Abstract

Plesiomonas shigelloides has been implicated as an etiological agent of human gastroenteritis for almost 40 years and has been isolated from the intestinal contents of dogs, cats, poultry and freshwater fish; river water and sludge (Arai et al, 1979). In May, 1985, P. shigelloides was diagnosed as the causative agent of gastroenteritis in the harbor seals at the California Marine Mammal Center (CMMC). P. shigelloides was found to not be part of the normal intestinal flora of these and other pinnipeds at CMMC in a limited study of enteric bacteria conducted from February through June 1985.

Isolation of P. shigelloides was performed by plating rectal swab samples onto MacConkey, Salmonella-Shigella and Xylose Lysine Desoxycholate agars. Suspected pathogens were transferred to Lysine Iron agar, Triple Sugar Iron agar and Motility Indole Ornithine medium, and final identification was made using conventional biochemicals (not API system), Antibiotic sensitivity tests (Kirby-Bauer) were conducted to ensure appropriate antibiotic therapy.

This presentation summarizes the findings of the microbiological testing, type of medical treatment employed, results of the intestinal flora study, and husbandry techniques undertaken to safeguard both pinnipeds and handlers from pathogenic organisms.

Introduction

Plesiomonas shigelloides is an oxidase-positive, gram-negative, nonsporeforming bacilli of the family Vibrionaceae. The role of P. shigelloides as a causative agent in food and waterborne gastroenteritis outbreaks in humans has been well documented (7). The organism has also been implicated as an etiological agent of opportunistic infections in immuno-compromised hosts and the very young (6). Although P. shigelloides has been isolated from cattle, swine, monkeys, fresh-water fish, poultry, dogs and cats, documentation as to its pathogenicity in these animals is fragmented (1).

In May of 1985 an outbreak of diarrheal disease in three Pacific harbor seals (Phoca vitulina richardsi) at CMMC was suspected to be caused by P. shigelloides. This report describes our bacteriological findings, as well as the medical treatments employed and measures taken to prevent future bacterial enteropathogen caused diseases among captive pinnipeds.

Materials and Methods

Case Histories and Clinical Features

On May 29, 1985 three Pacific harbor seal pups exhibited symptoms of a possible gastroenteritis. Symptoms included vomiting, non-bloody liquid stools, anorexia, lethargy and 'doughy' abdomen.

One of the animals (a female) was a full term birth admitted to CMMC at approximately two days of age. She had been receiving care at CMMC for 29 days as of May 29, 1985. The other two animals (both males) had been born prematurely (as indicated by the retainment of their lanugo coats) and were admitted to the facility at one day of age. When the first signs of a gastroanteritis appeared, they had been in captivity for 57 and 59 days.

Bacteriological Procedures

Rectal swabs were obtained from the three symptomatic harbor seals within 24 hours of the onset of vomiting and diarrhea, before any therapy was initiated. The swabs were inoculated directly onto Salmonella-Shigella (SS) agar, Xylose Lysine Desoxycholate (XLD) agar, MacConkey (MA) agar and alkaline peptone broth. Subcultures were made from the alkaline peptone broth to Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar following overnight incubation. All cultures were incubated aerobically at 37 degrees Celsius. Cultures were examined following overnight incubation as well as after 48 hours. Suspicious looking colonies were screened using Triple Sugar Iron (TSI) agar, Lysine Iron agar (LIA) and Motility Indole Ornithine (MIO) medium. Production of indole was observed on the MIO medium and the test for oxidase was performed on picks from LIA on filter paper with tetramethyl-p-phenylenediamine (4). Final identification of suspected pathogens was performed using standard biochemical tests (4), not API system.

Fecal Parasites

Fecal flotation tests were performed on stool samples of the three symptomatic harbor seal pups to identify any endoparasites (Ovatector, BGS Medical Products, Venice, FL).

Water Specimens

Two 600-700 ml samples of tap water and three 500 ml samples of pool water (from three different pools and originating from tap water) were each filtered through 0.45 mcg Millipore membranes. The filters were then placed in Gram Negative (GN) enrichment broth and incubated aerobically at 35 degrees Celsius overnight. Subcultures were made onto MA, XLD and Hektoen agars and incubated overnight at 35 degrees Celsius. Suspicious colonies were then screened and identified using procedures as described for the rectal swab samples.

