Introduction

Erysipelothrix rhusiopathiae is a well known cause of disease and mortality in dolphins. The exact route of infection is not known. Oral infection by eating contaminated fish and infection via scratch wounds are the most plausible ones.^1,4 Infection mostly results in septicaemia and mortality. Infected animals often die before symptoms are observed. Curative treatment of this acute form of erysipelas mostly comes too late. More rarely a subacute disease occurs, characterized by general illness, leukocytosis and rhomboidal skin lesions. Here, treatment by a combination of hyperimmune serum and antibiotics can be successful. Chronic infections, as in pigs or man, do not occur in dolphins.⁵

At least 15 different serotypes of Erysipelothrix rhusiopathiae have been described. Approximately 80% of all swine isolates belong to serotypes 1a, 1b and 2.⁷ Other serotypes isolated from swine are 4, 6, 7, 8, 9, 10, 15, 16, 20, N.^3,7 In dolphins, only 2 isolates seem to have been serotyped and were identified as serotypes 5 and 6,⁶ suggesting that other serotypes than those in swine could be responsible for erysipelas in dolphins. In pigs, prevention of infection is performed by vaccinating the animals with vaccines containing serotype 2 strains. In dolphins, vaccination is performed using swine vaccines, however, their efficacy against the Erysipelothrix rhusiopathiae strains infecting dolphins is not known.

The aim of this study was to determine if vaccination with serotype 2 strains could protect against mortality following infection with dolphin erysipelas isolates. Since challenge studies can not be performed in dolphins, a mice cross-protection model was used.

Materials and Methods

Animals

Thirty-one BALB/C mice were used. They were 12 weeks or older, were housed in isolation units and obtained food and water ad libitum.

Erysipelothrix rhusiopathiae isolates

The isolates used in this study were:

 5207 = an isolate from a dolphin of the Antwerp zoo that died from erysipelas septicaemia in 1990. It was a kind gift of Dr. W. Demeurichy (Antwerp Zoo)

 266/661 1= isolated from the liver of a dolphin that died from Erysipelas septicaemia in the dolphinarium of Brugge in 1987

 266/2344 = isolated from the kidney of a pig which has died of erysipelas in 1979. It was a kind gift of Dr. L. Devriese (Lab Vet. Mcrob. and Mycol., Faculty Vet. Med., RUG, Gent, Belgium).

The serotyping of these 3 strains is in progress.

Furthermore, a commercial serotype 2 swine vaccine was used.

Bacterial inoculum

Isolates were stored lyophilized at 4° C or in LYM medium (I volume Brain Heart Infusion (BHI) broth, 3 volumes inactivated horse serum and 0.5% (wt/vol) glucose) at ^-8° C. Lyophilized bacteria were suspended in sterilized phosphate buffered salt solution (PBS). Suspensions were inoculated on blood agar base plus 5% (vol/vol) whole defibrinated sheep blood and incubated during 2 days at 37° C. Subsequently, characteristic colonies were reinoculated in BHI broth and incubated for 48 to 72 hours at 37° C. Bacteria were harvested by centrifugation at 3000 g during 30 min, washed 3 times in PBS and re-suspended in PBS. The number of viable bacteria was determined by inoculating 10-fold dilutions in PBS on BHI agar, incubating the plates for 48 hours at 37° C and counting the number of colony forming units (CFU).

Lethal dose 50% in mice

The lethal dose 50% (LD50) of an isolate - the number of CFU which induces mortality in 50 percent of the inoculated mice - was determined by inoculating at least 5 different mice with tenfold dilutions of a suspension of this isolate. Mice were observed for mortality during 14 days. Severely ill mice were euthanatized.

The LD50 was calculated by the method described by Kärber (193 1)2 and was expressed as the number of colony forming units (CFU)/ml bacterial suspension.

Vaccination and challenge procedure

A first group of seven mice was vaccinated subcutaneously with 0. I ml of the commercial serotype 2 swine vaccine and a second group of seven mice was not vaccinated and served as control group.

In each group mice were challenge infected intraperitoneally with 300 to 10000 LD50 in 0.1 ml PBS at three and 24 weeks after vaccination and were observed during 14 days for signs of illness and mortality.

Samples

Blood samples were taken from mice via the tail vene just before vaccination or infection. Challenge infected mice which were euthanatized, were examined for Erysipelothrix rhusiopathiae septicaemia by attempting to isolate the bacteria from organs (spleen, liver) on blood agar base plus 5% (vol/vol) whole defibrinated sheep blood.

