Introduction

A survey of the normal microbial flora of the southern sea otters (Enhydra lutris), their food, holding and display tanks, the intertidal pool, and two other tanks within the Monterey Bay Aquarium was undertaken in order to identify the bacterial and fungal species present, including any potential pathogens of human and veterinary importance. Water and otter food samples were collected in June, July, August, and November 1986, and February 1987, Otter rectal swabs were obtained in July and November 1986, and February 1987. This report presents preliminary data, as samples will be collected in May 1987. This will provide a complete year of data, allowing analysis of microbial flora on a seasonal basis. Many organisms have been identified to the genus and species level, but some isolates remain unidentified. The most complete data have been obtained for the otters, their food and their tanks, so this report will focus on these data.

Materials and Methods

Otters. Rectal swabs for isolation of aerobic organisms were obtained from each of the four display otters (three females and one male) using swab transport packs with Amie's medium (Difco, Detroit, MI). Only one sample was collected on otter #1 (in July) as he died in October 1986. Only one sample was collected on otter #4 (in February), after he joined the display otters. Swabs were plated onto anaerobic media for isolation of obligately anaerobic organismsas soon as possible following collection. Inoculated anaerobic media were immediately placed in a Gas-Pak (BBL, Cockeysville, MD) jar and maintained under anaerobic conditions for 48 hours. Upon arrival at the lab, swabs were plated unto various elective, differential, enrichment, and non-selective media (Table 1 ) for isolation of aerobic bacteria and fungi.

Otter Food Items. A representative sample of each food item given to the otters was collected in sterile bags. Food items included shrimp, squid, abalone, clams (fresh in-shell), crab, smelt, cod (3 varieties), smelt, mussels (fresh in-shell), sea urchin, and kelp. The samples were transported to the lab in a cooler. If the samples were not processed immediately, they were kept at 4° C. Samples were homogenized in a sterile sorvall Omni-Mixer in 1% peptone water and then plated onto the same media as the otter samples. Anaerobic media were inoculated from the interiors of the squid, shrimp, and whole fish samples collected in July, August, and November.

Water. Water samples were collected in sterile one gallon containers, except in the Kelp Forest Tank. In this tank, a diver filled a sterile 500 ml bottle from a deep area of the tank with minimal water circulation. Unfortunately, a diver was unavailable for collection of the February sample, so a grab sample was obtained from this tank. Samples also were obtained prior to and following ultraviolet irradiation fo the water used in the otter tank. Deep water samples were obtained from the otter tank approximately two hours after the otters were fed using a pole and small bottle (approx. 100 ml) for every sampIing, except in February. Surface water samples were obtained from the otter tank just prior to the first feeding of the day, with the exception of February's sample, which was collected half an hour after the first feeding. Samples were kept in a cooler during transport to the lab, and were kept cool until processed. Samples were filtered through a Millipore filtration apparatus using .45 μm filters. For each sample, approximately 200 m were processed per filter. Each filter was placed onto a different medium (same as for the otters and food). Anaerobic media were inoculated from deep water samples obtained from the Kelp Forest and otter display tanks.

Identification of isolates. Initial attempts to identify the isolates utilizing commercial rapid identification systems were unsuccessful. Therefore, standard biochemicals were used to identify bacterial isolates. Various references were consulted in order to identify these organisms, including Cowan (1974), MacFaddin (1980), Kreig Holt (1984), Lennette, et al.,(1985) and Farmer et al., (1985). Fungi were identified using standard biochemicals and morphologic observations. Working and stock bacterial cultures were maintained in 61 motility agar stabs. Working and stock fungal cultures were maintained on Sabouraud dextrose agar slants.

Table 1. Media Used For Isolation of Bacteria and Fungi

 Eosin Methylene blue (EMB)--differential medium for isolation of Gram-negative bacilli; slightly selective.

 MacConkey--differential and moderately selective medium for isolation and differentiation of Enterobacteriaceae and other Gram-negatives.

 Hektoen Enteric Agar--moderately selective and differential medium for Isolation of Gram-negative enteric bacilli.

 Xylose-Lysine-Deoxycholate (XLD)--selective and differential medium for Gram-negative enteric bacilli.

 Bismuth Sulfite Agar--highly selective and differential medium for detection and isolation of Salmonella typhi and other Salmonella.

 Yersinia Selective Agar (CIN)--selective and differential medium for isolation of Yersinia enterocolitica.

