Infectious Bovine Keratocon junctivitis 1. Experimental Production P. S. G. Nayar and J. R. Saunders*

ABSTRACT One or both eyes of 20 calves were inoculated one or more times with various combinations of microorganisms (live or killed Moraxella bovis, infectious bovine rhinotracheitis virus, bovine adenovirus, bovine parainfluenza-3 virus and Mycoplasma bovoculi) by conjunctival instillation or direct inoculation of the conjunctiva or cornea. The eyes of all the calves received natural or artificial ultraviolet irradiation. Neither the adenovirus nor parainfluenza-3 virus became established in the eye or produced keratoconjunctivitis. Both M. bovis and infectious bovine rhinotracheitis virus became established in the bovine eye and produced disease. Subconjunctival or intracorneal inoculation of M. bovis caused a severe disease, simulating natural infectious bovine keratoconjunctivitis. Only the intracorneal inoculation

of mycoplasma produced severe keratoconjunctivitis. Eyes that on initial exposure to M. bovis became severely inflamed were more resistant to a second or third exposure to M. bovis, presumably by enhanced local defence mechanisms. RESUME Les auteurs ont utilise l'instillation de la ou l'injection au sein de cette membrane ou de la cornee pour introduire, 'a une

conjonctive

of Veterinary Microbiology, Westem College of Veterinary Medicine, University of Saskatchewan,

*Depart1ment

Saskatoon, Saskatchewan. Present address of senior author: Henderson Animal Clinic, 1432 Henderson Highway, Winnipeg, Manitoba. Submitted July 10, 1973.

22

ou plusieurs reprises, dans un seul ou dans les deux yeux de 20 veaux, differentes combinaisons des micro-organismes suivants: Moraxella bovis, vivant ou tue; le virus de la rhino-tracheite infectieuse bovine; un adenovirus bovin; le virus para-influenza 3 bovin et Mycoplasma bovoculi. Les yeux de tous ces veaux recurent une irradiation naturelle ou artificielle aux ultraviolets. Ni l'adenovirus ni le virus para-influenza 3 ne se developperent dans l'oeil ou produisirent la kerato-conjunctivite, contrairement a M. bovis et au virus de la rhino-tracheite infectieuse bovine qui declencherent une ophtalmie. L'inoculation de M. bovis sous la conjonctive ou dans la cornee produisit une maladie grave et semblable 'a la ke'rato-conjonctivite infectieuse bovine naturelle. Seule l'injection intracorneenne de M. bovoculi produisit de la kerato-conjonctivite grave. Les yeux qui developperent une inflammation marque'e 'a la suite d'un premier contact avec M. bovis, resisterent mieux a une seconde ou 'a une troisieme injection, probablement 'a cause d'une meilleure re'action de defense locale.

INTRODUCTION The bacterium, Moraxella bovis, is most often isolated from the ocular lesions of infectious bovine keratoconjunctivitis (IBK) and is considered to be the primary causative agent (12, 15, 16, 19). In addition, certain viruses (18, 20, 21), rickettsia (20) and mycoplasmas (4, 7, 8) have been assoiated with the disease. Many researchers have attempted to reproduce IBK experimenially (1, 2, 5, 6, 11, 13). The microbial agents, particularly M. bovis, were either instilled into the con-

Can. J. comp. Med.

junctival sac or inoculated directly into the conjunctiva or cornea. The role of ultraviolet radiation as an important predisposing factor in the pathogenesis of IBK was shown quite conclusively by Hughes et al (5, 11). The purpose of the present study was twofold: to produce IBK in calves using the basic experimental model of Hughes et al (5) and to study the local immunological response of the eyes of these calves to infection (9).

MATERIALS AND METHODS Four different exposure trials (Experiments I, II, III and IV) in 20 calves were planned on the basis of differences in inocula and route of administration. ANIMALS

Seven Holstein calves were purchased from the Department of Animal Science, University of Saskatchewan. Nine Hereford calves and four Aberdeen Angus calves were purchased from the Western Stockyards, Saskatoon. All the calves were four to six months old. The eyes of all the calves appeared clinically normal at the time of purchase and were culturally negative for M. bovis, mycoplasma and cytopathic viruses when sampled on two or more occasions prior to experimental infection. ANIMAL QUARTERS

The 12 calves in Experiments I, III and IV (numbers 1 io 5, 14 to 16, 17 to 20 respectively) were kept indoors in individual pens (separated by concrete walls five feet high) within a larger semi-isolation unit. This unit had no windows and filtered air was supplied through a mechanical ventilation system. Lighting was supplied by fluorescent lamps. The individual pens were equipped with separate feeders and automatic watering troughs. The eight calves (6 to 13) in Experiment II were initially housed in individual pens in the semi-isolation unit where they were maintained for 14 days following the first experimental infection. On the 15th day, these eight calves were put together in a fenced, grassed lot (115' x 40') in the WCVM Animal Paddocks and maintained there for the duration of the experiment.

