Bacterial Chlamydial Culture Results in Steller Sea Lions From the Gulf of Alaska and Southeast Alaska
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,
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
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
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
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
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.