Detection of Mycoplasma agassizii in the Texas Tortoise (Gopherus berlandieri )

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1 DOI: / Journal of Wildlife Diseases, 49(3), 2013, pp # Wildlife Disease Association 2013 Detection of Mycoplasma agassizii in the Texas Tortoise (Gopherus berlandieri ) Amanda L. Guthrie, 1,5 C. LeAnn White, 2 Mary B. Brown, 3 and Thomas W. demaar 4 1 Virginia Zoo, 3500 Granby St. Norfolk, Virginia 23504, USA; 2 USGS National Wildlife Health Center, 6006 Schroeder Road, Madison, Wisconsin 53711, USA; 3 University of Florida, Department of Infectious Diseases and Pathology, PO Box , 2015 SW 16th Ave., Gainesville, Florida 32608, USA; 4 Gladys Porter Zoo, 500 Ringgold St. Brownsville, Texas 78520, USA; 5 Corresponding author ( aguthrie0665@gmail.com) ABSTRACT: Mycoplasma agassizii causes upper respiratory tract disease (URTD) in Texas tortoises (Gopherus berlandieri). To determine exposure to and shedding of M. agassizii, we collected blood samples and nasal swabs from 40 free-ranging Texas tortoises on public and private lands in Texas, USA, from May to October We used an enzyme-linked immunosorbent assay (ELISA) to detect M. agassizii specific antibodies. Eleven (28%) tortoises were antibody positive, three (8%) were suspect, and the remaining 26 (65%) were negative. Nasal lavage samples were collected from 35 of the 40 tortoises for M. agassizii culture and PCR to detect shedding of M. agassizii. Current infection with M. agassizii was confirmed in one tortoise that had mild clinical signs of URTD and was positive by ELISA (antibody titer.512), PCR, and culture. The clinical isolate was confirmed as M. agassizii by restriction fragment length polymorphism and immunobinding. Key words: Antibodies, chelonian, clinical signs, exposure, reptile, shedding, upper respiratory tract disease. The Texas tortoise (Gopherus berlandieri) is one of five species of true tortoises native to North America (Murphy et al., 2011). All five species are members of the genus Gopherus, are closely related (Boyer and Boyer, 2006; Murphy et al., 2011), and receive some form of state or federally mandated protection because of declining population sizes (Judd and Rose, 2000). Upper respiratory tract disease (URTD) caused by Mycoplasma agassizii (Brown et al., 1994, 1999) is characterized by nasal discharge, ocular discharge, conjunctivitis, and palpebral edema (Brown et al., 1994, 1999) and has been observed in a variety of tortoise species (Wendland et al., 2006). Upper respiratory tract disease is suspected to cause morbidity and mortality in tortoises by hindering the ability to forage and causing altered behavior such as a diminished response to stimuli, possibly making a tortoise more susceptible to predation (McLaughlin et al., 2000). Diagnostic assays for URTD in tortoises include culture and direct isolation of M. agassizii, detection of mycoplasmal DNA by PCR (Brown et al., 2002), and detection of antimycoplasma antibodies by enzymelinked immunosorbent assay (ELISA; Brown et al., 2002; Wendland et al., 2007). Although direct isolation of the organism through culture or detection of mycoplasmal DNA from nasal secretions with PCR provides the most complete assessment of infection status, M. agassizii is fastidious and slow growing and the sensitivity of PCR can vary with the clinical disease state of the tortoise (Brown et al., 2002). The M. agassizii ELISA has high sensitivity (0.98) and specificity (0.99) (Wendland et al., 2007) and, although it has been validated only on desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises (Schumacher et al., 1993), the secondary antibodies used in this assay cross-react well with a variety of other chelonian species (Wendland, 2007; Wendland et al., 2007). Antibodies to M. agassizii were previously detected in 80% (12/15) of captive Texas tortoises that had been housed temporarily in a rehabilitation facility (Tristan, 2009). All free-ranging Texas tortoises (n539) in the study were antibody negative (Tristan, 2009). We examined free-ranging Texas tortoises from three southern Texas counties to determine exposure to and current infection with M. agassizii. 704

