Microbial Communities in the Nasal Passages of Healthy by wii13090

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									                       Microbial Communities in the Nasal Passages of Healthy and URTD Tortoises
                                                                                          Ashley M. Ordorica, F. Harvey Pough, and Charles E. Deutch
                                                               Department of Integrated Natural Sciences, Arizona State University West, Phoenix AZ
                                                                    PO Box 37100 Phoenix, AZ 85069-7100 Phone: (602)543-6939 FAX: (602)543-6073

                                                                                                                                                                                                                                     SUMMARY                                                                     CONCLUSIONS
                       ABSTRACT                                                                                                                                                                                                        TABLE
      Natural communities of microorganisms are believed to
                                                                                                                                                                                                                                                                                              • The total number of bacteria/ml in the nasal passages of desert
provide an important barrier against potential pathogens. We                                                                                                                                                Strain     Colony   Original       %    Shape     Gram       BBL Crystal™
                                                                                                                                                                                                                        Color   Number                        Stain      Identification
                                                                                                                                                                                                                                                                                                tortoises changes seasonally, increasing from the spring to the
characterized the microbial communities in the nasal passages of                                                                                                                                                                                                                                summer and decreasing in the fall.
desert tortoises (Gopherus agassizii) quantitatively to determine if                                                                                                                                        Ga 1     Rio        294.5.1    89      coccus      +      unknown
there are individual or seasonal variations, or differences between                                                                                                                                                  Fleece     559.6.1    26.9                                               • The microbial communities of both healthy and URTD tortoises
healthy tortoises and those with symptoms of upper respiratory                                                                                                                                                                  607.6.1    51.5                                                 are dominated by pigmented Gram-positive cocci.
tract disease (URTD). Twelve captive tortoises at the Adobe                                                                                                                                                                     294.6.1    100
                                                                                                                                                                                                                                                                                              • Communities in URTD tortoises are more variable and complex
                                                                                                                                                                                                                                360.6.1    73.7
Mountain Wildlife Rehabilitation Center in Phoenix, AZ were                                                                                                                                                                                                                                     than those in healthy tortoises.
                                                                                                                                                                                                                                607.7.1    96.4
divided into four groups: three healthy tortoises were sampled
                                                                                                                                                                                                                                                                                              • Thermal tolerances of bacteria showed optimal growth at about
monthly, three URTD tortoises were sampled monthly, three
                                                                                                                                                                                                                                                                                                35°C.
healthy tortoises were sampled bimonthly, and three healthy                                                                                                                                                 Ga 2     Lemon      624.5.4    50      coccus      +      unknown
tortoises were sampled once. At each sampling time, the tortoises                                                                                                                                                                                                                             • Seven out of the twelve most frequent isolates have been
                                                                                                                                                                                                                     Head       559.6.4    1
were examined for general health, and both nares were probed                                                                                                                                                                    559.7.3    20.9                                                 identified as Staphylococcus xylosus, Micrococcus species,
with moistened sterile swabs. The bacteria on the swabs were                                                                                                                                                                    294.7.2    2.8                                                  Kytococcus sedentarius, Corynebacterium
suspended in sterile saline, serially diluted, and plated on tryptic                                                                                                                                                            624.7.2    41.4                                                 pseudodiphtheriticum, Staphylococcus vitulinus, and
soy agar medium. Total bacterial counts/ml varied from tortoise to                                                                                                                                                                                                                              Staphylococcus sciuri.
tortoise, increasing from May to August and decreasing from                                                                                                                                                                                                                                   • Some of the frequent isolates tested showed resistance to
                                                                                                                                                                                                            Ga 3     Sun Rise   270.6.3    18.9    coccus      +      Staphylococcus
September to November. Total counts were usually higher and                                         RESULTS                                                                                                                     270.7.5    <1                         xylosus
                                                                                                                                                                                                                                                                                                antibiotics including Bacitracin, Rifampin, Nalidixic Acid,
more variable in URTD tortoises. The microbial communities                                                                                                                                                                                                                                      Norfloxacin, Vancomycin, Penicillin, and Triple Sulfa.
were dominated by pigmented Gram-positive cocci, but Gram-
positive rods, coryneforms, and Gram-negative rods also were
found. The proportions of different bacteria varied from month to                          G roup 1 -Healthy Tortoises                                                                                      Ga 4     Falling    360.6.2    24.1    coccus      +      Micrococcus
month and were more variable in diseased animals. These studies            Percent
                                                                                                                                                                                    TotalCount                       Star       360.7.1    10.4                       species                                 FUTURE STUDIES
suggest that a broader ecological and microbiological analysis of
these communities would be valuable.                                           100%                                                                                                                00E+08
                                                                                                                                                                                                  1.
                                                                                                                                                                                                            Ga 5     Vanilla    294.6.2    <1      coccus      +      Kytococcus              • Determine salt tolerances of bacterial isolates
                                                                                80%                                                                                                                00E+07
                                                                                                                                                                                                  1.                 Cream      352.7.6    8.6                        sedentarius
                                                                                                                                                                                                                                                                                              • Determine the nutrients found in nasal passages
                   INTRODUCTION                                                 60%                                                                                                                                                                                                           • Confirm identity of bacterial isolates using BBL Crystal
                                                                                                                                                                                                   00E+06
                                                                                                                                                                                                  1.
       One factor that limits the susceptibility of any organism to an          40%                                                                                                                                                                                                             System and FT-IR spectroscopy of pigments
infectious agent is the presence of a community of nonpathogenic                                                                                                                                   00E+05
                                                                                                                                                                                                  1.
microorganisms at the site of infection. Indigenous bacteria in the             20%
respiratory system, the gastrointestinal system, and the urogenital              0%                                                                                                               1.
                                                                                                                                                                                                   00E+04   Ga 6     Lemon      352.5.1    26      coccus      +      unknown
system are well adapted to these sites. They protect the host from
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other potentially pathogenic organisms by occupying physical sites                                                                                                                                                              559.10.2   51.5
on the host tissue, by effectively consuming the available nutrients,                                                                                                                                                           607.10.2   16.7                                                         ACKNOWLEDGEMENTS
and by producing proteins and other metabolic products that are
inhibitory to other microorganisms. Little is known about the                                                                                                                                               Ga 7     Squash     026.11.2   30      Coryn-      +/-    Corynebacterium
bacteria in the upper respiratory tract of desert tortoises (Gopherus                                                                                                                                                           360.7.3    16.2    eform              pseudodiphtheriticum      We would like to thank Daniel Martinez, Jenny Gomes, and
                                                                                          G roup 2 -URTD Tortoises                                                                                                                                                                              Albert DiOrio for their work on some of the experiments and
agassizii). These reptiles are of interest because the Mojave Desert                                                                                                                                                            624.9.2    <1
population in California, Nevada, and Utah is classified as                                                                                                                                                                                                                                     Sandy Cate of the Arizona Game & Fish Department for
Threatened by the U.S. Fish and Wildlife Service, and the Sonoran              Percent                                                                                               TotalCount                                                                                                 providing the captive desert tortoises. Support for this research
Desert population in Arizona is protected by state regulations                                                                                                                                                                                                                                  was provided in part by a grant from the National Institute of
                                                                               100%                                                                                                                00E+08
                                                                                                                                                                                                  1.                                                                                            Health through the Minority Access to Research Careers, ASU
(Howland and Rorabaugh, 2002). One factor that has impacted
                                                                                                                                                                                                            Ga 8     Cream      607.9.1    100     Coccus      +      Staphylococcus            School of Life Sciences Enrichment Program.
these organisms is the occurrence of Upper Respiratory Tract                   80%                                                                                                                                   Cake       294.9.8    <1                         vitulinus
Disease (URTD, Berry, 1997), which is now known to be caused by                                                                                                                                                                 352.9.1    43
Mycoplasma agassizii (Brown et al., 1994).      Dickinson et al. (1996,        60%                                                                                                                                              811.9.1    70.1
2001) reported the recovery of bacteria from the generic groups                                                                                                                                    00E+07
                                                                                                                                                                                                  1.                            026.9.1    47.4
Corynebacterium, Flavobacterium, Pseudomonas, Staphylococcus,                  40%                                                                                                                                                                                                                               REFERENCES
and Streptococcus from the nares of individual animals. We have
now quantitatively characterized the microbial communities in the                                                                                                                                           Ga 9     Cream      294.8.1    89.3    coccus      +      unknown                Berry, K. 1997. Demographic consequences of disease in two desert
                                                                               20%
nasal passages in healthy desert tortoises and those with symptoms                                                                                                                                                   Cake       559.10.1   50.4                                                 tortoise populations in California, U. S. A. Pages 91-99 in Van
of URTD through a complete activity season. The long term goals                                                                                                                                                                 352.10.1   10                                                   Abbema, J. (ed.) Proceedings: Conservation, Restoration, and
                                                                                0%                                                                                                                 00E+06
                                                                                                                                                                                                  1.                            352.9.1    43                                                   Management of Tortoises and Turtles — An International
                                                                                                                                                                                     N ovem ber
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of this project are to define the indigenous respiratory community
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                                                                                                                                                                                                                                325.10.1   56.4                                                 Conference. New York Turtle and Tortoise Society and the WCS
and to assess its role in preventing URTD.                                                                                                                                                                                      872.10.1   70.3                                                 Turtle Recovery Program, New York, NY.