Food Specimens

Samples of the animal feed, flash frozen Atlantic herring, were tested for the presence of pathogens, Twenty-five grams of emulsified fish samples were added to 225 ml of GN broth to create a 1:10 suspension. This mixture was incubated at 35 degrees Celsius and subcultured onto MA, XLD and Hektoen after 16 and 24 hours. These cultures were incubated aerobically at 35 degrees Celsius overnight, and suspicious colonies were then screened and identified by the methods described for rectal swab samples.

Extended Bacteriological Procedures

Following the identification of P. shigelloides as the suspected causative agent of the gastroenteritis in the three harbor seals, bacteriological investigation was extended to other pinnipeds in captivity at CMMC. Rectal swabs were collected from four adult California sea lions (CSL), Zalophus californianus one CSL pup, four Northern elephant seal (NES) yearlings, Mirounga angustirostris and four harbor seal pups. All animals were asymptomatic for gastroenteritis at the time of sampling and throughout their captivity at CMMC.

Antibiotic Sensitivity Tests

Sensitivity to different antibiotics was assessed via the KirbyBauer method (2). The following antibiotics were used in the form of discs obtained from BBL: tetracycline (30 mcg), streptomycin (10 mcg), ampicillin (10 mcg), carbenicillin (100 mcg), cephalothin (30 mcg), chloramphenicol (30 mcg), gentamicin (10 mcg), kanamycin (30 mcg), tobramycin (10 mcg), sulfisoxazole (300 mcg), sulfamethoxasole-trimethoprim (25 mcg), polymyxin-B (300 units).

Results

Of the three rectal swabs collected from the symptomatic harbor seals, one yielded almost pure P. shigelloides culture on XLD and SS (one Proteus mirabilis colony was observed on SS), The other two swabs yielded luxuriant growth of P. shigelloides an XLD , SS and MA in association with E.coli and Proteus mirabilis. P. shigelloides grew best on XLD in small, colorless, translucent, flat colonies and would not grow on TCBS. The biochemical characteristics of the P. shigelloides isolates are presented in Table 1.

Cultures from the tap water specimens showed no growth of any organisms. The cultures from pool water specimens revealed abundant growth of P. shigelloides and lesser growth of E. coli, Proteus mirabilis and Pseudomonas aeriginosa.

The cultures of the food specimens did not reveal P. shigelloides or any other potential pathogens.

The antimicrobial sensitivity pattern of P. shigelloides revealed susceptibility to chloramphenicol, sulfamethoxazole-trimethoprim, gentamicin, tobramycin, kanamycin, cephalothin and polymyxin B, while showing an intermediate resistance to ampicillin. The organism was resistant to carbenicillin, sulfisoxazole, streptomycin and tetracycline.

The results of the fecal flotation tests were negative.

Rectal swab cultures of the asymptomatic pinnipeds at CMMC yielded P. shigelloides isolates from all four NES's and all four harbor seals. The four adult CSL's and one CSL pup did not reveal P. shigelloides upon investigation.

Table 2 offers a partial presentation of the results of a limited enteric flora study performed from February 1985 through June 1985. Rectal swabs were collected from animals upon admission and throughout their stay at CMMC. The study was conducted to learn about pinniped intestinal flora upon admission at CMMC and while in captivity. Rectal swab collection and culture procedures were the same as those described previously. Identification of bacteria was carried out by API methods.

Clinical Treatment

Following the identification of P. shigelloides (day two of the onset of symptoms), the harbor seals were treated with intravenous chloramphenicol (20 mg/lb.) every eight hours for two days. These animals received nothing orally for this 48 hour period and were administered 100 ml Lactated Ringers with 5% Dextrose (D-5/LR) subcutaneously at eight hour intervals. They were closely monitored for signs of dehydration (i.e. PCV elevation, total protein elevation, lack of tearing, and curling of vibrissae). On the third day of treatment D5/LR was administered via gavage with oral chloramphenicol (20 mg/lb.) every eight hours. Vomiting was not observed for any of the animals and diarrheal stools decreased greatly. Over the next 48 hours emulsified herring was introduced into the animals' gavage treatment and amounts of D-5/LR were decreased. By the fourth day of treatment all three animals exhibited alert, aggressive behavior, good appetite, with no signs of vomiting and only occasional diarrhea. Five days following the first signs of gastroenteritis, all three animals were returned to their normal feed rations (3/4 lbs. of herring pieces three times daily) with the addition of 130 ml water given via gavage once daily. Oral chloramphenicol was discontinued following the seventh day of treatment. At this point the animals appeared clinically healthy, with firm stools, no vomiting, good appetites and normal hydration and activity levels.