2 SHORT COMMUNICATIONS 705 FIGURE 1. Map of south Texas showing locations of sampled Texas tortoises (Gopherus berlandieri). Inset boxes show concentrated area of tortoises in the Texas Parks and Wildlife Department, Los Palomas Wildlife Management Area, Longoria Unit, Santa Rosa, Texas (Cameron County) and southwestern Hidalgo County. Key: symbols represent positive, negative, and suspect animals by enzyme-linked immunosorbent assay (ELISA) for Mycoplasma agassizii. Blood was collected from 40 freeranging Texas tortoises in Cameron, Hidalgo, and Willacy counties (27u229 25u519N, 97u189 98u99W) from May to October 2009 (Texas Parks and Wildlife Department permit ZOO ). Tortoises were captured in the field or brought to the Gladys Porter Zoo by local residents. Each tortoise was given a thorough physical exam, weighed, and measured, and sex was determined using previously described techniques (Berry and Christopher, 2001). The capture location of each tortoise was recorded with a handheld global positioning system unit and coordinates were mapped (Arc- Map ArcGIS version 9.3, Esri, Redlands, California, USA; Fig. 1). Approximately ml of blood was collected from the dorsal venous sinus of each tortoise and placed in tubes containing lithium heparin. Samples were centrifuged and plasma was frozen in cryovials at 220 C in a manual defrost freezer for to up to 12 mo until shipment to the University of Florida for ELISA, culture, and PCR. The following previously established cutoff values for gopher and desert tortoises were used for the ELISA; titers of,32, 32 and $64 were considered negative, suspect, and positive respectively (Wendland et al., 2007). Thirty-five tortoises had a nasal flush performed aseptically with approximately ml of sterile saline in each naris. The samples were collected in a sterile container and 0.50 ml of an enrichment medium (SP4 Glucose Broth, Remel, Lenexa, Kansas, USA) was added before the samples were frozen at 220 C. Frozen samples were shipped to the University of Florida for Mycoplasma culture and PCR testing. The PCR and culture results were classified as positive or negative for the presence of mycoplasma (University of Florida Institutional Animal Care and Use Committee Study ). Plasma samples from 28% (11/40) of the sampled tortoises were antibody positive (titer $64), 8% (3/40) were suspect (titer 32), and 65% (26/40) were antibody negative (titer,32). All antibody-positive tortoises were located in Cameron and Hidalgo counties (Fig. 1). One tortoise

3 706 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 3, JULY 2013 FIGURE 2. Immunobinding assay blot showing recognition of the Texas tortoise (Gopherus berlandieri) isolate by polyclonal rabbit a Mycoplasma agassizii. Rabbit a M. agassizii was reacted with (1) sterile SP4 broth, a negative control for medium cross-reactions; (2) reference strain M. agassizii PS6, positive control; (3) Texas isolate #40; (4) reference strain Mycoplasma testudineum CB50; and (5) Mycoplasma pulmonis CT, control for nonspecific cross-reactivity. (Texas tortoise 40), an adult male with mild clinical signs of URTD including palpebral edema and mucoid nasal discharge, had the highest antibody titer ($512) of any of the tortoises and was the only animal positive by culture and PCR. To identify the Mycoplasma species, the isolate was grown to midlogarithmic stage in SP4 broth. M. agassizii PS6 reference strain served as a positive control; both Mycoplasma testudineum CB50 and Mycoplasma pulmonis CT served as a control for nonspecific crossreactivity; and sterile SP4 broth served as a negative control for medium crossreactions. Polyclonal rabbit antiserum to M. agassizii PS6 was commercially prepared (Pel-Freez Biologicals, Rogers, Arkansas). An immunobinding assay (Kotani and McGarrity, 1985; Brown et al., 1990; Takahata et al., 1997; Poumarat, 1998) was performed as previously described (Brown et al., 1990). Although the PCR was positive for mycoplasma, the restriction fragment length polymorphism (RFLP) analysis did not give the predicted pattern for M. agassizii. However, in the immunobinding assay (Fig. 2), the polyclonal rabbit a M. agassizii reacted with both M. agassizii PS6 and the Texas tortoise isolate #40, confirming that Texas tortoise isolate #40 was M. agassizii. No cross-reactions were observed with uninoculated culture medium, M. testudineum CB50, or M. pulmonis CT. Of the 11 ELISA-positive animals, six were male, three were female, and two were of unknown sex. Five (13%) of the 40 tortoises showed mild clinical signs of URTD, including conjunctivitis, palpebral edema, and nasal discharge. Four of the five tortoises with clinical signs were antibody negative for M. agassizii; no further diagnostics were performed to determine the underlying cause of the clinical signs. Twenty-eight percent of the Texas tortoises in this study had antibody to M. agassizii, and antibody-positive tortoises were distributed in at least two counties (Fig. 1). Tortoises in close proximity varied in antibody status, suggesting variable exposure or immune response to M. agassizii (Fig. 1). Only one antibody-positive tortoise in our study had clinical signs of URTD. Presence of antibody to M. agassizii may not always correlate with clinical disease (Lederle et al., 1997; Wendland, 2007). The time between infection and immune response can take up to 8 wk in desert and gopher tortoises. Tortoises infected with M. agassizii typically present with chronic or subclinical infection (Schumacher et al., 1997; Brown et al., 2002). The discrepancy between the PCR RFLP and the serological identification of M. agassizii was unexpected, but not unprecedented. Although uncommon, an altered RFLP pattern for M. agassizii isolates obtained from Testudo graeca was due to a point mutation in the 16sS rrna sequence (GenBank: AF ) and was confirmed by Volokhov et al. (2006) (GenBank: AY ). An immunobinding assay, as was used for serologic verification with the Texas tortoise isolate, is the most common method of verification and is recommended in the minimum standards for taxonomy (Brown et al., 2007). Based on the reactivity of the Texas tortoise isolate with polyclonal rabbit a M. agassizii reference serum and the high specific antibody titer in the serum of the animal, the isolate was determined to be M. agassizii.

