clinical isolate from a pneumonic foal and a laboratory passaged Congo red negative variant of this strain. Three nonimmunized foals of similar age acted as ...
Protection of Foals Against Experimental Rhodococcus equi Pneumonia by Oral Immunization J.M. Chirino-Trejo, J.F. Prescott and J.A. Yager*
equivalent servirent de temoins. Trois peak incidence of the disease, in six to ABSTRACT apres la derniere immunisa- eight week old foals, coincides with the Two groups of three one to three semaines tous les poulains subirent cinq time when maternally derived antition, week old foals were immunized orally infections de defi, en sept jours, au body is waning. Prescott and others (6) on four occasions over five weeks with moyen d'aerosols qui, a chaque occa- had evidence that oral challenge of two strains of Rhodococcus equi, a contenaient environ 1010 bacteries foals with R. equi led to immunization. sion, clinical isolate from a pneumonic foal de la souche isolee du poulain atteint Recently Takai and others (5) reported and a laboratory passaged Congo red de la pneumonie clinique. Les temoins that R. equi administered orally to negative variant of this strain. Three devinrent gravement malades et on en foals resulted in significant increase in nonimmunized foals of similar age effectua I'euthanasie. L'immunisation specific IgG levels in serum. acted as controls. Three weeks after avec I'une ou l'autre des souches The purpose of the present study the last immunization, all foals were precitees protegea egalement les pou- was to determine whether oral immuchallenged on five occasions over lains contre les infections de defi et nization with live R. equi would proseven days by aerosol infection with une clairance pulmonaire tect against experimental challenge, about 1010 of the pneumonic foal iso- favorisa L'immunisation orale peut par and to compare the protection afforded rapide. late on each occasion. Control foals proteger les poulains contre by two strains. One strain was a lowbecame seriously ill and were eutha- consequent une forte infection de defi avec R. equi. passage isolate from the lung of a foal nized. Immunization with either strain Les proteines associees a la coloration with pneumonia, and the other was a protected foals equally against the rouge Congo ne semblent pas im- variant of this strain which differed challenge, and resulted in rapid lung au dans I'immunite protectrice. only in the absence of two proteins, a pliquees clearance. Oral immunization can thus dominant 17.5kd and a minor 15kd protect foals against severe challenge Mots cles: Rhodococcus equi, poulains, protein. This strain, unlike its parent, with R. equi. The proteins associated immunisation. did not produce a red colony when with Congo red colony staining appear grown on media containing Congo red not to be involved in protective dye. In other facultative intracellular INTRODUCTION immunity. pathogens Congo red staining has Rhodococcus equi is an important been associated with virulence (7,8). Key words: Rhodococcus equi, foals, cause of suppurative bronchopneuimmunization. monia of foals in certain parts of the world. In Ontario it was reported to cause 10% of deaths of foals under six RESUME MATERIALS AND METHODS months of age (1). No effective immunoprophylactic Cette experience portait sur deux FOALS measures have been described and groupes de trois poulains ages d'une a Three groups of three one to three trois semaines que les auteurs immu- many aspects of immunity to R. equi niserent, par la voie buccale, a quatre pneumonia in foals remain unclear. week old crossbred pony foals were occasions en cinq semaines, avec deux While R. equi appears to behave as a kept, with their dams, on separate souches de Rhodococcus equi dont typical facultative intracellular para- pastures, which had been used for l'une provenait d'un poulain atteint site (2,3), in which protection would be horses for several years. Group CR+ d'une pneumonie clinique et l'autre, expected to be primarily by cell- (mean age 1.5 weeks) were immunized d'une variante de cette souche soumise mediated immune mechanisms, recent orally with a live Congo red positive a des passages au laboratoire et nega- studies (4,5) suggest that humoral variant of R. equi and group CRtive a la coloration au rouge Congo. immunity may also be important in (mean age 2.8 weeks) were immunized Trois poulains non vaccines et d'un age protection against the disease. The orally with a live Congo red negative
*Department of Veterinary Microbiology and Immunology (Chirino-Trejo, Prescott) and Department of Pathology (Yager), Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1. Reprint requests to Dr. J.F. Prescott. Submitted January 15, 1987.
