Abstract

Microbiol0gic sampling of feed fish was initiated at Shedd Aquarium following the loss of a Pacific white-sided dolphin (Lagenorhynchus obliquidens) to acute erysipelas septicemia in 19961. The presumptive route of infection was introduction of the organism through a breach in the gastrointestinal mucosa. In an attempt to assess the prevalence of E. rhusiopathiae in feed fish, surface samples from thawed fish were plated on MacConkey, CNA and blood agar and incubated at 95°F (35°C) for 48 hours. E. rhusiopathiae was not isolated among sample fish at that time.

In 2000, following the death of a beluga whale (Delphinapterus leucas) to erysipelas septicemia, testing of feed fish resumed. Through collaboration with National Veterinary Services Laboratories in Ames, Iowa a formula for enrichment media was established (Table 1) to augment the growth of E. rhusiopathiae. Subsequent to this change E. rhusiopathiae has been isolated, predominantly from capelin (Mallotus villosus) but also intermittently from each type of feed fish sampled. Results are presented from twice-weekly sampling and culture of feed fish through the first six months of 2000 (Table 2).

The prevalence of E. rhusiopathiae on feed fish suggests that ingestion is the predominant route of infection. The role of reservoir animals and the environment in exposure and susceptibility has also been discussed. A prospective microbiologic study is currently being undertaken at Shedd Aquarium. Samples are obtained once monthly from the habitat, feed-fish and animal sources and are cultured using the methodology described (Table 1).

This is a pilot study. The hypothesis is that the organism will be isolated with moderate frequency from feed fish, infrequently from collection animals and rarely from the habitat (water or substrate). This study will provide information that will assist in 1) standardizing our methodology in environmental and feed fish microbiologic testing 2) provide preliminary information regarding the source of infection and 3) inform further studies.

Table 1 - Protocol and Methodology for the Isolation and Identification of Erysipelothrix rhusiopathiae

1.  Goal
To isolate Erysipelothrix rhusiopathiae from the surface of feed fish, potential collection animal reservoirs and the Aquarium environment.

2.  Sample Sites and Sources

a.  Randomly selected, partially or completely thawed fish and squid at breakout

b.  Randomly selected feed fish at 5+ hours post thaw

c.  Gingiva of harbor seals (P. vitulina)

d.  Feces from harbor seals

e.  Feces from cetaceans (D. leucas, L. obliquidens)

f.  System water (standardized location)

g.  System substrate (standardized location)

3.  Equipment

a.  Enrichment media formulated by National Veterinary Services Laboratory (see VI)

b.  Sterile, cotton tipped swabs

c.  35°C incubator

d.  Blood agar and Columbia CNA agar plates

e.  TSI (Triple Sugar Iron) agar slants, OF glucose tubes, gelatin infusion medium, oxidase and catalase reagents

f.  API Coryne Strip for confirmation

4.  Procedure

a.  Inoculate media tubes (warmed to room temperature) with cotton tip sample swabs.

b.  Place labeled tubes in incubator for 48 hours

c.  Examine each tube for turbidity at the end of incubation and plate to blood and CNA agar plates. (Do not flame loop, as growth tends to be sparse).

d.  Incubate plates for 24 to 48 hours. Examine after 24 hours and note colony morphology, examine at 48 hours and note presence/absence of alpha hemolysis.

e.  Inoculate positive appearing growth on a TSI slant and an OF glucose and incubate for 48 to 72 hours. Inoculate a gelatin tube and incubate at room temperature for 48 to 72 hours. Check for hydrogen sulfide along stab line of TSI agar and "feather" growth along stab line of gelatin tube, as well as a weak acid reaction in OF.

f.  Confirm suspected positive colonies with an API Coryne strip.

g.  Inoculate positive isolates to another blood plate. Following culture, freeze entire growth in 2 ml skim milk in a cryovial. Store at -70°C.

5.  Differentiation of E. rhusiopathiae from other commonly encountered Gram positive, catalase negative bacteria (modified from, Wood, 1970).

6.  Selective Media for Growth of E. rhusiopathiae (Blair et al, 1970, NVSL, 2000)

Table 2

Presence (+) or absence (-) o f isolates of Erysipelothrix rhusiopathiae in culture o f feed fish at Shedd Aquarium during late 1999 and 2000. A blank indicates that no sampling occurred. In March 1997 enrichment media use was initiated. LH = large herring, SH = small herring, CAP = capelin, SQ = squid.

* Sample run in duplicate.
**Isolation followed 72 hours o f incubation.

References

1.  Blair JE, EH Lennette and JP Truant eds. 1970. Manual of Clinical Microbiology. American Society for Microbiology. P. 650.

2.  Wood, RL. 1970. In Manual of Clinical Microbiology. Blair JE, EH Lennette and JP Truant eds. American Society for Microbiology. Pp. 101,899.

3.  National Veterinary Services Laboratory, Ames, Iowa, USA. 2000. Personal communication.

Literature Cited

1.  Kinsel, M., J. Boehm, B. Harris, R. Mumane. 1997. Fatal Erysipelothrix rhusiopathiae Septicemia in a Captive Pacific White-sided Dolphin (Lagenorhynchus obliquidens). Journal of Zoo and Wildlife Medicine 28(4)" 494-497.