4 SHORT COMMUNICATIONS 707 These results represent the first documented detection of exposure to M. agassizii, the etiological agent of URTD, in free-ranging Texas tortoises. The positive culture and serologic reactivity with polyclonal rabbit a M. agassizii reference antiserum demonstrated active infection in one animal; this animal had clinical signs of URTD and a high antibody titer. The implications of these findings for the Texas tortoise population are unknown but further investigation may be warranted. This project was made possible through the David J. Morafka Memorial Research AwardgenerouslyprovidedbytheDesert Tortoise Council. Thanks to the Gladys Porter Zoo for their tremendous support throughout this project. Special thanks to USFWS Field Biologist Mitch Sternberg and TPWD Field Biologist Sam Patten for recommendations about tortoise locations. Use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US government. LITERATURE CITED Berry KH, Christopher MM Guidelines for the field evaluation of desert tortoise health and disease. J Wildl Dis 37: Boyer TH, Boyer DM Turtles, tortoises, and terrapins, In: Reptile medicine and surgery, Mader DR, editor. Elsevier Inc., St. Louis, Missouri, pp Brown MB, Gionet P, Senior DF Identification of Mycoplasma felis and Mycoplasma gateae by an immunobinding assay. J Clin Microbiol 28: Brown MB, Schumacher IM, Klein PA, Harris K, Correll T, Jacobson ER Mycoplasma agassizii causes upper respiratory tract disease in the desert tortoise. Infect Immun 62: Brown MB, Mclaughlin GS, Klein PA, Crenshaw BC, Schumacher IM, Brown DR, Jacobson ER Upper respiratory tract disease in the gopher tortoise is caused by Mycoplasma agassizii. J Clin Microbiol 37: Brown DR, Schumacher IM, Mclaughlin GS, Wendland D, Brown MB, Klein PA, Jacobson ER Application of diagnostic tests for mycoplasmal infections of desert and gopher tortoises, with management recommendations. Chelonian Conserv Biol 4: Brown DR, Whitcomb RF, Bradbury JM Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes). Int J Syst Evol Microbiol 57: Judd FW, Rose FL Conservation status of the Texas tortoise (Gopherus berlandieri). Occas Pap Mus Tex Tech Univ 196:1 11. Kotani H, McGarrity GJ Rapid and simple identification of mycoplasmas by immunobinding. J Immunol Methods 85: Lederle PE, Rautenstrauch KR, Rakestraw DL, Zander KK, Boone JL Upper respiratory tract disease and mycoplasmosis in desert tortoises from Nevada. J Wildl Dis 33: McLaughlin GS, Jacobson ER, Brown DR, McKenna CE, Schumacher IM, Adams HP, Brown MB, Klein PA Pathology of upper respiratory tract disease of gopher tortoises in Florida. J Wildl Dis 36: Murphy RW, Berry KH, Edwards T, Leviton AE, Lathrop A, Riedle JD The dazed and confused identity of Agassiz s land tortoise, Gopherus agassizii (Testudines, Testudinidae) with the description of a new species, and its consequences for conservation. ZooKeys 113: Poumarat F Identification of mycoplasmas by dot immunobinding on membrane filtration (mf dot). Methods Mol Biol 104: Schumacher IM, Brown MB, Jacobson ER, Collins BR, Klein PA Detection of antibodies to a pathogenic Mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease. J Clin Microbiol 31: Schumacher IM, Hardenbrook DB, Brown MB, Jacobson ER, Klein PA Relationship between clinical signs of upper respiratory tract disease and antibodies to Mycoplasma agassizii in desert tortoises from Nevada. J Wildl Dis 33: Takahata T, Kato M, Nagatomo H, Shimizu T A filter immunobinding technique for the rapid detection and simultaneous identification of avian and bovine mycoplasmas. J Vet Med Sci 59: Tristan T Seroprevalence of Mycoplasma agassizii in wild caught and rescue Texas tortoises (Gopherus berlandieri) in south Texas. J Herp Med Surg 19: Volokhov DV, George J, Liu SX, Ikonomi P, Anderson C, Chizhikov V Sequencing of the intergenic 16S 23S rrna spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing. Appl Microbiol Biotechnol 71: Wendland LD Epidemiology of mycoplasmal upper respiratory tract disease in gopher tortoises. PhD Dissertation, University of Florida, Gainesville, Florida, 171 pp.

5 708 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 3, JULY 2013 Wendland LD, Brown DR, Klein PA, Brown MB Upper respiratory tract disease (mycoplasmosis) in tortoises. In: Reptile medicine and surgery, 2nd Ed., Mader DR, editor. Saunders, Elsevier, St. Louis, Missouri, pp Wendland LD, Zacher LA, Klein PA, Brown DR, Demcovitz D, Littell R, Brown MB Improved enzyme-linked immunosorbent assay to reveal Mycoplasma agassizii exposure: A valuable tool in the management of environmentally sensitive tortoise populations. Clin Vaccine Immunol 14: Submitted for publication 8 July Accepted 19 February 2013.