 Thiosulfate Citrate Bile Salts Sucrose (TCBS)--selective and differential medium for isolation of Vibrio sp.

 Hajna Gram Negative Broth--enrichment broth for selective isolation of pathogenic enteric bacilli (especially Salmonella and Shigella)

 Alkaline Peptone Water--enrichment broth for selective isolation of Vibrionaceae

 Rimier-Shotts Medium--selective and differential medium for presumptive identification of Aeromonas hydrophila

 V-9 and/or Mycosel--selective media for isolation of pathogenic and saprophytic fungi from samples with mixed bacterial and fungal flora.

 Phenylethanol Agar (PEA)--selective medium for isolation of Gram-positive cocci from mixed culture specimens; inhibits Proteus swarming.

 Sheep blood (5%) agar--non-selective medium for isolation of many fastidious organisms.

 Brucella (anaerobic)--non-selective medium for isolation of anaerobic and facultatively anaerobic organisms.

 Anaerobic PEA--selective medium for isolation of obligately anaerobic.

 Laked Kanamycin Vancomycin (LKV) (anaerobic)--selective and differential medium for isolation of Gram-negative anaerobes; used to detect fluorescence of Bacteroides sp.

 Bacteroides Bile Esculin (BBE) (anaerobic)--selective medium for and presumptive identification of Bacteroides fragilis group; aids in differentiation of Bacteroides sp.

Results

Otters. Isolates identified to date are shown in Table 2. Many of these organisms are atypical in their biochemical reactions. Therefore, these data represent preliminary identifications. Additional batteries of biochemicals and special tests will be required to definitively identify some of these isolates. These data focus on organisms of potential medical and/or veterinary importance. Autochthonous marine organisms were not identified.

Table 2

Otter Food Items. Isolates identified to date are shown In Table 3. Due to space considerations, data have been grouped together for each sampling period. As with the otter rectal samples, many of these organisms are atypical in their biochemical reactions. Therefore, these data represent preliminary identifications.

Table 3

Water samples. Isolates identified to date are shown in Table 4. As with the otter rectal and food item samples, many of these organisms are atypical biochemically. Thus, these data are preliminary.

Table 4

Discussion

Although these data are preliminary, they indicate that the microbial flora of the otters, their food, and tanks show seasonal variations. In general, there was a larger variety of organisms present during the summer months. One exception to this is the post-uv sample from 2/87, in which there were more organisms present than in the previous samples. This may be related to the operation of the ultraviolet irradiation equipment and/or the more varied flora in the sample. The data for the other tanks sampled in the aquarium also show a seasonal variation in their flora. Both otter tanks (display and holding) are not covered. Thus, migrating and resident birds may affect the flora of these tanks. It was interesting to note the variations as well. Some of the food given to otters is obtained locally, while portions are commercially available foods for human consumption.

Much more work remains to be done in definitively identifying each of the isolates; it is possible that some isolates will not be identifiable. The Vibrionaceae isolated will be thoroughly characterized. This is especially important due to the increasing incidence of human disease due to the non-cholera vibrio species. These isolates will be used to measure the immune response of otters to various vibrio species. This will indicate whether these animals are immunologically challenged by these organisms, which are known to cause important diseases of veterinary and medical importance. The unique diet and habitat of sea otters makes this investigation of otter flora and immune system significant in terms of indicating how the otters handle pathogens which are typically concentrated in their food items (most of which are filter feeders). This study will aid in optimizing veterinary and husbandry care of these valuable animals by providing baseline data defining the normal microbial flora of otters (wild otters are also under investigation).

References

1.  Cowan, S.T. 1974. Cowan and Steel's Manual for indentification of Medical Bacteria, 2nd edition. Cambridge University Press. New York, New York.

2.  Farmer, J.J., et al. 1985. Biochemical Identification of New Species and Biogroups of Enterobacteriaceae Isolated from Clinical Specimens. J. Clin. Micro., 21:46-76.

3.  Krieg, N.R. and J.G. Holt, (eds.). 1984. Bergey's Manual of Systematic Bacteriology. Williams and Wilkins, Inc., Baltimore, MD.

4.  Lennette, E.H., et al. 1985. Manual of Clinical microbiology. American Society for Microbiology, Washington D.C.

5.  MacFadden, J.F. 1980. Biochemical Tests for Identification of Medical Bacteria. Williams and Wilkins, Inc. Baltimore, MD.