Bacterial Chlamydial Culture Results in Steller Sea Lions From the Gulf of Alaska and Southeast Alaska
IAAAM 1994
D.J. Bradley; T.R. Spraker; D. Calkins; T. Loughlin
Wildlife Pathology International, Ft Collins, CO; Department Of Pathology, College of Veterinary Medicine, Colorado State University, Fort Collins, CO; Alaska Department of Fish and Game, Anchorage, AK; National Marine Mammal Laboratory, NOAA, Seattle, WA

Steller Sea lions (Eunetopias jubatus) have been listed as threatened under the U.S. Endangered Species Act by the National Marine Fisheries Service (NMFS) and are under consideration for elevation to endangered status. This action was taken by NMFS because sea lion numbers have declined precipitously in the area from the Gulf of Alaska northwest to the Kenai Peninsula and the central Aleutians Islands and Bering Sea. During 1992 and 1993, the role of infectious disease in the decline of Steller sea lion populations was investigated. The results of bacterial and chlamydial cultures from Steller sea lions from the Gulf of Alaska and southeast Alaska and their role as disease agents were examined.

Steller sea lions prefer remote areas far from human populations for rookeries and haul-outs. It is often difficult to send collected samples to a laboratory in a timely manner from these remote locations. Bacterial cultures that remain in transport media over 24 hours can permit overgrowth of some types of bacterial, while more fastidious types are lost. Bacterial cultures in transport media stored at low temperatures select for species of bacteria that grow well at lower temperatures, while other species die. It was necessary to create a laboratory that was both portable and durable. Often work was accomplished on a rocking boat or on uninhabited islands with varying sources and access to power. Identification of bacteria was done using standard morphologic, descriptive and chemical methods.

The Steller sea lions cultured included aborted fetuses, live pups and live, anesthetized adults. There were eight aborted fetuses necropsied and sampled from Kayak Island, located in the Gulf of Alaska. There were two pups from Lowry Island in southeast Alaska and eleven from Marmot Island in the Gulf of Alaska that were sampled. The pups were manually restrained. One anesthetized adult from Hazy Island and eight from Lowry Island (both in southeast Alaska were sampled. One adult and one yearling from Kayak Island were also anesthetized and sampled. A total of 32 animals were sampled.

Bacterial culture sites from aborted fetuses included lung, heart, stomach, amniotic fluid, liver, kidney and placenta. Bacterial culture sites from pups and adults included eyes, nares vagina and rectum.

Abortions are known to be caused by certain species of bacteria and chlamydia in other animals. Media was chosen to facilitate growth, isolation and identification of known pathogens. The media used included tryptic soy 5% sheep blood agar. This agar supports the growth of most organisms. Columbia nutrient 5% sheep blood agar was used to differentiate gram positive organisms. MacConkey agar was used to isolate most gram negative organisms. TCBS agar was used to differentiate Vibrio species. Selenite broth inhibits coliform bacteria aiding in the isolation of Salmonella species. Coliforms will commonly overgrow the more fastidious Salmonella, making this potentially important organism difficult to detect unless present in high numbers. Using the salmonella/shigella agar further inhibits growth of organisms other than salmonellas and shigellas. Gram stains were prepared from each swab, stained and examined.

Gram positive bacteria cultured included Staphylococcus aureus and epidermidis. Both of these species can be pathogens of other animals. Several non-pathogenic species of Staph were cultured. Streptococcus or Enterococcus fecalis was isolated. This is a normal inhabitant of the rectum of warm blooded animals and rarely cases problems. Streptococcus zooepidemicus was isolated from the mouth of two adults. This Strep in the group of pyogenic Strep sp.. It can cause abscesses and generalized bacteremias associated with bite wounds. Corynebacterium aquaticum is found in marine environments and is not considered pathogenic. Other Corynebacterium sp. recovered are inhabitants of the soil and are considered non-pathogenic They can be opportunistic and can cause abscesses when damaged tissue is present.