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Can J Vet Res 1987; 51: 444-447
variant of the same strain R. equi. CLINICAL AND POSTMORTEM EXAMINATION Group C foals (mean age 1.6 weeks) Blood samples were taken before were nonimmunized controls. each vaccination and after challenge for hematological examination. FolBACT[ERIA lowing challenge, foals were examined Temperatures, pulse and respidaily. Rhodococcus equi strain 2523-85 rates were recorded. Foals in ratory was isolated from the lung of a pneuC were euthanized ten days after group monic foal and passaged three times in initial aerosol challenge, together with vitro before being stored at -70°C. one foal from each of groups CR+ and Colonies staining red on Congo red CR-. The remaining foals were euthacontaining media (8) are described nized on day 14. Quantitative bacteriohere as CR+. A Congo red negative variant of this strain, CR-, was ob- logical examination was done at the tained after passage in vitro 100 times. same four predetermined sites in the This strain was also stored at -700C. lungs of all foals. Sections of lung and Bacteria were grown in nutrient broth intestine were fixed in 10% buffered formalin immediately after death and for 48 h at 370C. histological sections examined using routine histopathological procedures. Sections were stained with hematoxIMMUNIZATION PROCFDURFS ylin and eosin (H & E) and Brown and Group CR+ foals were given 100 mL Brenn's modification of the Gram phosphate buffered saline, pH 7.2, stain. containing 109-10'0 CR+ R. equi and group CR- foals the same number of CR- bacteria, on four occasions. RESULTS Administration of this dose by stomach Foals in each of the groups remained tube was at the time of group separahealthy and no changes in total or tion, and again two, three and four differential white blood cell count weeks later. Following administration occurred during the immunization of bacteria the stomach tube was period. flushed with water to decrease the Following challenge, foals appeared chance of pharyngeal contamination alert and in good condition until seven when the tube was withdrawn. Foals after initial respiratory exposure. days were weaned three weeks after the last The control group became increasingly oral immunization and placed sepadepressed and dyspneic from this time, rately in their groups in isolation faciliand became anorexic. The CR- foals ties for experimental challenge with R. equi. EXPERIMENTAI. CHAl l ENGE
appeared somewhat depressed from days 7-9, but the CR+ group remained alert. Mean temperatures, pulse and respiratory rates of foals in the three groups are shown in Table I. These parameters were significantly elevated in the nonimmunized foals compared to immunized animals from day 7 after initial infection. For humane reasons, all nonimmunized foals were euthanized on day 10, as were, for control reasons, one foal from each of the two immunized groups. The remaining foals were euthanized 14 days after first infection.
Significant pathological changes only in the lungs. Non-
were observed
vaccinated animals had heavy, edematous, congested lungs with extensive consolidation. On cut section, the consolidated parenchyma comprised multiple coalescing tan-colored, fleshy foci with no suppuration. In less severely affected areas, these foci were centrilobular in distribution. The lungs of one CR+ foal, sacrificed at the same time as the controls, showed areas of patchy consolidation, which were considerably less than in control animals. The CR- foal sacrificed at the same time as the controls had lungs which were moderately congested and slightly firmer on palpation than normal lungs. The lungs of the two foals in each of the CR+ and the CR- groups killed 14 days after first aerosol challenge were congested but were otherwise normal.
TABLE I. Temperature, Pulse and Respiratory Rates in Immunized and Nonimmunized Foals Following Aerosol Challenge with Rhodococcus equi
All foals in each group were chal- Parameter; Days Following Challenge lenged by aerosol with the CR+ strain, Foal 1 5 3 7 9 11 using a method based on that described Groupa 13 by Martens and others (9). The aerosol Temperature (C) was generated by an ultrasonic nebu- CR+ 38.6 ±0.7 39.3 ±0.1 38.5 ±0.2 37.9 ±0.4 38.1 ±0.2 38.0 ±0.1 38.0 ±0.2 38.5 ±0.8 38.6 ±0.6 38.7 ±0.1 39.7 ±0.8b 40.5 ±0.3b lizer with a disposable drug vial Control _ _ 38.7 ±0.8 39.1 ±0.6 39.1 +0.6 38.0±0.2 38.2 ±0.2 38.1 ±0.1 38.3 (Hospal Medical Co., Montreal, Que- CRbec). Twenty milliliters of a suspension RespiratorY rate (respirations/ min) NDC ND ND 27 ±8 31±5 23 1 18 ±2 of 6.6 x 108 R. equi were placed in the CR+ Control ND ND ND 48 ±6h 67 ±30b disposable drug vial which was at- CRND ND ND 37 ±6 25 ±2 28 ±2 20 tached to an inhalation mask. Foals Pulse rate min) (beats/ were challenged for 15 min. During CR+ ND ND ND 71 ±10 61 ±7 64±4 76±4 this time about 18 mL of the suspen- Control ND ND ND 87 ±22h 99 ±23b sion was aerosolized, the majority of CRND ND ND 65 ± 10 61 ±8 75 ±5 72 which (1.2 x 1010 colony forming units) with immunized CR+ R. equi strain; Control. nonimmunized control; CR-, immunized with was judged to have been inhaled. This aCR+, CR- R. equistrain. Three foals per group day 0-10, two thereafter. Control foals euthanized day 10 dose was administered on days 0, 1, 2, hSignificantly different from other groups (p