Vol. 39 -January, 1975

FEEDING SYSTEM

The calves were fed two to three lbs of pelleis (20% calf starter/grower) daily, along with good quality alfalfa hay free choice. MICROORGANISMS

Three recently isolated strains (69333CIRI, 68153C3-41, FLA-64(6)') and a laboratory stock strain3 of M. bovis were used during this study. In addition infectious bovine rhinotracheitis (IBR) virus3, bovine adenovirus (BAV)3 parainfluenza-3 (PI3) virus3 and Mycoplasma bovocali' were also used. PREPARATION OF INOCULA

The M. bovis inoculum for exposing the animals was prepared according to the method described by Pugh (12). Strains of M. bovis were streaked on 5% sheep blood agar4 plates. The plates were incubated at 37°C for 24 hours and then the growth was scraped off with an inoculating loop and suspended in trypticase soy broth4 (TSB) prior to inoculation of the calves. The IBR virus was propagated in embryonic bovine kidney (EBK) cell cultures for 72-96 hours. Then the cell cultures were frozen at -60°C and later thawed at room temperature and used for inoculation. A similar procedure was used to prepare the PI3 and BAV inocula. After thawing, the infected cell cultures contained 107.7, 101. and 1062 TCID50 per ml of IBR virus, P13

virus and BAV respectively. Mycoplasma bovoculi, grown in liquid medium (7) incubated for 48 hours at 37°C was used as inoculum for some calves. MICROBIOLOGICAL AND SEROLOGICAL EXAMINATIONS For bacteriological and virological examinations two sterile, saline moistened, cotton tipped applicators were used to collect ocular

'Animal Diseases Research Institute (Western ), Lethbridge, Alberta. 2National Animal Disease Laboratory, U.S.D.A., Ames, Iowa.

3Department of Veterinary Microbiology, WCVM, University of Saskatchewan, Saskatoon, Saskatchewan. 4Difco Laboratories, Detroit, Michigan.

23

secretions (from each eye of each calf) as follows: (i) twice a week during the preexposure period of one to two weeks, (ii) within one hour postinoculation, (iii) on the first postinoculation day and every second day thereafter for 15 days following each exposure and (iv) at the termination of each experiment. One swab from each eye was streaked on the surface of a 5 % sheep blood agar plate and the plates were incubated at 37°C for 24 hours and then examined for M. bovis colonies. These were identified by biochemical techniques and immunofluorescence (10). For the isolation of mycoplasmas the same swab was used to inoculate a tube and a plate of bovine mycoplasma broth and agar respectively

(7).

For virological examination the second swab from each eye was placed in a sterile tube containing 2 ml of maintenance medium' and stored at -60°C. Later 0.1 ml of this medium was used to inoculate each of two tubes of EBK cells. The inoculated tubes were then incubated at 37°C and examined daily for a week for cytopathic changes (17). Lacrimal secretions for immunological studies were collected before as well as every week after inoculation and processed according to the method described elsewhere (9) and were stored at -60°C. Blood samples were collected from the calves at time of purchase, at two to three weeks after each experimental infection and at the end of the observation period. These sera were tested for precipitins against M. bovis (14) and for virus neutralizing antibody (Experiments I and II only) to IBR virus, PI3 virus and BAV by a microtitre test (17).

IRRADIATION In experiments I, III, IV and the first part of experiment II, a sunlamp6 was used for exposing the eyes of the calves to ultraviolet radiation. Each eye of each animal was irradiated separately for ten minutes at a distance of 20 inches (5). Irradiation was initially performed 24 hours prior to the exposure to cultures of microbial

agents, on the day of inoculation and once daily for five days following each exposure. The movement of the animal during irradiation was restricted by securing its head with a rope halter. The lamp was placed parallel to the plane of the cornea so that the eye was in the approximate center of the beam. INOCULATION PROCEDURES

