Experimental Production of Infectious Bovine -Keratocon junctivitis: Comparison of Serological and Immunological Responses Using Pili Fractions of Moraxella bovis G. W. Pugh, Jr. and D. E. Hughes*
ABSTRACT
RASUME
The effect of vaccinating cattle and mice onu the development of keratoconjunctivitis was studied. Cattle were vaccinated with whole cells, disrupted cells and pili fractions of three strains of Moraxella bovis. Mice were vaccinated with pili fractions of three strains. The resistance of all vaccinated animals was challenged with virulent cultures of M. bovis. In an attempt to correlate the response seen after vaccination and challenge with a pili fraction of M. bovis, vaccinated cattle and mice were grouped on the basis of signs of disease manifested and compared on the basis of serological responses. Serum samples were tested for antibodies by a gel diffusion precipitin test. A greater number of the sera of resistant cattle had antibodies to the homologous pili antigen than those of vaccinated nonresistant cattle. Cattle vaccinated with disrupted cells were not resistant to infectious bovine keratoconjunctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to challenge exposure by homologous than heterologous cultures. A greater number of the sera of resistant mice had antibodies to pili antigens than nonresistant mice.
Cette experience visait a etudier l'effet de la vaccination de veaux et de souris, sur le developpement de la kerato-conjonctivite. On vaccina les premiers avec des microbes entiers ou fragmentes, ainsi qu'avec des cils de trois souches de Moraxella bovis. Quant aux secondes, on ne les vaccina qu'avec des cils des trois mimes souches du microbe. On eprouva ensuite la resistance de tous les animaux vaccines avec des cultures virulentes de M. bovis. Afin d'etablir une relation entre la reaction immunologique consecutive a la vaccination et l'infection avec des cils de M. bovis, on groupa les veaux et les souris vaccines d'apres les signes cliniques qu'ils manifesterent et on les compara d'apres leur reaction immunologique. On proceda a la recherche d'anticorps seriques, a l'aide de l'epreuve de diffusion en milieu gelifie. Un plus grand nombre d'echantillons de serum provenant de veaux r6sistants recelaient des anticorps a l'endroit de l'antigene homologue a base de cils, comparativement aux veaux vaccines, mais non r6sistants. Les veaux vaccines avec des microbes fragmentes ne s'avererent pas r6sistants 'a la keratoconjonctivite et leur serum etait depourvu d'anticorps a l'endroit de l'antigiene prepare avec des cils. Les souris vaccin6es resisteirent mieux a l'infection par des cultures homologues qu'heterologues. Plus d'echantillons de serum des souris resistantes poss&daient des anticorps a l'endroit de l'antigene prepare avec des cils, comparativement aux souris non resistantes.
*?National Animal Disease
Center, North Central Region, Agricaitural Research Service, US. Department of Agrielture, Ames, Iowa 50010. Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suftable. Submitted June 6, 1975.
60
Can. J. comp. Med.
INTRODUCTION
Recent studies have shown that cattle be protected from subsequent infection and disease due to Moraxella bovis through vaccination with whole cell cultures (4-6). Also, cattle previously affected with infectious bovine keratoconjunctivitis (IBK) were somewhat resistant to infection and disease. These resistant cattle had antibodies to M. bovis (1, 4-6) but the specific antigenic stimulants, whether of extra- or intracellular origin, were not apparent. Workers thought that the nature of the antigenic stimulant should be determined to facilitate the development of a vaccine against IBK. We hypothesized that this could be partially determined from results obtained by vaccinating cattle and mice with a specific fraction of M. bovis and then by challenging their immunity and by making inferences based on serological reactions between the specific fraction and the sera of resistant cattle and mice using the gel diffusion precipitin test (GDPT). Others (2, 8) have suggested that the pathogenicity of M. bovis is associated with fimbriation (presence of pili). This suggested to us that the pili should be a good fraction with which to begin our study. This report describes a study to determine whether pili fractions of M. bovis could be correlated to a specific immune response in cattle and mice.
can
MATERIALS AND METHODS Animals Cattle: Ten calves were specifically used for the present study. The other 124 calves were part of allied studies (Hughes and Pugh, unpublished data, 1973) and will be mentioned only briefly. All calves were healthy, 70 to 90 days old and of mixed breeding. Their eyes were culturally negative for M. bovis. Their prevaccination sera were free of antibodies to M. bovis. The calves were reared in isolation quarters as previously described (4, 5). Mice: A total of 108 healthy mice weighing 16-20 gm were used. Their eyes were
Volume 40
-
January, 1976
normal and culturally negative for M. bovis. The mice were divided into three groups of 36 each. Twenty-seven mice in each group were vaccinated with each strain of pili vaccine. The other nine mice in each group served as unvaccinated controls. Animal quarters - The calves were quartered in indoor isolation units. The quarters were air conditioned and mechanically ventilated with filtered air. The room had individual stalls, each large enough to accommodate three calves. Calves were assigned to stalls according to the challenge exposure organism they were to receive. Flies were excluded, and complete insect control was maintained with pyrethrin aerosol spray. Of the six calves vaccinated with disrupted cells of strain Epp-63, three were placed in one stall and three in another. Because the calves vaccinated with pili fractions were tested at the same time as calves in another experiment (Hughes and Pugh, unpublished data, 1973), animals from both experiments were housed and grouped together. The mice were housed in mouse cages within a laboratory room. EXPERIMENTAL DESIGN Differences in the serological response of resistant and nonresistant cattle and mice were determined by using pili of strains of M. bovis as antigens in the GDPT. Pili antigen made from each of three strains of M. bovis were used to vaccinate cattle and mice (Tables I and III). Six calves were vaccinated with disrupted cells. Forty-five cattle were vaccinated with formalized whole cells prepared from M. bovis strain Epp-63, 24 from strain Fla-64 and 24 from strain IBH-64. Later they were challenge exposed by instilling the conjunctival sac with three virulent strains of M. bovis (Table III). Sera from the calves collected prior to exposure were grouped on the basis of signs of disease
developed. Organisms - Three strains of M. bovis, identified as Epp-63, Fla-64 and IBH-64 were used. These strains were isolated from cattle naturally affected with IBK and, on the basis of other studies, were found to be pathogenic for cattle (7, 9-11). Smooth' 'Smooth means the friable, flat, agar corroding, autoagglutinable colony of M. bovis typically found on bovine blood &-ar vlate.' on initial isolation from eyes of cattle affected with IBK.
