Effect of Brucella abortus transfer factor in preventing murine brucellosis.

Mice vaccinated with a protein extract of attenuated Brucella abortus strain 19 had increased resistance to infection with virulent B. abortus strain 2308 and had increased antibody responses to strain 2308. However, resistance to infection and antibody responses were not increased when nonvaccinated recipient mice were given transfer factor preparations that were obtained from either vaccinated donor mice or strain 2308-infected donor mice. Vaccination of mice with the strain 19 extract plus treatment with each transfer factor preparation also did not further increase resistance to infection or antibody responses when compared with mice that received the vaccine alone. These results suggest that transfer factor from mice that have either vaccine-induced protective immunity to B. abortus or active B. abortus infections does not enhance antibody responses and resistance to infection with B. abortus.

Unresponsiveness of vaccinated BALB/c mice to a second inoculation of lipopolysaccharide from Brucella abortus strain 2308.

A study was conducted to determine whether the protection induced in mice by a primary inoculation of lipopolysaccharide from Brucella abortus would be enhanced by a second inoculation given at different time intervals. Protection was challenged by exposure of the mice to a virulent culture of B. abortus strain 2308. Reduced mean viable count and/or splenic weights were the criteria of protection. There was no significant difference (P greater than 0.05) in the protective responses among mice given a single inoculation. Vaccinated mice were significantly (P less than 0.05) better protected than were nonvaccinated mice. Mice given vaccinal inoculations simultaneous with challenge exposure were less protected (P less than 0.001) than were mice vaccinated prior to challenge, but were better protected (P less than 0.010) than were nonvaccinated mice.

Role of immune responses to a GroEL heat shock protein in preventing brucellosis in mice vaccinated with Brucella abortus strain RB51.

Resistance to infection with virulent Brucella abortus strain 2308 and antibody and lymphocyte proliferative responses to a recombinant 60 kDa B. abortus GroEL heat shock protein were measured in mice vaccinated with attenuated B. abortus strain RB51. Mice at 12-20 weeks after vaccination with 5 x 10(8) colony forming units (CFU) of strain RB51 had increased resistance to infection with strain 2308 and increased antibody and lymphocyte proliferative responses to GroEL following challenge infection with 2308. However, these mice at 12-20 weeks after vaccination did not have greater resistance to infection than mice vaccinated with 5 x 10(6) CFU of strain RB51, which had no increased antibody or lymphocyte proliferative response to GroEL. These results indicate that mice vaccinated with strain RB51 can have antibody and cell-mediated immune responses to GroEL during infection with virulent strain 2308, although neither response appeared to have an essential role in vaccine-induced immunity to brucellosis.

Infectious bovine keratoconjunctivitis: comparison of infection, signs of disease and weight gain in vaccinated versus nonvaccinated purebred Hereford heifer calves.

To determine whether the carrier state of Moraxella bovis could be prevented under natural conditions of exposure by vaccination, purebred Hereford calves were vaccinated twice; once in June 1982, and again in September 1982. Eyes of calves were examined for signs of infectious bovine keratoconjunctivitis (IBK) and cultured for M. bovis in June 1982, September 1982, January 1983 and June 1983. When calves were compared on the basis of selection lines (four genetic groups), there were significant (P less than 0.05) differences in the percentages of infection, disease and weight gain between selection line calves, regardless of their vaccination status. Results suggest that vaccination, in conjunction with clinical infectious, bovine keratoconjunctivitis, reduces the carrier state of M. bovis in the herd but the immunity lasted less than 9 months. The disproportionate representation of the most genetically resistant calves among nonvaccinated calves probably accounted for the equivocal results between vaccinated and nonvaccinated calves.

Identification of bovine carriers of Moraxella bovis by comparative cultural examinations of ocular and nasal secretions.

The carrier state of Moraxella bovis was investigated, using bacteriologic examinations of ocular and nasal secretions from cattle under experimental and natural conditions of exposure and management. Moraxella bovis was isolated throughout the year from the ocular and nasal secretions of cattle naturally affected with infectious bovine keratoconjunctivitis. There was also 1 case of nasal transmission of M bovis without isolation of M bovis from ocular secretions and 1 case of M bovis isolation from the vagina of a calf contracted by contact with a calf affected with infectious bovine keratoconjunctivitis. The frequency of isolations and duration of infections, as determined by examination of ocular and nasal secretions, indicated that these secretions were comparable in the identification of M bovis carriers. The increased cultural isolations of M bovis from nasal secretions after shipment relative to the number of isolations before shipment indicated that shipment may serve as a stress factor causing an increase in the number of carriers.