                                                                                                                                                                                                            Ga 10 Wax           270.11.3   10      coccus      +      Staphylococcus         Brown, M. B., I. M. Schumacher, P. A. Klein, K. Harris, T. Correll,
        SAMPLING                               MICRO-                                                                                                                                                             Bean          607.11.1   100                        sciuri                    and E. R. Jacobson. 1994. Mycoplasma agassizii causes upper
                                                                                                                                                                                                                                270.8.1    51
        PROTOCOL                             BIOLOGICAL                                         G roup 3 - Heal Tortoi
                                                                                                              thy    ses
                                                                                                                                                                                                                                                                                                respiratory tract disease in the desert tortoise. Infection and
                                                                                                                                                                                                                                                                                                Immunity 62:4580-4586.
                                              ANALYSIS                                Percent                                                                              TotalCount                       Ga 11 Rio           352.8.2    <1      coccus      +      Staphylococcus
                                                                                                                                                                                                                  Fleece        559.8.2    17.5                       sciuri
                                                                                       100%                                                                                                                                     352.11.1   59                                                Brown, M. B., K. H. Berry, I. M. Schumacher, K. A. Nagy, M. M.
                                                                                                                                                                                              00E+08
                                                                                                                                                                                             1.
 •Weigh animals, check for health       •Color and texture of colonies                                                                                                                                                          559.11.1   95                                                   Christopher, and P. A. Klein. 1999a. Seroepidemiology of upper
 status and nasal discharge                                                             80%                                                                                                                                                                                                     respiratory tract disease in the desert tortoise in the western
                                                                                                                                                                                                            Ga 12 Lemon         559.10.2   21.2    coccus      +      unknown                   Mojave Desert of California. J. Wildlife Diseases 35:716-727.
                                                                                        60%                                                                                                                       Head          352.11.2   42
 •Swab both nares with sterile          •Bacterial shape, size, and motility                                                                                                                  00E+06
                                                                                                                                                                                             1.                                 607.10.2   16.7
 transurethral swabs                    by phase contrast microscopy                    40%                                                                                                                                     294.10.1   36.4                                              Dickinson, V. M, J. L. Jarchow, and M. H. Trueblood. 1996. Health
                                                                                                                                                                                                                                294.10.7   <1                                                   studies on free-ranging Sonoran desert tortoises in Arizona.
                                                                                        20%                                                                                                                                     325.10.3   3.6                                                  Arizona Game and Fish Department, Research Branch,
                                                                                                                                                                                                                                                                                                Technical Report #24, Arizona Game and Fish Department,
 •Suspend bacteria in 1.0 ml sterile    •Gram reaction, catalase and                     0%                                                                                                   00E+04
                                                                                                                                                                                             1.                                                                                                 Phoenix, AZ.
                                                                                                                                                                                                            Ga 13 Falling       360.7.1    10.4    bacillus     -     Stenotrophomonas
                                                                                                                                                                   N o ve m b e r
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 0.85% NaCl                             oxidase activities                                                                                                                                                        Star                                                maltophila

