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 N ovem ber Head 352.6.3 9.8 S e p te m b e r O c to b e r A ugust M ay June J u ly 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 S e p te m b e r O c to b e r of this project are to define the indigenous respiratory community A ugust M ay June J u ly 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 S e p te m b e r M ay Ju ly 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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