61
TABLE I. The Response of Calves Vaccinated with Pili Vaccine to Ocular Inoculation of Moraxella bovis
M. bovis Became Established Unilateral Bilateral None
Development
of Challenge Calf Developed Infectious Vaccine used Strain No. Antibodies Keratitis Pili of strain Epp-63 Epp-63 7261 Homologous Unilateral Pili of strain Fla-64 .Fla-64 7263 Homologous Unilateral IBH-64 Pili of strain IBH-64 7264 Homologous None Mixed pili of strains Epp-63, Fla-64 and IBH-64 7259 HeterologousFla-64 Bilateral Bilateral -Calf developed antibodies to pili antigen of each of the three strains but the lines were of nonidentity
hemolytic colonies of the three strains were used to prepare pili vaccines, to prepare test antigens, to prepare formalized whole cell vaccines and for challenge exposure. Frozen cultures for each strain representing the fourth bovine blood agar passage (BAP) were streaked on blood agar plates. The inoculated plates were incubated for 24 hours at 37°C and for 24 additional hours at 25°C, at which time typical smooth hemolytic colonies were picked and used to inoculate additional plates. The latter plates were incubated at 37°C for 24 hours and the cells were collected into sterile, distilled water and stored at -60°C until used to prepare pili antigens or vaccines. The pili fractions were prepared similarly to a method previously described (14). Sixteen grams (wet weight) of each culture (strain) were suspended in 100 ml of cold distilled water, washed once and then vigorously agitated in a virtis homogenizer2. The homogenate was differentially centrifuged3 and the final precipitate was dissolved in 3.0 ml of cold distilled water and dialyzed for three days with frequent water change in the cold in order to remove the MgCI2. The preparation was observed under an electron microscope to verify the presence of pili. The suspension of pili was diluted twofold with water and sonicated4 for 30 minutes at 1.25 amperes. The pili concentration of the three strains was standardized by dilution with water so that the pili preparation for each of the three strains gave 25 % light transmittance at
540 nm'. The preparation representing each strain was placed in vaccine bottles and stored at -60°C until used as antigen or in vaccine. All procedures were carried out at 40C. Disrupted cell fractions (DC-1) were prepared from typical smooth cultures of M. bovis strain Epp-63. Ninety gm of cells harvested from 24-hour BBA culture was suspended in 123 ml of cold distilled water. The cells were further diluted with water to facilitate the passing of the suspension into a fractionator6 as previously described (13). The effluent (DC-1) was collected, freeze-dried and stored at -60°C until used to vaccinate cattle. Cultures of the three strains for challenge exposure were grown as described for the cell fraction preparation except that trypticase soy broth (TSB) was used to harvest the cells. The harvested cells were stored at -600C until used to challenge expose cattle and mice.
Vaccination and challenge exposure Cattle: Each of four calves was vaccinated by injecting a pili preparation alternately into the right or left semitendinosus muscle. Calves were given three injections with two weeks between each injection. Calves 7261, 7263, 7264 and 7259 were vaccinated with pili strains Epp-63, IBH-64 and Fla64 and an equal mixture of the three preparations, respectively. The eyes of calves 7261, 7263 and 7264 were challenge exposed to virulent smooth hemolytic sixth BAP cultures representing the homologous strains used in the pili vaccine. Calf 7259 was challenge exposed to Fla-64 smooth
2Virtis Homogenizer, The Virtis Co. Inc., Gardiner, New York. 3Beckman Model L Preparative Ultracentrifuge, Beekman Instruments Inc., Palo Alto, California. 4M.S.E. Ultrasonic Disintegrator, Measuring and Scientific Equipment Ltd., Spenser St., London S.W.I., Great Britain.
62
5Spectronic 20 Colorimeter, Bausch and Lomb Inc., Rochester, New York. 6Sorvall Ribi Cell Fractionator, Model RF-I, Ivan Sorvall Inc., Norwalk, Connecticut.
Can. J. comp. Med.