Antibody ELISA

Briefly, 100µl of a mixture of 10⁷ bacteria /ml containing equal amounts of the isolates 5207 and 266/6611 suspended in 0.05 M carbonate-bicarbonate buffer (coating buffer: pH 9.4) was used for coating the wells of an ELISA plate. After an overnight incubation at 4° C, remaining binding sites were blocked with glycine 5% (wt/vol) in coating buffer during 1 hour at room temperature, where after the wells were washed 4 times with PBS supplemented with 0.05% (vol/vol) Tween20 (wash solution). Meanwhile, sera of mice were pretreated with kaolin to remove substances that might cause increases in the background responses in the ELISA. Subsequently, the sera were diluted 1/50 in PBS supplemented with 0.05% (vol/vol) Tween-20 and 5% (wt/vol) non fat dry milk powder and 100 VI of each diluted sample was brought into one of the coated wells. After 1 hour incubation at 37 °C, the wells were washed four times with the wash solution. Then, a rabbit anti-mouse immunoglobulin (whole molecule) antiserum conjugated with horseradish peroxidase was added to the wells in the appropriate dilution. Following incubation during 1 hour at 37° C, wells were washed four times again and an ABTS substrate solution was added. The absorbance was measured at 405 nm after 1 hour incubation at 37°C and is positively correlated with the Erysipelothrix specific antibody concentration.

Results

LD50

The results of the LD50 in mice show that both dolphin isolates are highly pathogenic for mice (Table 1).

Table 1.

Antibody response following vaccination

No antibodies against Erysipelothrix were detected in non-vaccinated mice. Three weeks after vaccination with the commercial serotype 2 vaccine, antibodies could be demonstrated. Antibodies were still present at 24 weeks after vaccination.

Challenge infection

All control animals died between one and three weeks post challenge. There was no difference in survival time between the three isolates.

All vaccinated animals survived the challenge, except for the mouse challenged 24 weeks after vaccination with the swine isolate. Erysipelothrix could be isolated from the organs of this mouse.

Table 2.

Discussion

Results of the present study show that vaccination of mice with a serotype 2 commercial vaccine can protect against challenge with dolphin Erysipelothrix rhusiopathiae isolates. These results indicate that serotype 2 vaccines most likely can induce a protective immunity in dolphins against erysipelas. In swine and mice, it has been observed that vaccination with serotype 2 strains protected against challenge exposure with swine Erysipelothrix isolates of serotypes 4, 6, 7, 9, 15, 16 and N. However, challenge with serotype 20 could still induce mortality in mice and challenge with serotypes 8, 10 and 20 still local lesions in swine similar to the lesions in non-vaccinated controls.³ These data show that the serotype 2 vaccine does not induce immunity against all pathogenic serovars. At this moment few data are available on the Erysipelothrix serotypes which induce disease in dolphins. Only two pathogenic isolates have been serotyped.⁶ Serotyping of both dolphin isolates of the present study is in progress. More isolates, however, should be serotyped and used in this mice model in order to better evaluate the immunity induced by vaccination with serotype 2 strains against dolphin isolates.

Furthermore, an optimal immunization scheme should be developed. Therefore, data are needed on the vaccine dose necessary to induce a protective immunity in dolphins, on the duration of this immunity and on the interference of maternal immunity on this vaccination.

Conclusion

A commercial serotype 2 swine vaccine can induce a protective immunity against Erysipelothrix rhusiopathiae isolates of dolphins.

Acknowledgements

This research was made possible thanks to several financial supports. The authors wish to thank: la Bourse de la Vocation, Belgium; Hoechst, Belgium; John G. Shedd Aquarium, USA; Ocean Park, Hong Kong; Zoo Marine, Portugal. Furthermore, Dr. W. Demeurichy (Antwerp Zoo) and Dr. L. Devriese (Lab Vet. Microb. and Mycol., Faculty Vet. Med., RUG, Gent, Belgium) are kindly thanked for the isolates.

References

1.  Geraci, J.R., R.M. Sauer, and W. Medway. 1966. Erysipelas in dolphins. American Journal of Veterinary Research 27:597-606.

2.  Kärber, G. 193 1. Bietrach zur kollktiven Behandlung pharmakologischer reichen-versuche. Archive für Experimentele Pathologie und Pharmacologie 162:480-487.

3.  Sawada, T., and T. Takahashi. 1987. Cross protection of mice and swine given live organism vaccine against challenge exposure with strains of Erysipelothrix rhusiopathiae representing ten serovars. American Journal of Veterinary Research 48:81-87.

4.  Suer, L.D., N.A. Vedros, J.P. Schroeder, and J.L. Dunn. 1988. Erysipelothrix rhusiopathiae. I. Isolation and characterisation from pinnipeds and bite/abrasion wounds in humans. Diseases of Aquatic Organisms.

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6.  Takahashi, T., T. Fujisawa, Y. Tamura, Sh. Suzuki, M. Muramatsu, T. Sawada, Y. Benno, and T. Mitsuoka. 1992. DNA relatedness among Erysipelothrix rhusiopathiae strains representing all twenty-three serovars and Erysipelothrix tonsillarum. International Journal of Systematic Bacteriology 42:469-473.

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