J.COMP.PATH.
MORAXELLA
1979. VOL.~~.
441
STUDIES ON THE PATHOGENICITY BOVIS IN RELATION TO BOVINE KERATOCONJUNCTIVITIS
OF INFECTIOUS
BY R. L.
P.J.
CHANDLER,
H.
and B. TURFREY
P. BAPTISTA*
Research Council, Institute for Research on Animal Comjton, Berkshire, England
Apkultural
Diseases,
INTRODUCTION
Infectious bovine keratoconjunctivitis has many synonyms, e.g. contagious ophthalmia, pink-eye, and New Forest eye disease. The term, in its broad sense, refers to an inflammatory lesion confined to the eye, usually affecting the conjunctiva or cornea and produced by an infectious agent. Several agents have the potential to produce such a condition; however, the commonest expression of the condition constitutes a well-recognized syndrome and suggests a common aetiology in the majority of cases.h!ioraxeZZuLouisis the species most commonly incriminated in outbreaks of the disease; however, transmission experiments with this bacterium have had a variable degree of success(Wilcox, 1968; Baptista, 1979). It was decided to investigate the pathogenicity of several strains of M. bouis by a series of transmission experiments in cattle. Calves were chosen since they would be least likely to have suffered prior exposure and there is some evidence that they are more susceptible than adults. Attention was paid to the appearance of the colonies produced by the different strains since such appearance has been related to pathogenicity (Pedersen, Fronholm and Bovre, 1972). It has also been suggested that the possessionof pili (fimbriae) by &I. bouis is related to pathogenicity (Pedersen et al., 1972; Sandhu, White and Simpson, 1974) ; the strains used in the present experiment were therefore examined by electron microscopy. MATERIALS
AND
METHODS
Moraxella bovis Strains The following strains, originally isolated from field casesof the disease,were used; they were characterized as Moraxella bouis by the methods and criteria of Cowan and Steel (1975). GS. Isolated, subinoculated once on blood agar and lyophilized. In a preliminary experiment this strain was reconstituted after 2 years’ storage, serially subinoculated 11 times on blood agar and used to inoculate 4 calves; clinical signs of the disease developed in all 4 calves (Chandler-unpublished). In the present studiesthe strain, similarly reconstituted, was subinoculated on blood agar four times before its first use * Present
address:
002 l-9975/79/030421
Instituto +08
de Pesquisas
son.oojo
Veterinarias
Desiderio 6
Finamor,
1979 Academic
Porto
Press Inc.
Alegre, (London)
Brazil Limited
442
R. L. CHANDLER
et al.
in Group I, second inoculation. It was recovered from one case produced in this group and subinoculated 3 to 6 times on blood agar for use in the second series of inoculations in Groups III and V. ACV. Isolated, subinoculated twice on:blood agar and lyophilized”; after 6 months storage the strain was subinoculated on blood agar 3 times before use as inoculum for Group III. C17.5. Isolated and lyophilizedt as strain ACV above; following 1 year storage this strain was subinoculated twice on blood agar before use as inoculum for Group IV. Vet RO, JVotts 40 and Cav 2. These 3 strains: had been stored in the lyophilized state for several years; they had been isolated from clinical cases but their cultural history prior to lyophilization is not recorded. For the purpose of the present study they were reconstituted, subinoculated twice on blood agar and passaged 3 times through mice by the intraperitoneal route. The strains were subsequently subinoculated 5 times on blood agar, a rough and a smooth variant of strain Cav 2 being obtained before use as inocula for Groups I, II, V and VI. GSjPB and ACV/PB. These represent the respective strains GS and ACV which! following reconstitution from the lyophilized state, were subinoculated 5 times on blood agar, passaged twice in mice by the intraperitoneal route, and subinoculated a further 5 times on blood agar before use as inocula for Groups II and IV: second inoculations. 456/PB. This strain was isolated from a field case, subinoculated 12 times on blood agar, passaged 3 times in mice and subinoculated a further 3 times on blood agar before use as inoculum for Group VI, second inoculation. Calves These were crossbreds, of each sex and were aged 2 to 4 months at the time of inoculation. They were housed in groups of 6 in loose boxes, and were exposed to neither direct sunlight nor to excessive dust or flies. They were fed on calf nuts and hay from troughs at floor level. Inocula and Inoculation
Procedure