Identification of colonies of Moraxella bovis by plate epi-immunofluorescence.

A modification of the plate epi-immunofluorescence test was found to be a reliable method for the positive identification of colonies of Moraxella bovis when fluorescein isothiocyanate-conjugated globulin prepared against pili from selected strains of M bovis was used. A cross-reaction with other members of the genus Moraxella or other moraxella-like organisms was not observed, except with an equine Moraxella sp, which produced a weak reaction.

Plasmid profiles of Moraxella bovis isolates.

Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

Experimentally induced infectious bovine keratoconjunctivitis: relationship of vaccination schedule to protection against exposure with homologous Moraxella bovis culture.

Calves were intramuscularly vaccinated with a formalin-killed Moraxella bovis culture. They were given 3 doses of the vaccinal culture at 3 different intervals between injections, as follows: 7-day intervals (group I), 14-day intervals (group II), an21-day intervals (group III); 14 days after the 3rd vaccinal dose was given, calves in all groups were challenge exposed to virulent homologous M bovis. The challenge bacterium became established in all (100%) of the control (group IV, N equals 8) and group I (N equals 8) calves, in 7 (87%) of the group II calves (N equals 8), and in 5 (62%) of the group III (N equals 8) calves. Signs of infectious bovine keratoconjunctivitis (IBK) developed in 8 (100%) of the controls, in 6 (75%) of the group I calves, in 1 (12%) of the group II calves, and in 2 (25%) of the group III calves. These results indicate that at least 14 days should be allowed between successive doses of this experimental vaccine.

Infectious bovine keratoconjunctivitis: a ring device designed for prolonged retention in the bovine eye.

A study was conducted to determine whether treatment devices could be designed for prolonged retention in the eyes of cattle. Experimental design ring devices were compared relative to their ease of insertion, induction of adverse tissue reaction, and period of retention. The ring devices were also compared with commercially available devices used in human eyes. Ring devices having a circumference smaller than that of the conjunctival sac, but larger than that of the globe of the eye, were easy to insert, caused a minimal tissue reaction, and were retained by the eye up to 19 days. The authors suggest that a development using the ring design and an erodable matrix containing an antibacterial agent could lead to a possible method for the treatment of obstinate infections, such as infectious bovine keratoconjunctivitis (IBK) and Moraxella bovis carrier states in cattle.

Chemical and protective properties of Brucella lipopolysaccharide obtained by butanol extraction.

Lipopolysaccharide (LPS) fractions were obtained from smooth cultures of Brucella abortus strains 2308 and S-19 by butanol extraction procedures. The LPS from the initial butanol extraction contained 10 to 15% protein and was reduced to less than 1% protein by treatment with proteinase K. The LPS fractions were identified and characterized on the basis of the chemical analysis, sodium dodecyl sulfate gel electrophoresis, cesium chloride gradients, electron microscopy, and gel immunodiffusion. Results indicated that the butanol procedure is a reliable method in the extraction of LPS from Brucella abortus cells. Proteinase K-treated LPS containing less than 1% protein from strain 2308 was used to vaccinate BALB/cByJ mice. Immune and protective criteria for vaccinated and nonvaccinated mice were increased immunoglobulin (IgG and IgM) titers in sera of prechallenge-exposed mice, reduced colony-forming units/spleen, and splenomegaly in post-challenge-exposed mice. Results indicated that proteinase K-treated LPS was immunogenic as well as protective for mice.

Duration of strain 2308 infection and immunogenicity of Brucella abortus lipopolysaccharide in five strains of mice.

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as between vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.

Infectious bovine keratoconjunctivitis: subconjunctival administration of a Moraxella bovis pilus preparation enhances immunogenicity.

To compare the immune response elicited by 3 routes of vaccination, 36 calves were randomly allotted to 4 groups of 9 calves each. Group I was vaccinated subconjunctivally only. Group II was vaccinated concomitantly, both subconjunctivally and in the dewlap. Group III was vaccinated in the dewlap only. Group IV was not vaccinated and served as a virulence control for the Moraxella bovis culture. Calves in groups I, II, and III were given 2 inoculations with 14 days between inoculations. Twenty-one days after the last inoculation, the ventral conjunctival sac of all calves was instilled with cells of a virulent M bovis strain. After challenge exposure, all vaccinated calves (groups I, II, and III) had evidence of enhanced resistance, compared with the nonvaccinated calves. The highest to lowest gradients of immune responsiveness were: Group I greater than or equal to group II greater than group III greater than group IV. When immune criteria, such as the percentage of diseased eyes, the mean duration of infection, the severity of corneal lesions, and the serologic response were compared, groups I and II were significantly (P less than 0.05) more resistant to challenge exposure than were groups III and IV. Group III was significantly (P less than 0.05) different from group IV in severity of lesions and in serologic response. Also, the mean duration of infection was shorter, and the percentage of diseased eyes was less in group III than in group IV (P greater than 0.10). Group I was more resistant than was group II (P less than 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)

Infectious bovine keratoconjunctivitis: enhancement of Moraxella bovis pili immunogenicity with diphtheria-tetanus toxoids and pertussis vaccine.

