Detection of antibodies to Erysipelothrix in stray dogs in Japan.

Erysipelothrix rhusiopathiae is considered as a pathogenic bacterium for many species of animals. The genus Erysipelothrix had been thought to contain a single strain. However, on the basis of genetic homology, the genus Erysipelothrix was found to constitute different strains, E. rhusiopathiae and a new strain, E. tonsillarum [9]. E. tonsillarum can be distinguished from E. rhusiopathiae by serology, the fermentation of saccharose, genetic homology and the lack of pathogenicity for pigs [9,10,12].

The classical host of the bacterium is the pig, but it can also induce a wide variety of disease conditions in other mammals and birds [4,11]. The bacterium has been isolated from dogs with endocarditis [1,3,4,7]. In an experimental infection study using an isolate from a dog, it was confirmed that dogs developed endocarditis after intravenous inoculation [2]. The isolates from dogs with endocarditis in Belgium were typed as serovar 7, which is one of the E. tonsillarum serovars [6,10]. Furthermore, the isolate was classified into genomic E. tonsillarum based on the characteristics and genetic homology [12]. Other strains isolated from several cases of erysipelas in dogs were also typed as serovar 7 [3]. These reports strongly indicated that E. tonsillarum was a canine pathogen.

However, there is no information about serological surveys in dogs to elucidate the epidemiological features of the disease in the field. Furthermore, there are few studies about the mechanism of erysipelas in dogs. As a causative factor of bacterial endocarditis, a pre-existing heart lesion has been suspected, but the relation between them is still obscure [2,3]. It has not been documented, whether erysipelas is actually caused by the mixed infection with Erysipelothrix and other organisms in dogs.

In this study, to search for the epidemiological features of erysipelas infection among dogs, we surveyed the levels and the distribution of anti-Erysipelothrix antibodies among dogs in the field.

The serum samples used in this study were obtained from 120 stray or homeless dogs in Tokyo metropolitan animal preservation center, during the period of April 1999 to March 2000. As negative samples, we also used the serum derived from 19 dogs of SPF beagles origin in our laboratory. The growth agglutination (GA) test has been generally applied for the assessment of immunity in the animals to erysipelas [14]. It is known that E. rhusiopathiae antigen in the GA test cross-reacts with E. tonsillarum [8,11]. In the present study, therefore, the GA test was carried out to quantify the antibody responses to Erysipelothrix in dog serum. The procedure was carried out by a method of [5] with some modifications. Two fold dilutions of the serum were prepared with tryptose phosphate broth (pH 7.6, Difco) containing 0.1% Tween 80, 25 μg/ml of gentamicin, and 250 μg/ml of kanamicin in 96 well, V-bottom plates. Overnight broth culture of the Marienfelde strain (serovar 1a of E. rhusiopathiae, and international standard strain for the GA test) was used as live antigen. Five μl of the culture was added to 100 μl of each serum dilution. The agglutination was read after incubation at 37 degrees Celsius for 24 h, and titres were expressed as the reciprocal of the highest serum dilution causing agglutination. In studies of Erysipelothrix infection in pigs and chickens, we previously described that the GA titre rose to 1:16 or higher in the serum experimentally infected with virulent Erysipelothrix strains [5,13]. Thus, in the present investigation, porcine serum that had GA titre 1:16 to 32 was used as positive control and GA titre of 1:16 or higher was considered to be positive.

The results of serological survey of GA test are shown in Table 1. In total, a GA titre of 1:16 or higher indicating possible Erysipelothrix infection was detected in 6 (5.0%) of 120 serum samples derived from dogs in the field. Of these positive sera, four (66.7%) had a GA titre 1:16, one (16.7%) had a GA titre 1:32, and one (16.7%) had a GA titre 1:128. In 19 serum samples derived from laboratory dogs, one sample had a GA titre 1:4, but a sample with GA titer of 1:16 or higher was not detected. As a result of the antibody investigation, we could demonstrate the incidence of dogs having the GA titre 1:16 or higher (suspected Erysipelothrix infection) in the field, but there was no statistically significant difference between the population of positive samples in field dogs and that in laboratory dogs (Fisher's exact test).

Table 1

GA antibody level to Erysipelothrix in Japanese dogs.

	No. of sera tested	No. of sera with anti-Erysipelothrix antibody at indicated titre							Proportion of positive samples (%)(≥16)
		<4	4	8	16	32	64	128
Field dogs (%)	120	70	31	13	4	1	0	1	5.0
Laboratory dogs (%)	19	18	1	0	0	0	0	0	0

Erysipelothrix has been isolated from several cases of endocarditis and septicaemia in dogs [1,3,4,7] and it has been demonstrated that the bacterium could cause endocarditis and arthritis in dogs by the intravenous injection [2]. There are hardly any reports that had examined epidemiological investigation and mechanism of the erysipelas infection in dogs. This is the first report on the existence of dogs having the positive level of antibodies against Erysipelothrix with 5% prevalence, even if it is a low proportion, indicating there was a certain risk of Erysipelothrix infection among dogs in the field. From only the present data, it is difficult to know whether Erysipelothrix is able to cause the endocarditis absolutely or secondary to other organisms. In any case, further investigations are needed to make clear the mechanism of erysipelas infection in dogs.