These were prepared by the method of Hughes and Pugh (1971). A 24 h growth on blood agar plates was scraped off and suspended in Tripticase Soy Broths, the complete growth from each plate being suspended in 1 ml of diluent. Viable counts were made by the method of Miles, Misra and Irwin (1938) after dilution in 10 per cent magnesium chloride according to Pugh and Hughes (1970)) and ranged from 3 x 10s to 8 :r: log per ml. 0.5 ml of the suspension was instilled into the conjunctival sac according to the method of Hughes and Pugh (1971), by a plastic 1 ml syringe with the needle removed; the eyelids were held in the closed position for 1 min after instillation. Observations on Calves The eyes of the calves were swabbed and examined bacteriologically at least twice before each inoculation; all calves were free of Moraxella bovis infection in both eyes, After inoculation the eyes were examined bacteriologically for 3 successive days and then at Ieast twice weekly. The calves were observed daily for the development of clinical signs for at least 1 month after inoculation. * t $ 5
Strain supplied by courtesy of Mr C. L. Wright, Auchincruive Veterinary Investigation Centre. Strain supplied by courtesy of Mr A. J. E. Woods, A.D.A.S., Coley Park, Reading. Strains supplied by courtesy of I)r J. Grant, London School of Hygiene and Tropical Medicine. Baltimore Biological Laboratories.
PATHOGENICITY
OF
lt!hZX&
443
b0Vi.C
Electron Microscopy Sample colonies of the strain grown overnight on blood agar plates were suspended in saline, transferred to electron microscope grids, fixed in osmium vapour and metal-shadowed with gold-palladium. The preparations were examined with a Philips EM300 electron microscope at an instrumental magnification of 6800 and an accelerating voltage of 80 kV; representative fields were examined for each strain and a comparison of numbers of pili for each strain was made from photomicrographs.
Plansof Experiments An outline of the experiments is shown in Table 1. Thirty-six calves were divided into 6 groups and used to estimate the pathogenicity of6 M. bovis strains in the first series of inoculations. The calves of each group were held together in the same pen during each experiment. Calves were inoculated by instillation of the requisite strain into the left eye; individual calves inoculated with suspensory vehicle alone acted as controls for the production of symptoms by the vehicle itself and, together with uninoculated calves, acted as controls for contact infection. Towards the end of the first series of inoculations it was apparent that the suspensory vehicle was innocuous and the use of the respective control was discontinued; the use of unin-
oculated controls was similarly discontinued in later experiments since relevant data had accumulated and there was need to conserve experimental calves. The 6 strains chosen for these first inoculations
did not produce
clinical
signs (with
one exception). A secondseriesof inoculations was therefore made 5 to 6 weekslater, with
different
strains
from
those of the first series, and the right eye in each case. M. bovisonly if the bacterium had neither become established nor produced signs in the first inoculation; animals that had acted as controls in the first series were either used again as controls or assigned
Animals were used for second inoculation with for inoculation with M. bovis. RESULTS
TransmissionExperiments The results of the transmission experiments are summarized in Table 1. In the first series of inoculations strains Vet RO and Notts 40 failed to become established and strains ACV and Cl75 became established only transiently in 1 of 4 and 2 of 4 cases respectively. The rough variant of the strain Cav 2 became
established
transiently
in one calf,
the smooth
variant
became
estab-
lished in 3 out of 5 calves and produced lachrymation in one of these. In the second series of inoculations strain GS consistently produced lesions, e.g. 5 of 5 in one group and 2 of 3 and 3 of 4 in others, with establishment of infection in all cases. After passage through mice as strain GS/PB, this pathogenicity was greatly reduced and no lesions were produced. Similarly, strains ACV and 456 after passage through mice failed to produce lesions. Infection was considered to be established when ;21. bovis was present on at least two consecutive examinations. With the strain GS, infection was usually detectable 24 h after inoculation and in all cases48 h after inoculation. The earliest clinical signs were observed, in about
half
the cases,
24 h to 48 h after
infection,
lachrymation
and
often
shallow cornea1 ulcers, diameter approximately 3 mm, being noted. In other cases the earliest clinical signs were observed up to 8 days after inoculation.