Following the isolation of P. shigelloides from other pinnipeds at CMMC, all animals were given chloramphenicol orally (10 mg/lb., every 8 hours) for a period of one week. Although these animals were asymptomatic, the risk of infecting now arrivals to the center, especially pups and greatly debilitated animals, necessitated facility-wide treatment.

All animals, including the three symptomatic harbor seals, were retested for P. shigelloides two weeks after the cessation of antibiotic treatment. All animals were found to be shedding P. shigelloides in large numbers in their feces. Antimicrobial sensitivity was rerun and the identical antimicrobial susceptibility pattern was found. All CMMC animals were treated with oral sulfamethoxazole-trimethoprim (10 mg/lb every 12 hours) for ten days. Animals were again rechecked for the presence of P. shigelloides in their feces and only one animal, a consistently asymptomatic harbor seal, was still shedding the organism.

Hygienic conditions were maintained throughout CMMC with chlorhexidine diacetate (Nolvasan, Aveco, Fort Dodge, Iowa) footbaths at pen gates and hospital building entrances; the wearing of protective foot and hand gear; daily cleaning of pen floors and pools with chlorhexidine solution; adequate drainage facilities. Additionally, caretakers of harbor seal pups were required to clean gloves with chlorhexidine after handling each animal and were not permitted to work with adult harbor seals or other pinniped species on site.

Discussion

This is the first reported case of diarrheal disease in Pacific harbor seals suspected to be caused by P. shigelloides. Bacteriological testing of the symptomatic seals did not reveal any enteropathogens and P. shigelloides was the only suspect etiological agent isolated. A limited enteric flora study of pinnipeds at CMMC did not detect this organism prior to the outbreak of the gastroenteritis. The three harbor seals which exhibited the symptoms of gastroenteritis did not have the organism among their intestinal flora when admitted to CMMC nor did any other pinniped cultured upon admission. It is doubtful that P. shigelloides is part of the normal pinniped intestinal flora due to its intolerance of saline conditions (9,10). Though ubiquitous in the environment, it is a freshwater inhabitant and has been reported to die in 22 to 25 hours in sea water (9,10). The use of freshwater pools and close interactions with humans at CMMC offer a more plausible explanation for pinniped exposure to P. shigelloides. Such conditions are not uncommon among other rehabilitation facilities, aquaria and zoos, and hygienic conditions should be maintained as discussed previously to reduce the risk of infection. Due to the suspected vulnerability of infection by P. shigelloides to immunocompromised hosts and the very young (6), special treatment of water may be warranted. Chlorination has been shown to kill the organism (if done satisfactorily - 8) as does the addition of salt. Though not tested here, certain types of filtration (i.e. ozone) could potentially remove P. shigelloides from a water supply.

Accurate identification of P. shigelloides should include the biochemical tests presented in Table 1. The organism's resemblance to members of the family Enterobacteriaceae and others within its own family (Vibrionaceae) has accounted for numerous recognition failures in human cases of diarrheal outbreaks, The importance of introducing the oxidase test at the preliminary stage of identification, and also the introduction of a few other biochemical tests (asterisked tests in Table 1) for the final characterization of this organism is emphasized.

Though usually a self-limiting diarrheal disease in most adult humans, more severe infections have been reported in very young patients (1,7). Due to the young age of the symptomatic harbor seals and their developing immune systems, antibiotic therapy was implemented. Asymptomatic animals that tested positive for the organism were included in the antibiotic therapy to reduce the risk of infection to medically compromised individuals and incoming animals.

The isolation of a potential human pathogen in captive pinnipeds demonstrates the need for a more complete understanding of pinniped flora and effective hygienic techniques in captive situations. Our ability to identify P. shigelloides and treat the associated gastroenteritis at CMMC proves that such bacteriological investigations can promote more effective medical treatment of pinnipeds, create a safer working environment for handlers and advance our knowledge of pinniped bacteriology.

Table 1. Biochemical Characteristics of Plesimonas Shigelloides Isolated

* = tests required to accurately identify P. shigelloides
+ = positive - = negative GR = growth A = acid

Table 2. Aerobic Bacterial Isolates from Pinnipeds at CMMC (February 1965 - June 1985)

Acknowledgement

The authors wish to thank Dr. Neylan Vedros, School of Public Health, University of California, Berkeley; and Sharon Abbott, California certified microbiologist for their technical assistance and advice in this report.

References

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