TABLE II. Persistency of Moraxella bovis Strains and Development of Antibodies in Mice Vaccinated with Pili Vaccines
Vaccine Used Epp-63
Epp-63
Challenge Straina Epp-63 Fla-64 IBH
Period M. bovis Recovered 2 days 14 days No. mice No. eyes No. mice No. eyes 9 16 1 1 7 11 1 1 5 7 0 0 5 9 0 0 9 16 0 0 8 14 0 0 4 5 0 0 9 16 4 3 4 8 0 0 9 15 0 0 6 12 2 2 1 1 0 0
Epp-63 Control Epp-63 Fla-64 Fla-64 Fla-64 Epp-63 Fla-64 IBH-64 Control Fla-64 IBH-64 IBH-64 IBH-64 Epp-63 IBH-64 Fla-64 Control IBH-64 Nine mice were used for each challenge bSera with 2 lines had one line of identity
hemolytic sixth BAP culture. All eyes were exposed by instilling 1.0 ml (1.7 x 106 to 2.5 x 106 organisms) of TSB suspension of the respective culture into the ventral conjunctival sac of each eye. Six calves were vaccinated by injecting 0.045 g of DC-1 suspended in 1.0 ml of distilled water alternately into the right or the left semitendinosus muscle. Calves were given three injections, with two weeks between each injection. Fourteen days after the last injection the calves were challenge exposed by instilling 0.5 ml of M. bovis Epp-63 (1.7 x 10,6 to 2.5 x 108 cell) TSB suspension into the ventral conjunctival sac of each eye. The cattle whose sera were used to test against the pili antigens were vaccinated with formalized vaccines made of EPP63, Fla-64 and IBH-64. They were injected with 1.0 ml of the vaccine alternately into the right or left semitendinosus muscle. Calves were given three injections with two weeks between each injection. Fourteen days after the last injection the calves were challenge exposed by instilling 0.5 ml suspended in TSB of a sixth passage BAP hemolytic culture into the ventral conjunctival sac of each eye. Mice: Mice were injected with 0.1 ml of the respective vaccine alternately into the right or left hip. Mice were given three injections with two weeks between each injection. Fourteen days after the last injection each mouse was challenge exposed by smearing the eyeballs, conjunctivae and associated surfaces with a cotton tipped
Volume 40
-
January, 1976
Development of Antibodies Number of Linesb Epp-63 Fla-64 IHB-64 1 1 0 1 2 0 1 1 0 0 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 2 0 0 2 0 0 2 1 0 0 0
applicator saturated with a broth suspension of M. bovis cells.
Irradiation - All cattle were given ultraviolet radiation to enhance the effects of M. bovis infection of the eye (4, 5, 9). All eyes were irradiated for ten minutes each day on five successive days each week, beginning the day before challenge exposure and continuing for four weeks. The eyes of mice were not irradiated.
Bacteriological examination - Eye secretions of all calves were examined for M. bovis seven times during the period before challenge exposure. After calves were challenge exposed, examinations were made each day for approximately one month and then examinations were made at least five days per week. Sterilized cotton tipped applicators were used to collect secretions. To prevent drying, the applicators were placed in TSB immediately after the sample was collected. Samples were streaked on the surface of 5 % bovine blood agar and incubated at 37°C for 24 hours and at 25°C for 24 additional hours before being examined for M. bovis. Secretions from the eyes of mice were collected with sterilized cotton tipped applicators three times before exposure and for 14 consecutive days after exposure. The secretions were cultured for M. bovis in the same manner as those from cattle.
Serology - Blood samples were taken from the calves before they were vaccinated and the day they were challenge exposed to M.
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bovis. The sera from the calves were treated individually. The sera were checked for precipitins as evidenced by a line of identity as previously described (12). Blood samples were taken from the mice only once, two weeks after their challenge exposure to M. bovis. The sera from mice in each group were pooled and considered one sample. Clinical examination - All eyes were examined daily starting two days before challenge exposure and continuing for approximately one month and then examinations were made at least five days per week. Eyes were examined individually under oblique illumination from a hand held flashlight. The clinical condition was assessed by looking for photophobia, lacrimation iridospasm and blepharitis. A diagnosis of IBK was made only when a definite corneal lesion was observed.
RESULTS The results for the four calves vaccinated with pili preparations are summarized (Table I). All the calves developed antibodies against pili antigen as evidenced by lines in the GDPT. The serum of calf 7263 had two lines against the homologous pili. Numerous bacteriophage-like bodies were seen in this pili antigen when it was examined with the electron microscope. The results for mice vaccinated with pili preparations are summarized (Table II). The serological and immunological results with pili fractions of cattle vaccinated with formalin-killed cells are summarized (Table III). All calves vaccinated with the disrupted cell preparation became infected and developed IBK. Five had bilateral and one unilateral IBK. The calf that had unilateral IBK did not become infected in the eye that remained normal.
DISCUSSION Although our results are inconclusive, they do, when taken in conjunction with those of other studies (3, 5), indicate that
Volume 40 - January, 1976
the immune response seen in cattle after vaccination is probably due in part to surface components of M. bovis. In the present study the calves vaccinated with disrupted cells of Epp-63 were highly susceptible (100%) to infection and disease but the calves vaccinated with formalized whole cells of Epp-63 had only about 50% infection and disease. In another study, calves vaccinated with heat killed vaccines were as susceptible to challenge exposure as were nonvaccinated controls (5). We assume that heat destroys the surface antigens but formalin leaves their antigenicity unimpaired. The immunological results with pili vaccines were equivocal although the serological results with pili antigens indicated a tendency for antibodies to be produced against the homologous strain. This would suggest that for each strain of M. bovis, the pili might be specific and that the immunogenicity and possibly of pathogenicity of a strain are associated with pili antigens. Therefore, single strain bacterins may not be of value in protecting against IRK under field conditions. Others (2, 8) have suggested that the pathogenicity of M. bovis is associated with fimbriation (presence of pili). The facts that (i) each pili vaccine induced antibody formation against homologous and sometimes heterologous pili and (ii) the calf vaccinated with IBH-64 pili was resistant to infection and disease could be the basis of additional study. Similarly, the presence of two distinct lines in sera of vaccinated mice, the presence of phagelike particles seen under the electron microscope (A. E. Ritchie and Pugh, unpublished data, 1973), and the low infectivity of strain IBH-64 suggest further study on its pathogenicity and immunogenicity. The presence of a phage might explain the low infectivity (culture destroyed by a phage) of certain strains of M. bovis. Significantly, the infectivity of the three strains of M. bovis in cattle and mice in the present study parallel their relative virulence as seen in an earlier study (10). The pathogenic effect of a culture could be initiated by the presence of a virulent phage, as seen with virulent Corynebacterium diphtheriae cultures. However, its effect may have been neutralized in the calf (vaccinated with IBH-64 pili) in the present study because the phage was also present in the vaccine. Phage-like particles were also seen in the pili preparations of strain Epp-63 and Fla-64 but they were in-
65
ferior quantities. Although the finding of the phage-like particles under microscopy indicates the presence of a phage, we have been unable to isolate a Moraxella bovis phage by conventional methods. The death of mice during the vaccination supports the results of another study that M. bovis cultures and fractions are toxic (13). Although our results fail to establish that the pili fraction of M. bovis is responsible for the apparent immune response seen in vaccinated calves, they suggest when considered with others (5) that the response is related to surface antigens.
ACKNOWLEDGMENTS The technical assistance of Mr. Victor D. Schulz is appreciated. REFERENCES 1. BARNER, R. D. A study of Moraxella bovis and its relation to bovine keratitis. Am. J. vet. Res. 13:
132-144. 1952. 2. BOVRE, K. and L. 0. FROHOLML Variation of colony morphology reflecting fimbriation in Moraxella bovis and two reference strains of M. nonliquefaciens. Acta path. microbiol. scand. S.B. 80: 629-640. 1972.
66
3. HENSON, J. B., L. C. GRUMBLES and J. W. DOLLAHITE. Evaluation of experimental bacterins for infectious bovine keratoconjunctivitis. SWest Vet. 13: 213-216. 1960. 4. HUGHES, D. E. and G. W. PUGH, JR. Experimentally induced bovine infectious keratoconjunctivritis: Effectiveness of intramuscular vaccination witb viable Moraxella bovis culture. Am. J. vet. Res. 32: 879-886. 1971. 5. HUGHES, D. E. and G. W. PUGH, JR. Experimentally induced infectious bovine keratoconjunctivitis: Vaccination with nonviable Moraxella bovis culture. Am. J. vet. Res. 33: 2475-2479. 1972. 6. HUGHES, D. E. and G. W. PUGH, JR. Experimentally induced infectious bovine keratoconjunctivitis: Relationship of vaccination schedule to protection against exposure with homologous Moraxella bovis culture. Am. J. vet. Res. 35: 263-265. 1975. 7. HUGHES, D. E., G. W. PUGH, JR. and T. J. McDONALD. Experimental bovine infectious keratoconjunctivitis caused by sunlamp irradiation and Moraxella bovis infection: Resistance to re-exposure with homologous Moraxella bovis. Am. J. vet. Res. 29: 829-833. 1968. 8. PEDERSON, B., L. 0. FROHOLM and K. BOVRE. Fimbriation and colony type of Moraxella bovis in relation to conjunctival colonization and development of keratoconjunctivitis in cattle. Acta path. microbiol. scand. S. B. 80: 911-918. 1972. 9. PUGH, G. W., JR. and D. E. HUGHES. Experimental bovine infectious keratoconjunctivitis caused by sunlamp irradiation and Moraxella bovis infection: Correlation of hemolytic ability and pathogenicity. Am. J. vet. Res. 29: 835-839. 1968. 10. PUGH, G. W., JR. and D. E. HUGHES. Comparison of the virulence of various strains of Moraxella bovis. Can. J. comp. Med. 34: 333-340. 1970. 11. PUGH, G. W., JR., D. E. HUGHES and T. J. McDONALD. The isolation and characterization of Moraxella bovis. Am. J. vet. Res. 27: 957,962. 1966. 12. PUGH, G. W., JR., D. E. HUGHES and T. J. McDONALD. Bovine infectious keratoconjunctivitis: Serological aspects of Moraxella bovis infection. Can. J. comp. Med. 35: 161-166. 1971. 13. PUGH, G. W., JR., D. E. HUGHES and V. D. SCHULZ. The pathophysiological effects of Moraxella bovis toxins on cattle, mice and guinea pigs. Can. J. comp. Med. 37: 70-78. 1973. 14. SANDHU, T. S., 'F. H. WHITE and C. F. SIMPSON. Association of pili with rough colony type of Moraxella bovis. Am. J. vet. Res. 35: 437-439. 1974.
Can. J. comp. Med.
ABSTRACT
RASUME
The effect of vaccinating cattle and mice onu the development of keratoconjunctivitis was studied. Cattle were vaccinated with whole cells, disrupted cells and pili fractions of three strains of Moraxella bovis. Mice were vaccinated with pili fractions of three strains. The resistance of all vaccinated animals was challenged with virulent cultures of M. bovis. In an attempt to correlate the response seen after vaccination and challenge with a pili fraction of M. bovis, vaccinated cattle and mice were grouped on the basis of signs of disease manifested and compared on the basis of serological responses. Serum samples were tested for antibodies by a gel diffusion precipitin test. A greater number of the sera of resistant cattle had antibodies to the homologous pili antigen than those of vaccinated nonresistant cattle. Cattle vaccinated with disrupted cells were not resistant to infectious bovine keratoconjunctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to challenge exposure by homologous than heterologous cultures. A greater number of the sera of resistant mice had antibodies to pili antigens than nonresistant mice.
Cette experience visait a etudier l'effet de la vaccination de veaux et de souris, sur le developpement de la kerato-conjonctivite. On vaccina les premiers avec des microbes entiers ou fragmentes, ainsi qu'avec des cils de trois souches de Moraxella bovis. Quant aux secondes, on ne les vaccina qu'avec des cils des trois mimes souches du microbe. On eprouva ensuite la resistance de tous les animaux vaccines avec des cultures virulentes de M. bovis. Afin d'etablir une relation entre la reaction immunologique consecutive a la vaccination et l'infection avec des cils de M. bovis, on groupa les veaux et les souris vaccines d'apres les signes cliniques qu'ils manifesterent et on les compara d'apres leur reaction immunologique. On proceda a la recherche d'anticorps seriques, a l'aide de l'epreuve de diffusion en milieu gelifie. Un plus grand nombre d'echantillons de serum provenant de veaux r6sistants recelaient des anticorps a l'endroit de l'antigene homologue a base de cils, comparativement aux veaux vaccines, mais non r6sistants. Les veaux vaccines avec des microbes fragmentes ne s'avererent pas r6sistants 'a la keratoconjonctivite et leur serum etait depourvu d'anticorps a l'endroit de l'antigiene prepare avec des cils. Les souris vaccin6es resisteirent mieux a l'infection par des cultures homologues qu'heterologues. Plus d'echantillons de serum des souris resistantes poss&daient des anticorps a l'endroit de l'antigene prepare avec des cils, comparativement aux souris non resistantes.
*?National Animal Disease
Center, North Central Region, Agricaitural Research Service, US. Department of Agrielture, Ames, Iowa 50010. Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suftable. Submitted June 6, 1975.
60
Can. J. comp. Med.
INTRODUCTION
Recent studies have shown that cattle be protected from subsequent infection and disease due to Moraxella bovis through vaccination with whole cell cultures (4-6). Also, cattle previously affected with infectious bovine keratoconjunctivitis (IBK) were somewhat resistant to infection and disease. These resistant cattle had antibodies to M. bovis (1, 4-6) but the specific antigenic stimulants, whether of extra- or intracellular origin, were not apparent. Workers thought that the nature of the antigenic stimulant should be determined to facilitate the development of a vaccine against IBK. We hypothesized that this could be partially determined from results obtained by vaccinating cattle and mice with a specific fraction of M. bovis and then by challenging their immunity and by making inferences based on serological reactions between the specific fraction and the sera of resistant cattle and mice using the gel diffusion precipitin test (GDPT). Others (2, 8) have suggested that the pathogenicity of M. bovis is associated with fimbriation (presence of pili). This suggested to us that the pili should be a good fraction with which to begin our study. This report describes a study to determine whether pili fractions of M. bovis could be correlated to a specific immune response in cattle and mice.
can
MATERIALS AND METHODS Animals Cattle: Ten calves were specifically used for the present study. The other 124 calves were part of allied studies (Hughes and Pugh, unpublished data, 1973) and will be mentioned only briefly. All calves were healthy, 70 to 90 days old and of mixed breeding. Their eyes were culturally negative for M. bovis. Their prevaccination sera were free of antibodies to M. bovis. The calves were reared in isolation quarters as previously described (4, 5). Mice: A total of 108 healthy mice weighing 16-20 gm were used. Their eyes were
Volume 40
-
January, 1976
normal and culturally negative for M. bovis. The mice were divided into three groups of 36 each. Twenty-seven mice in each group were vaccinated with each strain of pili vaccine. The other nine mice in each group served as unvaccinated controls. Animal quarters - The calves were quartered in indoor isolation units. The quarters were air conditioned and mechanically ventilated with filtered air. The room had individual stalls, each large enough to accommodate three calves. Calves were assigned to stalls according to the challenge exposure organism they were to receive. Flies were excluded, and complete insect control was maintained with pyrethrin aerosol spray. Of the six calves vaccinated with disrupted cells of strain Epp-63, three were placed in one stall and three in another. Because the calves vaccinated with pili fractions were tested at the same time as calves in another experiment (Hughes and Pugh, unpublished data, 1973), animals from both experiments were housed and grouped together. The mice were housed in mouse cages within a laboratory room. EXPERIMENTAL DESIGN Differences in the serological response of resistant and nonresistant cattle and mice were determined by using pili of strains of M. bovis as antigens in the GDPT. Pili antigen made from each of three strains of M. bovis were used to vaccinate cattle and mice (Tables I and III). Six calves were vaccinated with disrupted cells. Forty-five cattle were vaccinated with formalized whole cells prepared from M. bovis strain Epp-63, 24 from strain Fla-64 and 24 from strain IBH-64. Later they were challenge exposed by instilling the conjunctival sac with three virulent strains of M. bovis (Table III). Sera from the calves collected prior to exposure were grouped on the basis of signs of disease
developed. Organisms - Three strains of M. bovis, identified as Epp-63, Fla-64 and IBH-64 were used. These strains were isolated from cattle naturally affected with IBK and, on the basis of other studies, were found to be pathogenic for cattle (7, 9-11). Smooth' 'Smooth means the friable, flat, agar corroding, autoagglutinable colony of M. bovis typically found on bovine blood &-ar vlate.' on initial isolation from eyes of cattle affected with IBK.
61
TABLE I. The Response of Calves Vaccinated with Pili Vaccine to Ocular Inoculation of Moraxella bovis
M. bovis Became Established Unilateral Bilateral None
Development
of Challenge Calf Developed Infectious Vaccine used Strain No. Antibodies Keratitis Pili of strain Epp-63 Epp-63 7261 Homologous Unilateral Pili of strain Fla-64 .Fla-64 7263 Homologous Unilateral IBH-64 Pili of strain IBH-64 7264 Homologous None Mixed pili of strains Epp-63, Fla-64 and IBH-64 7259 HeterologousFla-64 Bilateral Bilateral -Calf developed antibodies to pili antigen of each of the three strains but the lines were of nonidentity
hemolytic colonies of the three strains were used to prepare pili vaccines, to prepare test antigens, to prepare formalized whole cell vaccines and for challenge exposure. Frozen cultures for each strain representing the fourth bovine blood agar passage (BAP) were streaked on blood agar plates. The inoculated plates were incubated for 24 hours at 37°C and for 24 additional hours at 25°C, at which time typical smooth hemolytic colonies were picked and used to inoculate additional plates. The latter plates were incubated at 37°C for 24 hours and the cells were collected into sterile, distilled water and stored at -60°C until used to prepare pili antigens or vaccines. The pili fractions were prepared similarly to a method previously described (14). Sixteen grams (wet weight) of each culture (strain) were suspended in 100 ml of cold distilled water, washed once and then vigorously agitated in a virtis homogenizer2. The homogenate was differentially centrifuged3 and the final precipitate was dissolved in 3.0 ml of cold distilled water and dialyzed for three days with frequent water change in the cold in order to remove the MgCI2. The preparation was observed under an electron microscope to verify the presence of pili. The suspension of pili was diluted twofold with water and sonicated4 for 30 minutes at 1.25 amperes. The pili concentration of the three strains was standardized by dilution with water so that the pili preparation for each of the three strains gave 25 % light transmittance at
540 nm'. The preparation representing each strain was placed in vaccine bottles and stored at -60°C until used as antigen or in vaccine. All procedures were carried out at 40C. Disrupted cell fractions (DC-1) were prepared from typical smooth cultures of M. bovis strain Epp-63. Ninety gm of cells harvested from 24-hour BBA culture was suspended in 123 ml of cold distilled water. The cells were further diluted with water to facilitate the passing of the suspension into a fractionator6 as previously described (13). The effluent (DC-1) was collected, freeze-dried and stored at -60°C until used to vaccinate cattle. Cultures of the three strains for challenge exposure were grown as described for the cell fraction preparation except that trypticase soy broth (TSB) was used to harvest the cells. The harvested cells were stored at -600C until used to challenge expose cattle and mice.
Vaccination and challenge exposure Cattle: Each of four calves was vaccinated by injecting a pili preparation alternately into the right or left semitendinosus muscle. Calves were given three injections with two weeks between each injection. Calves 7261, 7263, 7264 and 7259 were vaccinated with pili strains Epp-63, IBH-64 and Fla64 and an equal mixture of the three preparations, respectively. The eyes of calves 7261, 7263 and 7264 were challenge exposed to virulent smooth hemolytic sixth BAP cultures representing the homologous strains used in the pili vaccine. Calf 7259 was challenge exposed to Fla-64 smooth
2Virtis Homogenizer, The Virtis Co. Inc., Gardiner, New York. 3Beckman Model L Preparative Ultracentrifuge, Beekman Instruments Inc., Palo Alto, California. 4M.S.E. Ultrasonic Disintegrator, Measuring and Scientific Equipment Ltd., Spenser St., London S.W.I., Great Britain.
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5Spectronic 20 Colorimeter, Bausch and Lomb Inc., Rochester, New York. 6Sorvall Ribi Cell Fractionator, Model RF-I, Ivan Sorvall Inc., Norwalk, Connecticut.
Can. J. comp. Med.
TABLE II. Persistency of Moraxella bovis Strains and Development of Antibodies in Mice Vaccinated with Pili Vaccines
Vaccine Used Epp-63
Epp-63
Challenge Straina Epp-63 Fla-64 IBH
Period M. bovis Recovered 2 days 14 days No. mice No. eyes No. mice No. eyes 9 16 1 1 7 11 1 1 5 7 0 0 5 9 0 0 9 16 0 0 8 14 0 0 4 5 0 0 9 16 4 3 4 8 0 0 9 15 0 0 6 12 2 2 1 1 0 0
Epp-63 Control Epp-63 Fla-64 Fla-64 Fla-64 Epp-63 Fla-64 IBH-64 Control Fla-64 IBH-64 IBH-64 IBH-64 Epp-63 IBH-64 Fla-64 Control IBH-64 Nine mice were used for each challenge bSera with 2 lines had one line of identity
hemolytic sixth BAP culture. All eyes were exposed by instilling 1.0 ml (1.7 x 106 to 2.5 x 106 organisms) of TSB suspension of the respective culture into the ventral conjunctival sac of each eye. Six calves were vaccinated by injecting 0.045 g of DC-1 suspended in 1.0 ml of distilled water alternately into the right or the left semitendinosus muscle. Calves were given three injections, with two weeks between each injection. Fourteen days after the last injection the calves were challenge exposed by instilling 0.5 ml of M. bovis Epp-63 (1.7 x 10,6 to 2.5 x 108 cell) TSB suspension into the ventral conjunctival sac of each eye. The cattle whose sera were used to test against the pili antigens were vaccinated with formalized vaccines made of EPP63, Fla-64 and IBH-64. They were injected with 1.0 ml of the vaccine alternately into the right or left semitendinosus muscle. Calves were given three injections with two weeks between each injection. Fourteen days after the last injection the calves were challenge exposed by instilling 0.5 ml suspended in TSB of a sixth passage BAP hemolytic culture into the ventral conjunctival sac of each eye. Mice: Mice were injected with 0.1 ml of the respective vaccine alternately into the right or left hip. Mice were given three injections with two weeks between each injection. Fourteen days after the last injection each mouse was challenge exposed by smearing the eyeballs, conjunctivae and associated surfaces with a cotton tipped
Volume 40
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January, 1976
Development of Antibodies Number of Linesb Epp-63 Fla-64 IHB-64 1 1 0 1 2 0 1 1 0 0 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 2 0 0 2 0 0 2 1 0 0 0
applicator saturated with a broth suspension of M. bovis cells.
Irradiation - All cattle were given ultraviolet radiation to enhance the effects of M. bovis infection of the eye (4, 5, 9). All eyes were irradiated for ten minutes each day on five successive days each week, beginning the day before challenge exposure and continuing for four weeks. The eyes of mice were not irradiated.
Bacteriological examination - Eye secretions of all calves were examined for M. bovis seven times during the period before challenge exposure. After calves were challenge exposed, examinations were made each day for approximately one month and then examinations were made at least five days per week. Sterilized cotton tipped applicators were used to collect secretions. To prevent drying, the applicators were placed in TSB immediately after the sample was collected. Samples were streaked on the surface of 5 % bovine blood agar and incubated at 37°C for 24 hours and at 25°C for 24 additional hours before being examined for M. bovis. Secretions from the eyes of mice were collected with sterilized cotton tipped applicators three times before exposure and for 14 consecutive days after exposure. The secretions were cultured for M. bovis in the same manner as those from cattle.
Serology - Blood samples were taken from the calves before they were vaccinated and the day they were challenge exposed to M.
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bovis. The sera from the calves were treated individually. The sera were checked for precipitins as evidenced by a line of identity as previously described (12). Blood samples were taken from the mice only once, two weeks after their challenge exposure to M. bovis. The sera from mice in each group were pooled and considered one sample. Clinical examination - All eyes were examined daily starting two days before challenge exposure and continuing for approximately one month and then examinations were made at least five days per week. Eyes were examined individually under oblique illumination from a hand held flashlight. The clinical condition was assessed by looking for photophobia, lacrimation iridospasm and blepharitis. A diagnosis of IBK was made only when a definite corneal lesion was observed.
RESULTS The results for the four calves vaccinated with pili preparations are summarized (Table I). All the calves developed antibodies against pili antigen as evidenced by lines in the GDPT. The serum of calf 7263 had two lines against the homologous pili. Numerous bacteriophage-like bodies were seen in this pili antigen when it was examined with the electron microscope. The results for mice vaccinated with pili preparations are summarized (Table II). The serological and immunological results with pili fractions of cattle vaccinated with formalin-killed cells are summarized (Table III). All calves vaccinated with the disrupted cell preparation became infected and developed IBK. Five had bilateral and one unilateral IBK. The calf that had unilateral IBK did not become infected in the eye that remained normal.