Cl75
I\-
v.i. control = Uninoculated. Vehicle control = Inoculated &ok: The controls remained * infection developed ** clinical syndrome
\.I
CAV 2 smooth CA\’ 2 rough
AC\r
III
\.
4”
Vet RO Notts 40
I’1
2*
1
1
;
v.i. control
1
1
1
1 1
Vehicle control
‘P itransient)
114 (transirntj 214 (transient) 415
O/4 014
iconsistent
Established injectiom
EXPERIiMENTS
exceptions
First inoculalionc
OF TRANSMISSION
with vehicle only, uninfected. with the following in one control calf. developed in each control call’.
4
5
4
4
Inoculated nmraxella
Strain
croup
RESULTS
1
with
1N CALVES
TAR1.E
contact
WITH
infection)
4x/I%
GS
,4C\‘/PR
GS
Strnhl
MORAXELLA
:
STRAINS
4
3
4
4
5
5
Inoculated moraxella
ROWS
2
2**
1** 1
2l.i. control
214 (transient)
313
o/4
5/j z/5 (transient) 414
Established infections
Second ittoculations
014
213
o/4
314
515 O/5
Clinical syndromes
PATHOGENICITY
OF
fb!OrcZXe&Z
b#ViS
445
(In group V 3 calves were killed 5 days after inoculation with this strain; 2 showed clinical signs at that time and it is likely that the clinical state would have developed later in the third animal.) The early clinical signs progressed over 2 to 3 days, photophobia, blepharospasm, oedema of the eyelids, and more evident conjunctivitis and keratitis being seen. In the majority of cases the intensity then declined and recovery, albeit with minor scarring, took place; in certain cases there was an increase in intensity of the lesion, including leucoma and blindness, and no evidence of recovery before slaughter took place. In Group I infection and clinical signs were noted in an uninoculated control calf; the incubation period indicated cross-infection with the strain GS. Similarly, in Group III, infection and the development of clinical signs was noted in 2 control calves, the time of development again indicating crossinfection. In most cases the infection was established and clinical signs developed only in the inoculated eye. However, both eyes became affected in 2 calves in Group I and 3 calves in Group III, subsequent to strain GS infection. hl. bovis was recoverable from the eyes of all affected calves that had been infected with strain GS and which subsequently developed clinical signs, up to the time of termination of the experiments. In Group V, following the inoculation with the smooth variety of strain Cav 2, infection was established in 4 of the 5 calves inoculated but clinical signs, represented by moderate lachrymation on one day of observation only, were seen in only one case. Cross-infection to an uninoculated control occurred 7 days after the inoculation of its companions, but no clinical signs were observed. Colonial Morphology Three colonial
types were observed,
as outlined
in Table 2:
A. Smooth, slightly raised, 1 to 3 mm in diameter with regular edges and leaving concavities in the solid medium when removed. This type was observed with strain GS and the smooth subline of strain Cav 2, and resembles the SCa form described by Bnvre and FrDnholm (1972). U. Smooth, convex, mucoid, 2 to 4 mm in diameter, leaving few or no concavities on the substrate when removed. This type was characteristic of the majority of the strains studied and resembles the N form described by Bavre and Fronholm (1972). C. Flattened, with a dense centre and slight or markedly irregular edge. This type was seen characteristically with the rough subline of Cav 2 strain, and resembles the SCb form of Bavre and Franholm (1972). Light microscopy of the strains confirmed that M. bouis has a considerable degree of pleomorphism. The usual appearance was of plump diplobacilli with rounded ends and some short chains. Other forms had square ends; diplococcal appearance was sometimes seen and slender filamentous forms occasionally occurred.