Micrococcus and Bacillus sp. are normally found in the environment and rarely cause problems. The Listeria species isolated was not monocytogenes or ivanovii, two species associated with bacteremia and abortion. This bacteria was present in low numbers in the nares of one adult and the rectum of another. The significance and pathogenic potential of this organism in Steller sea lions is unknown.

Gram negative organisms cultured and isolated included Pseudomonas aeruginosa and Ps. fluorescens. Ps. aeruginosa can cause abscesses and generalized bacteremia. Ps. fluorescens is commonly found in soil and rarely causes problems in mammals. This bacteria grows well at low temperatures and is often found in cultures that have been held in transport media for long periods before planting. Pseudomonas pseudomallei and Ps mallet are known pathogens of marine mammals. These species were not found. Moraxella sp. was isolated from the nares of three animals, the vagina of two animals and the pharynx of one animal. Possible diseases resulting from this organism are conjunctivitis and pneumonia in animals under stress or in combination with other infections. Enterobacter, Provenclencia, Kluyvera Edwardsiella and E. cold all belong to a family of bacteria called Enterobacteriaceae. This family is normally found in the lower intestinal tract of warm blooded animals, especially carnivores. As long as there are no concurrent problems that allow for overgrowth or invasion into the blood stream, these bacteria are considered normal.

Plesiomonas shigelloides and Proteus penner) are both commonly found environmental bacteria.. Proteus mirabilis is an opportunistic pathogen that can cause bacteremia if injury or concurrent problems are present. Hafnia alvei and Vibrio fulvallis are normally found in marine environments are not considered pathogenic but may be opportunistic. Pathogenic species of Vibrio were not found. Salmonella Saint-Paul was isolated from the rectum of one adult. Salmonella usually begins as an enteric infection and may generalize after entry into the bloodstream. Conditions seen with salrnonellosis include septicemia, meningitis, arthritis, pneumonia' abortion or any combination of these diseases. This particular serotype is known in other species to cause problems in young animals or animals stressed by other factors.

Examining the gram stains revealed a few bacteria that could be seen but were not grown. Campylobacters are comma shaped gram negative bacteria that often require near anaerobic conditions and enriched media to grow. They are known to cause enteritis and abortion in some species of domestic animals. Several types of spirochetes were also seen but did not grow. Culturing spirochetes is difficult, requiring special media and growth media, especially in the presence of enteric bacteria. These spirochetes are probably Treponema or Serpulina sp.. These bacteria are found in the intestinal tracts of other animals. Some species of Treponema are pathogens of domestic animals, but most are non-pathogenic. Another spirochete, Leptospira interrogans has been implicated in causing abortion in California sea lions. Based on serology done in 1975-78 and 1985-86 by Calkins and Goodwin, leptospirosis is probably not a significant problem in Steller sea lions. Leptospiral cultures were done on the aborted fetuses, all were negative. Bacteria isolated from animals from the Gulf of Alaska and the animals from southeast Alaska did not differ. All cultures from the clinically normal animals contained normal flora for mammals and environmental bacteria. No known abortifacient bacteria were isolated from the fetuses. The significance of bacterial pathogens in the population decline of the Steller sea lions was not demonstrated.

Chlamydia are small bacteria that live as obligate parasites of cells. They must be grown in cell culture. Conditions caused by Chlamydia in other animal species include conjunctivitis, polyarthritis, enteritis, placentitis, and abortion. Chlamydia culture sites were lung in aborted fetuses, eye, vagina and rectum. Transport media was frozen and cell culture done. The use of ELISA testing for Chlamydia to facilitate screening of large numbers of animals is being evaluated. Chlamydia psittaci was isolated from the lung of an aborted fetus. All other cultures were negative. Serologic evidence of Chlarrrydia was found by Calkins and Goodwin (1988) in adult populations in the Gulf of Alaska. Premature pupping continues to occur at a high rate at Cape St. Elias. The relationship of chlamydiosis and reproductive failure to the population decline remains obscure.

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D. J. Bradley

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