not cause any noticeable illness or lesions. The relation of 44-M-E482 to the viruses which cause bovine papular stomatitis and pseudocowpox is discussed.

Immunoglobulin concentrations and bovine enterovirus inhibitors in fetal bovine fluids.

Fluids from 53 bovine fetuses ranging in age from 90 to 240 days were examined for immunoglobulin G (IgG) and immunoglobulin M (IgM) and neutralizing activity to ten bovine viruses. Non-specific inhibitors to bovine enteroviruses were found in serum, allantoic, and amniotic fluids of most samples tested. In most cases, serum IgG were within normal values. Neither IgG nor IgM was detected in amniotic fluids, whereas 2 samples of allantoic fluid contained traces of IgG.

Antibody class and complement requirement of neutralizing antibodies in the primary and secondary antibody response of cattle to infectious bovine rhinotracheitis virus vaccine.

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.

Serological evidence for the association of bovine respiratory syncytial virus with respiratory tract disease in Alabama cattle.

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.

Antibody to viruses affecting cattle in commercial tissue culture grade fetal calf serum.

Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum.