II.

Infectious Bovine Keratocon junctivitis Antibodies in Lacrimal Secretions of Cattle Naturally or Experimentally Infected with Moraxella bovis P. S. G. Nayar and J. R. Saunders*

ABSTRACT Preinfection, acute and convalescent phase lacrimal secretions from 20 cattle that had mild to severe keratoconjunctivitis (associated with natural or experimental Moraxella bovis infection) were tested for specific antibody to M. bovis as well as for immunoglobulins, particularly of the A class. The convalescent phase secretions from all four natural cases but from only seven of the 16 experimental cases contained increased amounts of immunoglobulins, predominantly of the A class. Only the secretions from cattle having severe keratoconjunctivitis contained precipitins specific for Moraxella bovis. Therefore, M. bovis specifically stimulates the appearance in tears of immunoglobulins that are important in the age associated, acquired resistance to reinfection with M. bovis.

RESUME Les auteurs ont recueilli des larmes, avant l'infection, au cours de la phase aigue de la maladie et durant la convalescence de 20 bobins souffrant de kerato-conjonctivite dont la gravite variait d'un sujet it l'autre. Cette condition r6sultait d'une infection naturelle ou experimentale avec Moraxella bovis. Ils rechercherent ensuite dans ces secretions des anticorps specifiques pour M. bovis, ainsi que des immunoglobulines, en particulier celles de la classe A.

*Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan. Present address of senior author: Henderson Animal

Clinic, 1432 Henderson Highway, Winnipeg, Manitoba. Submitted September 17, 1973.

32

Les larmes recueillies durant la convalescence des quatre cas d'infection naturelle, mais de seulement sept des 16 cas d'infection experimentale, contenaient une plus grande quantite d'immunoglobulines, surtout de la classe A. Seulement les larmes des sujets chez qui la kerato-conjonctivite etait plus marquee contenaient des precipitines specifiques 'a M. bovis. Par consequent, M. bovis suscite specifiquement l'apparition dans les larmes d'immunoglobulines qui s'averent importantes en ce qui concerne la resistance acquise et reliee a l'age, face a une reinfection.

INTRODUCTION Limited information is available on the amount and protective benefit of immunoglobulins (Ig) in external secretions of cattle (2, 7). However, ihe importance of locally secreted antibodies against infections of the respiratory tract has been notably established in man (5). It has also been adequately demonstrated that a few bovine viral infections stimulate the production of protective local antibodies (21). The degree of protection resulting from paraininfluenza-3 virus infection is reflected more by the level of nasal antibody than by serum antibody (10). Duncan (5) in studies on Pasteurella hemolytica in cattle reported that the degree of protection from locally produced antibodies might be superior io that produced by parenteral vaccination. It has been shown that circulating antibody has relatively little significance or is indirectly related to resistance in certain infections (22). In such infections, where the causative organisms are generally noninvasive, the secretory antibodies are considered to be very important defence mechanisms (22). Immunoglobulin

Can. J. comp. Med.

A (SIgA) has been found to be the major Moraxella bovis CULTURES class of Ig in lacrimal as well as other Three recently isolated strains (6933Cexternal secretions (2, 6, 10). In regards to infectious bovine kerato- IR, 68153C3-4' and FLA-642) and a labconjunctivitis (IBK) the literature deals oratory stock strain3 were used during this mostly with the etiology and pathology study. Antisera were prepared against all (9, 19, 24). In one study subcutaneous in- strains. The FLA-64 strain was used for oculation of M. bovis bacterins did not the preparation of soluble antigens and for protect cattle against IBK (12). More re- absorbing lacrimal secretions. cently, partial success has been achieved in immunizing cattle against IBK by a PRODUCTION OF ANTISERA series of intramuscular inoculations with Specific antisera against the four strains viable M. bovis cultures (13). In view of the above, a need was rec- of M. bovis were prepared in rabbits4 by a (20), ognized for basic research on the role of modification of the procedure of Pugh densitythe that being difference the of infections in local defence mechanisms the eye. This need in the case of IBK is standardized suspension of heat killed bacparts of suggested by these facts: a) the disease terial cells was mixed with equal is recognized as a localized rather than a incomplete Freund's adjuvant5 prior to subsystemic infection (24), b) M. bovis bac- cutaneous inoculation of 0.2 ml doses into anterins or vaccines given parenterally do each of six sites on the backs of thewere rabbits the days 45-50 After imals. not adequately protect the animal from subsequent infections (12, 13) clinical in- anesthetized by intravenous injection of fections generally induce resistance to re- sodium pentabarbitone' and exsanguinated infection (4, 15), c) in localized infections by cardiac puncture. The serum was subcaused by noninvasive organisms (as in sequently stored at -60°C for later use. IBK) the development of secretory antibodies appears to be an important specific PRODUCTION OF M. boris SOLUBLE ANTIGEN defence mechanism (23). The FLA-64 strain of M. bovis was used The primary objective of this study, to prepare the soluble antigen (20) and for of lacrimal therefore, was the examination use in the immunoelectrophoresubsequent secretions (preinfection to convalescent procedures. This and immunodiffusion sis phase) from cases of natural and experiwas stored without preservative in mental IBK, for specific antibody to M. antigen bovis as well as for Ig, particularly SIgA. small vials at -60°C.

MATERIALS AND METHODS

PRODUCTION OF BOVINE IMMUNOGLOBULINS AND MONOSPECIFIC ANTISERA

EXPERIMENTAL DESIGN

Lacrimal secretions collected from 20 cattle naturally or experimentally infected with M1. boc-is were to be tested by electrophoresis and immunodiffusion for SIgA as well as for precipitating antibodies to soluble antigens of the bacteria. The performance of these tests required: (i) the production in rabbits of antisera to M. bovis, (ii) the preparation of soluble bacterial antigens and (iii) the preparation or acquisition of purified bovine immunoglobulins (SIgA, IgG, IgM) in addition to their specific antisera (anti-IgA, anti-IgG, anti-IgM). The results of these tests would then be compared to the clinical findings in the animals for possible correlations.

Vol. 39- January, 1975

An initial and naive idea was that quantitation of SIgA in lacrimal secretions (from cases of IBK) was of major importance but

'Animal Diseases Research Institute (Western), Lethbridge, Alberta.

2National Animal Disease Laboratory, USDA, Ames, Iowa.

:'Department

of Veterinary Microbiology, WCVM, University of Saskatchewan, Saskatoon, Saskatchewan. 'Obtained from Animal Resources Center, University of Saskatchewan, Saskatoon, Saskatchewan. 5Bacto-Incomplete Freund's Adjuvant, Difco Lab., Detroit, Michigan. 60btained from the Central Supply Pharmacy, WCVM, University of Saskatchewan, Saskatoon, Saskatchewan.

33

quantitation of IgG and IgM was not of importance since SIgA was by far the predominant Ig in tears (15). On that basis and because of technical problems involved in preparation of such reagents, 5 ml of guinea pig antibovine SIgA (anti-IgA) prepared by the procedure of Duncan et al (6) was obtained7 for use in this study. This anti-IgA was monospecific and did not react with free secretory component (FSC). When this study was nearly completed it; was realized that some testing of lacrima] secretions for IgG and IgM should be done. Unfortunately lacrimal secretions from only three of the experimental calves were still available for further testing. At this time the senior author was involved in a collaborative study8 on the quantitation of Ig's in bovine colostrum, for which IgG and IgM were isolated from bovine serum and monospecific antisera subsequently prepared in rabbits according to the method of Butler and Maxwell (3). Small volumes of these reagents were kindly provided8 for this study. SOURCE, COLLECTION AND HANDLING OF LACRIMAL SECRETIONS

purchased from the Western Stockyards, Saskatoon. For the collection of lacrimal secretions the animals were tightly restrained by a rope halter. Using sterile disposable rubber gloves a sterile cotton ball was placed in the eye at the lateral canthus under the lower eyelid. The eyelids were kept closed with digital pressure for three to five minutes. The cotton ball, now completely soaked with tears, was taken out and squeezed into a vial. By repeating this procedure two or three times one could easily collect 3-5 ml3 of tears. These samples were then filtered through a 0.45 mu pore size millipore filter and stored at -60°C for later use. ABSORBED LACRIMAL SECRETIONS A dense suspension of live M. bocis cells (taken directly from a sheep blood agar plate) was added to approximately 1 ml of each lacrimal sample, the resulting optical density being equal to that of a MacFarland Nephelometer number 2. The mixture was then incubated for one hour at 37°C. Following centrifugation at 2000 rpm for ten minutes the clear supernatant was used as the absorbed laCrimal secretion.

In a previously reported study (17) the single or repeated inoculation of M. bovis (alone or in combination with mycoplasma and/or viruses) into the eyes of 19 calves TEST PROCEDURES resulted in clinical IBK in eight calves and mild or inapparent infection in the Polyacrylamide gel electrophoresis remaining 11 calves (Table I). Samples (PEP), immunoelectrophoresis (1EP), of lacrimal secretions (from 16 repre- double immunodiffusion (DID) and radial sentative experimental calves), collected immunodiffusion (RID) were the four proprior to initial inoculation and weekly cedures used for the detection of immunothereafter until the termination of the globulins and specific antibody. The RID experiments, were used in this study. In and PEP procedures were considered to be addition, lacrimal secretions were collected the most useful and results are mainly reweekly from four natural cases of IBK, corded on that basis. from the onset of acute clinical disease until two months later (recovery phase). POLYACRYLAMIDE GEL ELECTROPHORESIS These four natural cases of IBK studied included (a) two calves and one cow from procedure as previously described the feed lot (Department of Animal (14) The was used for the electrophoresis of Science, University of Saskatchewan, lacrimal secretions. The electrophoresis where there was an epizootic of IBK in chamber, power unit, chemicals for the the summer of 1971) and (b) one calf that preparation of the separating gel (7%) was developing acute signs of IBK when and the buffer were commercially obtained. Following a running time of three hours 7Dr. J. R. Duncan, New York State Veterinary College. Ithaca, New York. the gels were fixed in 12.5% trichloroacetic 8Dr. S. D. Acres, Department of Vet. Clinical Studies and Dr. B. T. Rouse, Department of Vet. Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan.

34

'Difco Laboratories, Detroit,

Michigan.

Can. J. comp. Med.

TABLE I. Summary of the Response of Experimental Calves to Ocular or Intramuscular Inoculation of Moraxella bovis

Inoculated with M. bovis at weeks 7 4 0

Calf 1 2

.................

....

y y

.................

4

y

y

5 ........

y

y

6 7

y yb

y y

...............

........

9. 10 il

y................ ... ...................

...-

..................

12 ..

...

13 ........ 14 15

...

...

RE

y

..

y

..

.............

y

y

y

yC

y

.

yb

y y

..

y

y

..

y

y

y

y

y

yb y

.............

19LE RE .............. 2OdLE ................ RE .

.

.. ..

ybc yb

yb ..

y yb

..

+

M. boris at weeks 7 0 4 -

_

_

..

.

ybe

..............

... .. 16 ... 17LE RE 18LE ................

..

..

y

...............

+ +

..

..

Serum Antibody to

Severity of Lesions Following Exposure 1 2 3

.. yb

y

y

y y

+

-

+

-

++

-

-

+

-

+

++ -

++

++-

+

-

.

-

. .. _

+

_

+

+

_

+

-

+

+

+

±

+

+

+++ _ + ++ -

-

-

..

y

..

..

..

y

..

..

++ + + +++ + +++ +++ +++

-

+

-

+ -

+ +

-

±

-

_

+ _

-

aCalves 1 to 16 were exposed to viable M. bovis by conjunctival instillation of both eyes. Calves 17 to 20 were exposed to viable M. bovis by intraconjunctival or intracorneal inoculation. Exceptions are indicated by footnote bHeat-killed organisms cIntramuscular inoculation dHad antibody at ten weeks = Yes Abbreviations: y = No treatment + = Mild disease or antibody present + + = Moderate disease + ++ = Severe disease - = No response or negative = Left eye LE = Right eye RE

acid for two to three hours, stained for one to two hours with Comassie blue'0 stain and then destained by transferring to 10% trichloroacetic acid. The stained protein bands were then scanned using a Gelscan Automatic Recording and Integrating Scanner". IM MUNOELECTROPHORESIS

The basic procedure followed was that described by Brighton (1), using in this case the deluxe electrophoresis chamber'2 and immunoframes" for six microscope slides. The "Canalco Inc., Rockville, Maryland. "Gelman Instrument Co., Ann Arbor, Michigan. 12Gelman Instrument Co., Ann Arbor, Michigan.

Vol. 39 -January, 1975

lacrimal secretions were tested both with anti-IgA and M. bovis antigen. DOUBLE IMMUNODIFFUSION

The DID plates were prepared according to the procedure described by Pugh (19) except for the use of different patterns of wells. Absorbed and unabsorbed lacrimal sceretions from cases of natural and experiment IBK were tested against both M. bovis antigen and anti-IgA. RADIAL IMMUNODIFFUSION The procedure for the preparation and use of RID plates was that of Mancini et al (16), except that Hyland Immunoplates'3

l:3Hyland

Laboratories, Los Angeles, California.

35

(78 mm x 132 mm) were substituted for glass slides. Fifteen ml of 1.5% agar gel containing the respective antisera were delivered with a preheated pipette onto the plate. Separate plates were made by incorporating anti-IgA and M. bovis antigen in the agar. After the agar was hardened on a level table, ten rows of six wells were cut 12 mm apart using LKB well cutters"4 with a 3 mm bore. The lacrimal secretions, both absorbed and unabsorbed, from cases of IBK (natural and experimental) were tested to detect any postinoculation increase in SIgA content and also to see whether antibody specific for M. bovis was present. Doubling dilutions of each sample from 1:1 to 1:32 were tested in RID plates containing anti-IgA or the M. bovis antigen. In the case of 3 calves (# 18, 19, 200) preinoculation and convalescent phase lacrimal secretions were also tested for IgM and IgG.

RESULTS The antisera from rabbits inoculated with M. bovis contained precipitating antibodies (by DID) as early as one week after the first injection of antigen. At 45-50 days sera from these rabbits had precipitin titres satisfactory for reference antisera. The results obtained in the PEP, DID and RID tests by reacting the unabsorbed and absorbed (with M. bovis) lacrimal secretions against anti-IgA and M. bovis antigen are summarized in Tables II and III. Of the 16 experimental calves included here, only four (#17, 18, 19, 20) had developed typical IBK, another four (#6, 8, 12, 16) had developed a moderately severe infection, seven had developed a mild infection and the remaining one had remained essentially normal (Table I). By PEP increased amounts of a protein band (shown to be SIgA by extracting and testing against anti-IgA in DID) were detected in convalescent phase secretions of all four naturally infected animals but in only six of the 15 experimentally infected calves that had developed lesions. Absorption of these secretions with M. bovis cells, prior to electrophoresis, partially or completely removed this protein band. Following absorption with M. bovis cells the secretions from calves that developed 14Gelman Instrument Co., Ann Arbor, Michigan.

36

mild IBK were unchanged in PEP with respect to this protein (SIgA) band. When the gels were subsequently scanned and the results plotted graphically a definite peak (corresponding to this particular protein band) was observed in the unabsorbed, convalescent sample gels. The magnitude of this peak was reduced or eliminated by absorption of the same samples with cells of M. bovis (Figs. 1, 2, 3). There was no apparent difference between absorbed and unabsorbed preinfection samples from experimental calves in regards to the size of this peak (Figs. 3, 4). Observation and measurement of the area under this peak indicated that the samples (convalescent or postinfection) from natural cases contained more of this protein than did those from experimentally infected calves. The samples collected from the third to the fourth week postinfection contained more of this protein than did those from the first week. The lacrimal secretions when tested against anti-IgA by RID formed precipitin rings. There was an increase in the diameters of the precipitin rings formed by secretions from diseased eyes as compared to those formed by secretions from normal eyes. The unabsorbed samples from the four natural cases. and from eight experimental calves (,#6, 8, 12, 16, 17, 18, 19 and 20) had more SIgA antibodies than similar samples from other experimental calves as indicated by the ring diameters produced by the undiluted secretions. Samples from these same animals produced precipitin rings even at a dilution of 1:16 or 1:32. The secretions from these animals (except for calf 8) when absorbed once with M. bovis cells had their SIgA content reduced, as shown by a two to fourfold reduction in titre as well as by a reduction in the ring diameters formed with undiluted secretions. No significant change was evident with samples from the other experimental calves on absorption with M. bovis. RID with M. bovis antigen (incorporated in the agar) resulted in the formation of clear precipitin rings with samples from the four natural cases and from two experimental calves 17 and 19. None of the absorbed (M. bovis) samples produced

precipitin rings. The results of IEP and DID tests (Figs. 5, 6, 7, 8) were similar to those of the other tests (PEP, RID) and supported the contention that immunoglobulin increased with infection and could be partially re-

Can. J. comp. Med.

TABLE II. Results of the Immunological Tests for SIgA of Convalescent Phase Lacrimal Secretions from Twenty Cattle Experimentally or Naturally Infected with Moraxella bovis

Severity of Disease

Calf #

Exp. 1.+

2 .. ..

NC .. ... 4.........

6..... 7.. 8......±.. 9. 12 ... 13

..

......

1 2 .

+ ++ + + +

NC

NC

1:32

.......

+ ++

+++ +++ +++ +++ +++

+++

....

+++ +++

1:16

NC NC

NC NC NC

++

3...... 4

+

...

.. 14 15 .. 16 .. 17 ..... 18 ................ 19 .. 20 ....

Nat.

RID Ppt Ring Difference Between Unabs. Abs.

NC NC 1:32 1:32

1:16 1:8

1:16 1:32

1:8 1:16

1:16

1:8

1:32

1:8

1:32 1:32 1:32

1:16

1:16 1:8

PEP Increased Amount of SIgA Abs. Unabs.

P P P P P P P P P P P P P P P P

NC NC NC R

P P P P

R R R R

NC

NC NC NC NC NC NC R R R R R

aSamples tested were collected from the most severely affected eye at 2 to 4 weeks after the last inoculation in the case of experimental calves or 4 weeks after the acute phase of disease in the naturally infected cattle Abbreviations: P = Present Ppt = Precipitin Exp. = Experimental = Reduced intensity R NC = No significant change Nat. = Natural Unabs. = Unabsorbed sample of lacrimal secretion = Lacrimal secretion absorbed with Al. bovis Abs. = No sign of disease + = Mild disease ++ = Moderate disease +++ = Severe disease (typical acute IBK)

moved by absorption with M. bovis. IEP of convalescent phase lacrimal secretions with M. bovis antigen gave precipitin bands in a few unabsorbed samples but all of the absorbed samples were negative. DID against M. bovis antigen of unabsorbed, convalescent phase secretions from the four natural cases of IBK and from six experimental calves (#6, 16, 17, 18, 19, 20) resulted in precipitin bands that were reduced or eliminated by absorption with M. bovis. Samples from four calves (#6, 16, 18, 20) did not give precipitin rings by the RID test. Retrospectively, when RID was done with preinfection and convalescent phase lacrimal secretions from three calves (#18, 19, 20) against monospecific anti-IgM and anti-IgG (reactive with both IgG, and IgG2 ) antisera, both globulins were detected in all samples. The levels of IgG were slightly greater than those of IgM with the mean ratios of SIgA :IgG :IgM being approximately 10:2:1 on the basis of ring diameters. There was no consistent increase in

Vol. 39 -January, 1975

IgG and IgM in convalescent samples as compared to preinfection samples.

1 ...........

N1lA4& 18K CAWf3If[KS 9M7iER /NFKZWg-IMW Fig. 1. Scanned gel plotted on graph (samp'e from calf 2, natural IBK, three weeks postinfection). Un-

atbsorbed.

Fig. 2. Scanned gel plotted on graph (same sample Fig. 1). Absorbed with M. bovis.

as

37

TABLE III. Results of the Immunological Tests for Antibody to Moraxella boris of Convalescent Phase Lacrimal Secretions from Twenty Cattle Experimentally or Naturally Infected with M. boris

Calf Exp. 1 2 4 6 7 8 9 12 13 14 15 16 17 18 19 20

Severity of Disease #a + + + ++ + + + ++ ...+ ++ +++ . +++ +++ +++

....

Nat.

1 2 3 4

..

..

DIP Presence of Ppt Band Unabs. Abs. N N N N N N N P N N N N N N N N N N N N N N N P P R N P P N P R

+++ +++ +++ +++

P P P P

R R R R

RID Presence of Ppt Ring Unabs. Abs. N N N N N N N N N N N N N N N N N N N N N N N N N P N N N P N N

P P P P

PEP Increased Amount of SIgA Unabs. Abs.h P NC P NC P NC P R NC P P NC P NC P NC P NC P NC P NC R P P R R P P R P R

P P P P

N N N N

R R R R

aSamples tested were collected from the most severely affected eye at 2 to 4 weeks after the last inoculation

in the case of experimental calves or 4 weeks after the acute phase of disease in the naturally infected cattle bResults as in Table II Abbreviations: P = Present (positive) NC = No significant change N = Negative (absent) R = Reduced intensity = No sign of disease = Mild disease + = Moderate disease ++ + + + = Severe disease Unabs. = Unabsorbed sample of lacrimal secretion = Lacrimal secretion absorbed with M. bovis Abs.

DISCUSSION

EX?ERYtNTAL CAF PAWNFTk1M-dMAUfIP Fig. 3. Scanned gel plotted on graph (sample from experimental calf 2, preinfection). Unabsorbed.

E,A'PFRfLWTL Cf /'Rf/?NPFC7T/OA/-RSfORRfZ Fig. 4. Scanned gel plotted on graph as Fig. 3). Absorbed with M. bovis.

38

(same sample

The results of this study indicated that there was a moderate increase in the IgA class of Ig in lacrimal secretions of animals that had severe IBK. There was no increase in this Ig in the secretions of calves having milder signs of the disease. The presence of this class of Ig in normal lacrimal secretions has already been reported by Mach and Pahud ( 15). The absorption of the secretions with M. bovis cells resulted in a reduction in the amount of SIgA present. This was confirmed qualitatively in the RID test by the two to fourfold reduction in maximum titres and quantitatively by the reduction in ring diameters. Such a reduction was not achieved with secretions from those calves with milder signs of IBK. This might suggest that the increase in the SIgA content (specific for M. bovis) accompanied the development of severe IBK. According

Can. J. comp. Med.

0

%

The present study also supported the concept that severe ocular infection due to M. bovis stimulated the production of specific secretory antibodies. More of these antibodies were apparently induced by the na^

_ Fig. 5. Immunoelectrophoresis of unabsorbed lacrimal secretions (calf 4, natural IBK, four weeks postinfection). Precipitin bands formed both with antihovine

IgA and M. bovis antigen.

tural infection than by the experimental infection. This could be due to the generally more severe nature of the natural disease. The exact protective role of the secretory antibodies specific for M. bovis is not clearly understood. Since the lacrimal secretions of convalescing cattle contained locally secreted antibodies specific for the rec-

ognized

pathogen M.

bovis,

one is led to

1__

Af.'J W..

Fig. 6. Immunoelectrophoresis of absorbed (with M. bovis) lacrimal secretions (same sample as Fig. 5). Precipitin band formed with antibovine IgA only.

to Table III it is clear that all the secretions from natural cases of IBK and from

j , .] . * acute (with cases a few of the experimental . infections) contained aniibodies to M. bovis antigens. This specific antibody was partially or completely removed by absorption with M. bovis cells. Secretions from severely affected animals formed precipitin bands both with M. bovis antigen and antiIgA in IEP, DID and RID, thus emphasizing the importance of severity of the lesion in enhancing the production of antibodies, probably at the local level. These findings are in agreement with the observations of

previous researchers (8, 9, 20), namely, that animals developing only a mild disease could subsequently be reinfected while those developing acute clinical disease could not be reinfected. On PEP of samples from these severely affected animals, intensely stained protein bands (of the SIgA class of Ig) were seen and were reduced in size or intensity after absorption with M. bovis cells'.

Vol. 39 -January, 1975

Fig. 7. Double immunodiffusion of unabsorbed lacrimal secretions from four naturally infected cattleand(N1 to E1s), N4) and experimental calves 18 and 19 (E1s four weeks postinfect:on against antibovine IgA. Dis~~tinct precipitin bands formed.

t4

N2

Fig. 8. Double immunodiffusion of unabsorbed lacrima [s secretions (same samples as Fig. 7) against M. bovis antigen. Precipitin bands formcd.

39

the hypothesis that the disease may be best prevented by some more refined method of local vaccination. In the present experimental studies (17) calves that developed a severe disease after the first inoculation with M. bovis were more resistent to a second or third inoculation with that organism. These calves also developed specific antibodies to M. bovis in their lacrimal secretions. The precise nature of this mechanism of increased resistance, developed as a result of the disease, is unknown but one can postulate that it is due primarily to secretory antibody specific for the pathogen M. bovis. The possibility of mechanisms of cellular immunity also has to be considered (2, 23). It should be reemphasized that this study incompletely assessed the significance of IgM and IgG in the lacrimal secretions and as indicated by Duncan et al (6) selective transport mechanisms for Ig may operate in the eye. It has been shown that serum derived antibodies in lacrimal secre-

tions are important in resistance to chlamydial infections of the eye in man (11). Further studies on the pathogenesis of M. bovis-induced IBK are necessary to determine which bacterial antigens stimulate protective immunity. This might be done in laboratory animals (19). Increased local resistance might also be achieved by local or parenteral administration of M. bovis vaccines or bacterins or purified M. bovis antigen (2) with adjuvanis. Suggested approaches as a result of this study might parallel the approach of Provost and Vickers (18) who studied the possible use of a chlamydial vaccine in stimulating local ocular immunity to trachoma in baboons. They considered the local application of a trachoma strain of low virulence as a possible prophylactic measure. Similarly, the use of locally administered M. boris vaccines, bacterins or antigens for possible protection against IBK has not been explored and deserves attention.

REFERENCES 1. BRIGHTON, W. D. Gel diffusion and immunoelectrophoresis methods. In Progress in Microbiological Techniques. Edited by C. H. Collins. p. 140. New York: Plenum Publishing Corporation. 1967. 2. BUTLER, J. E., A. J. WINTER and G. G. WAGNER. Symposium: Bovine Immune System. J. Dairy Sci. 54: 1309-1314. 1971. 3. BUTLER, J. E and C. F. MAXWELL. Pr eparation of bovine immunoglobulins and free secretory component and their specific antisera. J. Dairy Sci 55: 151-164. 1972. 4. BITTLER, J. E., C. F. MAXWELL, C. S. PIERCE, M. B. HYLTON, R. ASOFSKY and C. A. KIDDY. Studies on the relative synthesis and distr ibution of IgA and IgG in various tissues and body fluids of the cow. J. Immun. 109: 38-46. 1972. 5. DUNCAN, J. R. Immune response to bacterial r-espiratory infections. Symposium: Bovine Immune Svstem. .1. Dairy Sci. 54: 1335. 1971. 6. DUNCAN, J. R., B. N. WILKIE, F. HIESTAND and A. J. WINTER. The serum and secretory immunoglobulins of cattle: Characterization and quantitation. J. Immun. 108: 965-976. 1972. 7. DUNCAN, J. R., B. N. WILKIE and A. J. WINTER. Natural and immune antibodies for Vibrio fetus in serum and secretions of cattle. Infection & Immunity 5: 728-733. 1972. 8. FARLEY, H., I. 0. KLIEWER, C. C. PEARSON and L. E. FOOTE. Infectious keratitis of cattle - a preliminary report. Am. J. vet. Res. 11: 17-21. 1950. 9. FORMSTON, C. Infectious keratoconjunctivitis of cattle (new forest disease). Vet. Rec. 66: 522-527. 1954. 10. FRANK, G. H. and R. G. MARSHALL. Relationship of serum and nasal secretion - neutralizing antibodies in protection of calves against parainfluenza-3 virus. Am. J. vet. Res 32: 1707-1713. 1971. 11. HANNA, L., E. JAWETZ, 0. BRIONES, H. B. OSLTER, H. KESHISHYAN and C. R. DAWSON. Antibodies to TRIC agents in matched human tears and sera. J. Immun. 110: 1464-1469. 1973. 12. HENSON, J. B., L. C. GRUMBLES and J. B.

40

13.

14.

15. 16.

17.

18. 19. 20. 21.

22. 23.

24.

DOLLAHITE. Evaluation of experimental bacterins for infectious hovine keratoconjunctivitis. SWest. Vet. 13: 213-216. 1960. HUGHES, D. E. and G. W. PUGH. Experimentally induced bovine infectious keratoconjunctivitis: effectiveness of intramuscular vaccination with viable Moraxella bovis culture. Am J. vet. Res. 32: 879886. 1971. IVOR-SMITH. Chromatographic and electrophoretic techniquLes. Vol. 2, p. 365. Interscience Publishers. 1968. MACH, J. P. and J. J. PAHUD. Secretory IgA. a major immunoglobulin in most bovine external secretions. J. Immun. 106: 552-563. 1971. MANCINI, G., A. 0. CARBONARA and J. F. HEREMANS. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2: 235-254. 1965. NAYAR, G.P.S. and J. R. SAUNDERS. Infectious bovine keratoconjunctivitis I. Experimental pro(luction. Can. J. comp. Med. 39: 22-31. 1975. PROVOST, P. J. and J. H. VICKERS. Attempted immunization against trachoma infection in baboons. Am. J. vet. Res. 33: 599-605. 1972. PUGH, G. W., Jr. Characterization of Moraxella bovis and its relationship to bovine infectious keratoconjunctivitis. Thesis, Iowa State Univ. 1969. PUGH, G. W., Jr. and D. E. HUGHES. Infectious bovine keratoconjunctivitis induced by different experimental methods. Cornell Vet. 61: 2345. 1971. TODD, J. D. Bovine immune response to respiratory infections of viral etiology. Symposium: Bovine Immune System. J. Dairy Sci. 54: 1334. 1971. TOMASI, T. B,, Jr. and J. BIENENSTOCK. Secretory Immunoglobulins. In Advances in Immunology. Vol. 9, p. 1. Edited by F. J. Dixon, Jr. and H. G. Kunkel. 1968. WATSON, R. R., A. B. MACDONALD, E. S. MURRAY and F. Z. MODABBER. Immunity to infections of the eye. III. Presence and duration of delayed hypersensitivity to guinea-pig inclusion conjunctivitis. J. Immun. 111: 618-623. 1973. WILCOX, G. E. Infectious bovine keratoconjunctivitis: A review. Vet. Bull. 38: 349-360. 1968.

Can. J. comp. Med.

Infectious bovine keratoconjunctivitis II. Antibodies in lacrimal secretions of cattle naturally or experimentally infected with Moraxella bovis.

II. Infectious Bovine Keratocon junctivitis Antibodies in Lacrimal Secretions of Cattle Naturally or Experimentally Infected with Moraxella bovis P...
1MB Sizes 0 Downloads 0 Views