A study was conducted to determine whether diphtheria-tetanus-toxoids and pertussis vaccine (DPT) would enhance the immunogenicity of homologous Moraxella bovis pili fractions. Thirty-six calves were divided into 4 groups (I, II, III, and IV) of 9 calves each. Calves in group I were not vaccinated and served as controls. Calves in group II were vaccinated with pili fractions only. Calves in group III were vaccinated with DPT only. Calves in group IV were vaccinated with DPT and pili. Vaccination consisted of 2 inoculations, 21 days apart. Fourteen days after the last vaccinal inoculation was done, the eyes of all calves were exposed to a hemolytic homologous strain of M bovis. The percentage of eyes with disease was significantly less in calves given DPT and pili (P less than 0.001) and calves given pili only (P less than 0.05) than in calves given DPT only or nonvaccinated calves. The lesions were less severe in calves vaccinated with pili only than in calves not vaccinated with pili. Serologic results also showed a positive relationship between the development of serum antibodies against pili and immunity. The results indicate that DPT enhanced the immune response and if used as an adjuvant, might be useful in the development of a vaccine against infectious bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis: effects of vaccination on Moraxella bovis carrier state in cattle.

A study was conducted to determine whether vaccination of cattle while they were undergoing an acute episode of infectious bovine keratoconjunctivitis (IBK), would cause vaccinated cattle to abort Moraxella bovis infection sooner than nonvaccinated cattle. Fourteen calves were allotted into two groups of seven calves each, and the eyes of each calf were exposed to a virulent culture of M bovis. Twenty days after calves were infected and showing signs of IBK, seven calves were vaccinated with M bovis pilus vaccine made from the exposure strain. Ocular and nasal discharges were collected and examined for M bovis for 64 days. Most calves developed signs of IBK after exposure and all but one calf (nonvaccinated) developed ocular infection with M bovis. The mean number of days (33 and 33.3, respectively) of ocular infection was not significantly (P = 0.05) different in the vaccinated calves than in the nonvaccinated calves. The eyes of the calf that did not become infected with M bovis were infected with nearly a pure culture of Escherichia coli. This calf failed to develop ocular infection, but had M bovis in its nasal discharge throughout the study (64 days). Moraxella bovis was isolated from the nasal discharge of other calves only when there were concurrent ocular infections.

Wavelength of ultraviolet radiation that enhances onset of clinical infectious bovine keratoconjunctivitis.

Cellulose acetate filtered ultraviolet (UV) radiation and unfiltered UV radiation were used on calves that were subsequently challenge exposed with Moraxella bovis. The onset, course, and severity of infectious bovine keratoconjunctivitis (IBK) were studied. Ten calves irradiated with unfiltered UV had the disease 1 to 2 days after M bovis challenge exposure; all 10 calves had moderate to severe IBK. Ten calves irradiated with filtered UV and 10 calves not irradiated manifested IBK in a similar manner. The date of onset and severity of the disease varied. Evidence is presented to support the contention that the wavelengths (around 270 nm) which are eliminated by cellulose acetate enhance the course of IBK. The effects on IBK of environmentally increased solar UV radiation is also discussed.

Serologic response of vaccinated cattle to strains of Moraxella bovis isolated during epizootics of keratoconjunctivitis.

In studies to determine whether there were antigenic differences between strains (isolates) of Moraxella bovis, the sera from vaccinated calves were tested with isolates of M bovis while the calves were experiencing epizootics of infectious bovine keratoconjunctivitis (IBK). Before the epizootics of IBK, the calves were intramuscularly vaccinated with a formalin-killed autogenous M bovis bacterin. During the epizootics, the eyes were examined by cultural technique, and isolates which were obtained were categorized by catalase activity, source (diseased or nondiseased eyes), and reactivity with the various sera. The serum reactivity of the isolates was compared with that of the vaccinal strain. The vaccinal strain and 8 of the 1 5 selected isolates obtained during the 1974 epizootic were catalase negative. Seven of the 15 isolates from the 1974 epizootic and all of the selected isolates from the 1975 epizootic were catalase positive. A significantly higher (P less than 0.01) percentage of calf sera were serologically reactive with the vaccinal strain and other catalase-negative isolates (45.0%) than with catalase-positive isolates (34.8%). The results, although not definitive, suggest that there may be antigenic differences among strains of M bovis. These differences should be considered when cattle are vaccinated against IBK under natural conditions of exposure.