446
et
R. L. CHANDLER TABLE COLONIAL
Strain
MORPHOLOGY lNFECTI”ITY
Colonial (ype
Vet RO Notts 40 ACV Cl75 Cav 2 smooth Cav 2 rough
B B B B A c A B B B
LgPB ACVjPB 456/PB
Colonial
@pe:
AND AND
al.
2
DEGREE OF PILIATION IN RELATION I’ATHOGENICITY IN CALVES
Piliation
ZnfectiviQ
:
0 0 t
G i+ 0 Tf
t :-
6 0
0 t
TO
Pathogenici&
see text.
Piliation: + + profuse; + few; 0 no pili observed. Znfec/iui~: + ability to establish in eye; t transient infection Pathogenicity: + + ability to produce typical disease. + ability to produce lachrymation.
only.
Electron Microscopy The degree of piliation of the strains is outlined in Table 2. Profuse pili (fimbriae) were observed in preparations of strain GS and the smooth variant of strain Cav 2. Very few pili, apparently separated from the cell bodies were seen with strains ACV and GS/PB and no pili were observed with the other strains. DISCUSSIOiK
The results of the first series of experiments in calves showed that the strains used were either of minimal pathogenicity or non-pathogenic. Two strains which had been isolated several years previously and had also undergone passage in mice failed to become established; the rough variant of a similar strain became established only transiently in one case. However, the smooth variant of the latter strain became established in 4 out of 5 calves inoculated and lachrymation was observed in one case, * furthermore, there was evidence of cross-infection in one uninoculated calf. Two strains which had been more recently isolated became established only transiently in a minority of cases and produced no clinical signs. The second series of inoculations, into the uninoculated eye of calves that had been previously inoculated but failed to develop either infection OI symptoms, together with certain of the control calves that had not been inoculated, utilized the pathogenic strain GS and the latter and two other strains that had undergone passage in mice. The prior exposures during the first series of inoculations did not appear to have produced either immunity or hypersensitivity. Strain GS became established in all cases and consistently produced lesions; there was also evidence of cross-infection to control calves held in contact. Only transient infections in a minority of cases, and no pro-
PATHOGENICITY
OF
Moraxella
bovis
447
duction of the clinical state, were produced by the strains that had undergone mouse passage. Mouse passage had been introduced as an attempt to maintain or enhance the pathogenicity by in vivo passage but appeared detrimental in the case of strain GS, ineffective with strain ACV and either ineffective or detrimental regarding strain 456/PB. It is possible that the components responsible for pathogenicity of many M. bouis strains are very labile under laboratory conditions. Strains Vet RO and Notts 40 had the longest history of laboratory storage and maintenance and showed no evidence of infectivity; however, it must be noted that they had also undergone mouse passage. Strain GS had a 2 year history and was infective and pathogenic, so clearly its inherent pathogenicity had survived laboratory maintenance; but its pathogenicity was lost following mouse passage as strain GS/PB. Whereas mouse passage appears detrimental there was evidence suggesting lability due to laboratory storage and maintenance alone, as in the case of strains ACV and c 175. A direct association between loss of pathogenicity and subinoculation on blood agar was not evident however, e.g. the latter two strains had undergone 4 to 5 subinoculations but strain GS was still pathogenic following 12 subinoculations. Rough and smooth sublines of strain Cav 2 were obtained by simple selection during a series of 5 subinoculations, and these had very different levels of infectivity; fortuitous selection of sublines of strains, having varying pathogenicity, may well occur during serial subinoculation. The studies on colonial morphology indicated that colonies of the smooth, corroding type were associated with t.he pathogenic strain GS and with the smooth variant of strain Cav 2 which became established in the majority of calves; strains producing colonies of this type should be selected in searches for strains likely to be pathogenic. Electron microscopy showed ample pili in the two strains which became established in the eye, i.e. GS and the smooth variant of Cav 2. Pili may well be an important factor in the establishment of infection; however, this Cav 2 strain did not produce clinical signs. A further pathogenicity factor may be necessary for production of the disease syndrome. It is of interest that strain GS showed a reduced degree of piliation and also reduced pathogenicity following mouse passage; strain ACV also showed loss of piliation following mouse passage. The course of the disease and the nature of the lesions produced with strain GS closely resembled infectious bovine keratoconjunctivitis as seen in the field. This condition was obtained without recourse to exposure to artificial sources of ultraviolet irradiation (Hughes, Pugh and McDonald, 1965; 1968). The pathogenicity factors for M. bouis have yet to be defined; strain GS represents a strain for further studies in this respect. However, it would be advisable in selecting strains for transmission experiments and other investigations, such as vaccine studies, to take into account the extent of their maintenance in vitro, their colonial morphology and their possession of pili. These studies indicate that selected M. bouis strains can reproduce the disease syndrome, in the apparent absence of other pathogens. This does not, however, preclude the possibility that, under field conditions, and with less pathogenic strains of izI. bouis, other pathogens may play an additional role.