Instillation: One-half ml of the appropriate inoculum was placed in the conjunctival sac and the eyelids were held closed manually for one to two minutes. Subconjunctival inoculation: A sterile one ml syringe with 25 G 5/ " needle was used for inoculating 0.2 ml of the inoculum subconjunctivally. The head of the animal was well secured using a rope halter. The lower eyelid was pulled down carefully to expose the ventral conjunctival membrane into which the microbial suspension was inoculated. Intracorneal inoculation: The calf was anaesthetized by intravenous injection of 20 to 25 ml of a 2.5% solution of Sodium Thiamylol7 (Surital). Then 0.2 ml of the inoculum was inoculated intracorneally at the limbus with a tuberculin syringe and a 25 G 5/8 " needle. Intramuscular injection: Five ml of the inoculum was inoculated into the gluteal muscles. EXPERIMENTAL DESIGN

A summarized experimental design with results is given in Tables I through III. Experiment I (calves 1 to 5) was designed to determine whether M. bovis, IBR virus, Pb virus and BAV would become established in the eye and produce IBK. An additional objective of Experiments II (calves 6 to 13). III (calves 14 to 16) and IV (calves 17 to 20) was a comparison of the effect of routes of administration on the severity of lesions produced by M. boris alone, with those produced by a combination of M. bovis, IBR virus and Mycoplasma bovoculi. An additional aim was to determine whether the calves developed increased resistance to subse-

5Hank's lactalbumin medium (Gibco Laboratories, New York) with 3% volume calf serum, penicillin and streptomycin pH 7.2.

6General Electric Co., Ltd., Canada.

24

7Parke, Davis, & Co., Ltd. Brockville, Ontario.

Can. J. comp. Med.

quent exposures, particularly to M. bovis. In ihis regard viable and killed M. bovis cells were tested in different calves in order to detect any differences in resistance to reexposure. Before and after inoculation of the calves recognized standard precautions to prevent possible spread of microorganisms by personnel or fomites were used as: (i) separate pens, no direct contact except in second part of Experiment II when calves were together in paddock (ii) individual halters and other equipment for each calf and (iii) washing of boots and hands, changing coveralls when going from pen to pen. In addition to sampling procedures outlined above the calves were observed daily and rectal temperatures were taken once daily during the experiments. At the end of the experiments the calves were sent to slaughter, the heads being collected for gross and possible microscopic examination of the eyes.

RESULTS Tables I, II and III summarize the experimental treatments as well as the clinical and laboratory findings for Experiments I, II, III and IV respectively. Clinical signs

and ocular lesions that developed following inoculation were graded as to severity by these designations: (i) - (negative or normal eye) (ii) + (mild disease) (iii) + + (moderate disease) and (iv) + + + (severe disease) as shown in Figs. 1 to 5. A microorganism was considered to have established infection of the eye only if it could be reisolated from the eye for longer than one day following inoculation. EXPERIMENT I

All five calves were serologically negative for antibody to M. bovis, IBR virus and BAV prior to inoculation but had low titres (up to 1:16) against PI3 virus. In calf 1 (see Table I) the conjunctival instillation of a laboraiory stock strain of M. bovis resulted in a mild conjunctivitis and slight corneal opacity (Fig. 2). Reexposure to M. bovis four weeks after initial exposure resulted in a mild conjunctivitis only. Calf 2 also developed a mild disease after exposure to a freshly isolated strain of M. bovis. Conjunctival instillation of IBR virus at day 28 resulted in mild conj unctivitis while intramuscular inoculation of IBR virus at day 35 resulted in an elevated rectal temperature and a leuko-

TABLE I. The Response of Calves to Conjunctival Installation of Moraxella bovis and Three Bovine Viruses (Experiment I) Calf 1

Mb

2

Mic

3 4

+

+

Days Organisms Reisolated after Exposure 2 1 7 13

+

+

(M) 13

Organisms Instilled Into Both Eyes at Severity of Lesions Following Exposure Days 1 2 35 28 0 Mb

IBR

P13 Vc

. . .d

(P13) 0

...d

Mc

+

-

BAV

1\jc

(IBR)3

(M) (BAV) (M) (BAV)

15

1

0

Serum Antibody to Organisms at Days 28 50 0 (M) -

-

(M)--

(IBR)-

(PI3) +

(M)

+

+

+

-

-

-

(BAV) -

-

-

at for seven irradiation received each in isolation exposures days sunlamp pens -These Holstein calves housed 1 and 2 bovis M. of bLaboratory stock strain (nonhemolytic) cStrain 68153C3-4 of M. bovis dCalves 3 and 4 inoculated intramuscularly with IBR virus and PI3 virus respectively ... = No treatment M = M. bovis Abbreviations: - = No response or negative IBR = Infectious bovine + = Mild disease or antibody present rhinotracheitis PI3 = Parainfluenza 3 virus BAV = Bovine adenovirus, serotype 3 D

Mc

..

BAV

Vol. 39 -January, 1975

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13

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