DISCUSSION Although our results are inconclusive, they do, when taken in conjunction with those of other studies (3, 5), indicate that
Volume 40 - January, 1976
the immune response seen in cattle after vaccination is probably due in part to surface components of M. bovis. In the present study the calves vaccinated with disrupted cells of Epp-63 were highly susceptible (100%) to infection and disease but the calves vaccinated with formalized whole cells of Epp-63 had only about 50% infection and disease. In another study, calves vaccinated with heat killed vaccines were as susceptible to challenge exposure as were nonvaccinated controls (5). We assume that heat destroys the surface antigens but formalin leaves their antigenicity unimpaired. The immunological results with pili vaccines were equivocal although the serological results with pili antigens indicated a tendency for antibodies to be produced against the homologous strain. This would suggest that for each strain of M. bovis, the pili might be specific and that the immunogenicity and possibly of pathogenicity of a strain are associated with pili antigens. Therefore, single strain bacterins may not be of value in protecting against IRK under field conditions. Others (2, 8) have suggested that the pathogenicity of M. bovis is associated with fimbriation (presence of pili). The facts that (i) each pili vaccine induced antibody formation against homologous and sometimes heterologous pili and (ii) the calf vaccinated with IBH-64 pili was resistant to infection and disease could be the basis of additional study. Similarly, the presence of two distinct lines in sera of vaccinated mice, the presence of phagelike particles seen under the electron microscope (A. E. Ritchie and Pugh, unpublished data, 1973), and the low infectivity of strain IBH-64 suggest further study on its pathogenicity and immunogenicity. The presence of a phage might explain the low infectivity (culture destroyed by a phage) of certain strains of M. bovis. Significantly, the infectivity of the three strains of M. bovis in cattle and mice in the present study parallel their relative virulence as seen in an earlier study (10). The pathogenic effect of a culture could be initiated by the presence of a virulent phage, as seen with virulent Corynebacterium diphtheriae cultures. However, its effect may have been neutralized in the calf (vaccinated with IBH-64 pili) in the present study because the phage was also present in the vaccine. Phage-like particles were also seen in the pili preparations of strain Epp-63 and Fla-64 but they were in-
65
ferior quantities. Although the finding of the phage-like particles under microscopy indicates the presence of a phage, we have been unable to isolate a Moraxella bovis phage by conventional methods. The death of mice during the vaccination supports the results of another study that M. bovis cultures and fractions are toxic (13). Although our results fail to establish that the pili fraction of M. bovis is responsible for the apparent immune response seen in vaccinated calves, they suggest when considered with others (5) that the response is related to surface antigens.
ACKNOWLEDGMENTS The technical assistance of Mr. Victor D. Schulz is appreciated. REFERENCES 1. BARNER, R. D. A study of Moraxella bovis and its relation to bovine keratitis. Am. J. vet. Res. 13:
132-144. 1952. 2. BOVRE, K. and L. 0. FROHOLML Variation of colony morphology reflecting fimbriation in Moraxella bovis and two reference strains of M. nonliquefaciens. Acta path. microbiol. scand. S.B. 80: 629-640. 1972.
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3. HENSON, J. B., L. C. GRUMBLES and J. W. DOLLAHITE. Evaluation of experimental bacterins for infectious bovine keratoconjunctivitis. SWest Vet. 13: 213-216. 1960. 4. HUGHES, D. E. and G. W. PUGH, JR. Experimentally induced bovine infectious keratoconjunctivritis: Effectiveness of intramuscular vaccination witb viable Moraxella bovis culture. Am. J. vet. Res. 32: 879-886. 1971. 5. HUGHES, D. E. and G. W. PUGH, JR. Experimentally induced infectious bovine keratoconjunctivitis: Vaccination with nonviable Moraxella bovis culture. Am. J. vet. Res. 33: 2475-2479. 1972. 6. HUGHES, D. E. and G. W. PUGH, JR. Experimentally induced infectious bovine keratoconjunctivitis: Relationship of vaccination schedule to protection against exposure with homologous Moraxella bovis culture. Am. J. vet. Res. 35: 263-265. 1975. 7. HUGHES, D. E., G. W. PUGH, JR. and T. J. McDONALD. Experimental bovine infectious keratoconjunctivitis caused by sunlamp irradiation and Moraxella bovis infection: Resistance to re-exposure with homologous Moraxella bovis. Am. J. vet. Res. 29: 829-833. 1968. 8. PEDERSON, B., L. 0. FROHOLM and K. BOVRE. Fimbriation and colony type of Moraxella bovis in relation to conjunctival colonization and development of keratoconjunctivitis in cattle. Acta path. microbiol. scand. S. B. 80: 911-918. 1972. 9. PUGH, G. W., JR. and D. E. HUGHES. Experimental bovine infectious keratoconjunctivitis caused by sunlamp irradiation and Moraxella bovis infection: Correlation of hemolytic ability and pathogenicity. Am. J. vet. Res. 29: 835-839. 1968. 10. PUGH, G. W., JR. and D. E. HUGHES. Comparison of the virulence of various strains of Moraxella bovis. Can. J. comp. Med. 34: 333-340. 1970. 11. PUGH, G. W., JR., D. E. HUGHES and T. J. McDONALD. The isolation and characterization of Moraxella bovis. Am. J. vet. Res. 27: 957,962. 1966. 12. PUGH, G. W., JR., D. E. HUGHES and T. J. McDONALD. Bovine infectious keratoconjunctivitis: Serological aspects of Moraxella bovis infection. Can. J. comp. Med. 35: 161-166. 1971. 13. PUGH, G. W., JR., D. E. HUGHES and V. D. SCHULZ. The pathophysiological effects of Moraxella bovis toxins on cattle, mice and guinea pigs. Can. J. comp. Med. 37: 70-78. 1973. 14. SANDHU, T. S., 'F. H. WHITE and C. F. SIMPSON. Association of pili with rough colony type of Moraxella bovis. Am. J. vet. Res. 35: 437-439. 1974.
Can. J. comp. Med.