448
R.
L.
CHANDLER
et
d.
SUMMARY
The pathogenicity of nine strains of Moraxella bouis, in relation to infectious bovine keratoconjunctivitis, was investigated in a series of transmission experiments in calves. One strain consistently reproduced the condition, one strain became established but produced a minimal clinical state and the other strains failed to establish or produce the disease. Subsidiary morphological studies were made. Factors associated with the pathogenicity of field isolates 01 M. bovis may be labile under laboratory conditions of maintenance. ACKNOWLEDGMENTS
We wish to thank Dr J. Grant of the London School of Hygiene for his help and advice in these studies; and Messrs K. Smith, G. Osborn and J. Fogerty of this Institute for their specialized assistance. The work was supported in part by a grant
from the Ministry grant
of Overseas Development and Dr Baptista was in receipt of a
from the British
Council. REFERENCES
Baptista, P. J. H. P. (1979). Infectious bovine keratoconjunctivitis: a review. British Veterinary Journal. In press. Bavre, K., and Franholm, L. 0. (1972). V ariation of colony morphology reflecting fimbriation in Moraxella bovis and two reference strains of Moraxella nonliquefaciens. Acta Pathologica et Microbiologica Scandinavica 80b, 629-640. Cowan, S. T., and Steel, K. J. (1975). Manualfor the IdentQ’ication of Medical Bactel-ia. 2nd edit. Cambridge University Press. Hughes, D. E., and Pugh, G. W. (1971). Experimentally induced bovine infectious keratoconjunctivitis : effectiveness of intramuscular vaccination with viablr Moraxella bovis culture. American Journal of Veterinary Research, 32, 879-886. Hughes, D. E., Pugh, G. W., and McDonald, T. J. (1965). Ultraviolet radiation and Moraxella bouis in the etiology of bovine infectious keratoconjunctivitis. American Journal of Veterinary Research, 26, 133 1-l 338. Hughes, D. E., Pugh, G. W., and McDonald, T. J. (1968). Experimental bovine infectious keratoconjunctivitis caused by sunlamp irradiation and Morasella bovis infection : resistance to rr-exposure with homologous and heterolngous Moraxella bovis. American Journal of Veterinary Research, 29, 829-833. Miles, -4. -4., Misra, S. S., and Irwin, J. 0. (1938). Th c estimation of the bactcriocidal power of the blood. Journal of Hygiene, Cambridge, 38, 732-749.
Pedersen, K. B., FrDnholm, L. O., and Bovre, K. (1972). Fimbriation and colon) type of Moraxella bovis in relation to conjunctival colonization and development of keratoconjunctivitis in cattle. Acta Pathologica et Microbiologica Scandinavica, 80b, 91 l-918. Pugh, G. W., and Hughes, D. E. (1970). Inhibition of autoagglutination of Noraxella bovis by 10% magnesium chloride. Applied Microbiology, 19, 201L203. Sandhu, T. S., White, F. H., and Simpson, C. F. (1974). association of pili with rough colony type of Moraxella bovis. American Journal of Veterinary Research, 35, 437-439. Wilcox, G. E. (1968). Infectious bovine keratoconjunctivitis; a review. Veterinary Bulletin, 38, 349-360. [Received for publication,
November 27th, 19781