                                                                                                                                                                                                                                                                                             Dickinson, V. M., T. Duck, C. R. Schwalbe, J. L. Jarchow, and M. H.
 •Prepare 6 serial 1/10 dilutions     •Aerobic and anaerobic growth                                                                                                                                         Ga 14 Torchlight 360.8.5       1       bacillus     -     Bacillus sphaericus
                                                                                                                                                                                                                                                                                                Trueblood. 2001. Nasal and cloacal bacteria in free-ranging
 and spread 0.1ml of each dilution                                                                                                                                                                                                                                                              desert tortoises from the Western United States. J. Wildlife
 on duplicate tryptic soy agar plates                                                                                                                                                                                                                                                           Diseases 37:252-257.
                                                                                                                  orm
                                                                                                            Corynef s
                                                                                                                                                                                                            Ga 15 Angel         360.9.5    1       bacillus    +      unknown
                                                                                                            G ram negatve
                                                                                                                       i                                                                                          Food          270.10.2   1                                                 Homer, B. L., K. H. Berry, M. B. Brown, G. Ellis, and E. R.
 •Count plates after 3 days at room     •Growth on Mannitol Salt Agar                                                                                                                                                                                                                          Jacobson. 1998. Pathology of diseases in wild desert tortoises
 temperature and determine total                                                                                      i      lus
                                                                                                            G ram postve bacil                                                                                                                                                                 from California. J. Wildlife Diseases 34:508-523.
 bacteria/ml                                                                                                                                                                                                Ga 16 Teresa        270.8.4    30      Bacillus     -     Misc. Gram Negative
                                                                                                                      i      -nonpi ent
                                                                                                            G ram postve cocci    gm ed
                                                                                                                                                                                                                                                                      Bacilli
                                                                                                                                                                                                                                                                                             Howland, J. M. and J. C. Rorabaugh. 2002. Conservation and
                                                                                                                      i      -pi    ed
                                                                                                            G ram postve cocci gm ent                                                                                                                                                          protection of the desert tortoise. Pages 334-354 in Van Devender,
                                                                                                                                                                                                                                                                      Pseudomonas
 •Assess community structure by         •Phenotypes in API Staph-                                                                                                                                                                                                                              T. R. (ed.). The Sonoran Desert Tortoise: Natural History,
                                                                                                            Tot Count
                                                                                                              al    s                                                                                                                                                 flurescens
 calculating bacteria/ml for each       identification strip tests                                                                                                                                                                                                                             Biology, and Conservation. The University of Arizona Press,
 major colony type                                                                                                                                                                                                                                                                             Tucson, AZ.

								
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