Experimentally induced infections bovine keratoconjunctivitis: effectiveness of a pilus vaccine against exposure to homologous strains of Moraxella bovis.

A study was conducted to determine whether vaccination with sonicated pili of Moraxella bovis would protect cattle from subsequent infection and disease when experimentally challenged by exposure to homologous cultures of M bovis. Some calves were intramuscularly inoculated 2 times with pili of M bovis suspended in water, and others were subcutaneously inoculated 2 times with pili of M bovis suspended in oil; 21 days were allowed between inculations. Controls were nonvaccinated calves. Fourteen days after the last inculation, all calves were exposed to virulent homologous cultures of M bovis. The results indicated that vaccination with sonicated pili of M bovis may induce protective immunity against homologous strain challenge exposure. Vaccines in oil that were injected subcutaneously protected to a greater extent than did vaccines in water that were injected intramuscularly. The development of inflammatory nodules at the site of inoculation was associated with resistance to infection and disease. Only 1 of the vaccinated calves that resisted disease lacked precipitating antibodies against sonicated pili at the time of the challenge exposure. This calf had antibodies 2 weeks later.

Induced infectious bovine keratoconjunctivitis: vaccination with whole cell bacterins of Moraxella bovis mixed with Freund's incomplete adjuvant.

Our studies on vaccination against infectious bovine keratoconjunctivitis were extended to include the use of adjuvant bacterins. Seventeen calves were given 2 doses of killed Moraxella bovis mixed with Freund's incomplete adjuvant; 15 calves were given 2 doses of whole cell bacterin without adjuvant. Four weeks later, all vaccinated and non-vaccinated (control) calves were challenge exposed by the conjunctival administration of virulent M bovis. The incorporation of Freund's incomplete adjuvant did not enhance the immunogenicity of M bovis bacterin for protection of calves against infectious bovine keratoconjunctivitis. Results indicated that the use of Freund's incomplete adjuvant does not compensate for a reduction in the number of doses which are required for protective vaccination of cattle.

Isolation of Moraxella bovis ribosomes and their subsequent use in a vaccine against infectious bovine keratoconjunctivitis.

A study was conducted to determine whether a Moraxella bovis ribosomal vaccine would protect calves from infectious bovine keratoconjunctivitis (IBK). Each of 16 calves were given 2 inoculations 21 days apart. Twenty-one days after the 2nd inoculation, 8 of the calves were challenge exposed with a homologous strain culture and 8 calves were challenge exposed to a heterologous strain culture of M bovis. Sedimentation velocity analysis of the ribosomes used in this study indicated that they were mostly 30S and 50S subunits. Chemical assays indicated that the ribosomes were composed of 64% to 65% RNA and 35% to 36% protein. The cesium chloride buoyant density of the ribosomes was 1.62 g/ml. Ribosomes used as antigen gave 1 line of precipitation in a gel-diffusion precipitin test with hyperimmune serum against the whole-cell antigen of the homologous strain of M bovis. The eyes of all the experimentally exposed calves became infected and all calves developed clinical signs of either unilateral or bilateral IBK. None of the sera of the vaccinated calves had detectable precipitins against the ribosomal antigen at the time they were challenge exposed, but most of the sera had precipitins against whole-cell and pilus antigens. The results indicate that M bovis ribosomes, although similar to other bacterial ribosomes, did not protect cattle against IBK.

Experimental infectious bovine keratoconjunctivitis: efficacy of a vaccine prepared from nonhemolytic strains of Moraxella bovis.

Calves vaccinated with a bacterin prepared from 4 nonhemolytic strains of Moraxella bovis were challenge exposed with a virulent hemolytic strain of M bovis. Ten calves were given 2 vaccinal inoculations 21 days apart. Twenty-one days after the last vaccinal inoculation, the eyes of the 10 vaccinated and the eyes of the 12 non-vaccinated calves were exposed to a hemolytic heterologous strain of M bovis. The percentage of eyes with infectious bovine keratoconjunctivitis was lower, the lesions were less severe, and the duration of infection was shorter in vaccinated calves than in nonvaccinated calves. Seemingly, the resistance induced by nonhemolytic strains of M bovis was at least comparable with the induced by hemolytic virulent strains of that organism. The nature of the immune response in vaccinated calves is discussed.