SHORT COMMUNICATIONS DOI: 10.7589/2017-07-153 Journal of Wildlife Diseases, 54(2), 2018, pp. 000–000 Ó Wildlife Disease Association 2018

Identification of Hemotropic in an Eastern Box Turtle (Terrapene carolina carolina) and a Yellow-Bellied Slider (Trachemys scripta scripta) from North Carolina

Jo Jarred,1 Gregory Lewbart,1,2 Kelsey Stover,1 Brittany Thomas,1 Ricardo Maggi,1 and Edward B. Breitschwerdt1 1Department of Clinical Sciences and the Comparative Medicine Institute, North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina 27607, USA; 2Corresponding author: (email: [email protected])

ABSTRACT: spp. are known from and infertility (Messick 2004). Although it has several chelonian and other reptilian . We been determined that hemoplasmas share determined if turtles obtained by the Turtle phenotypic and genetic similarities with other Rescue Team at North Carolina State University are carriers of hemotropic Mycoplasma or Bar- Mycoplasma spp., variability in the genomes tonella spp. Spleen samples were collected at of these continue to complicate exact necropsy during May through July, 2014 from 53 classification at the species level (Guimaraes turtles of seven species. All turtles were dead or et al. 2014). Different hemoplasma species are were euthanized upon arrival due to severe traumatic injuries, or they died shortly after zoonotic and cause the same effects on red beginning treatment. We used PCR amplification blood cells in human patients as they do in for both bacterial genera; Bartonella spp. DNA other mammals (Sykes et al. 2010; Steer et al. was not amplified. Based upon sequencing of the 2011). Clinical signs and pathogenic proper- 16S rRNA subunit, one eastern box turtle ties (if any) associated with hemoplasmas in (Terrapene carolina carolina) and one yellow- bellied slider (Trachemys scripta scripta) were reptiles are unknown. infected with Mycoplasma spp. that have genetic The North Carolina State University Col- similarities to strains that infect other animals. lege of Veterinary Medicine Turtle Rescue Key words: Chelonian, hemoplasma, hemo- Team (TRT) is a student-run organization of tropic mycoplasma, PCR, wildlife. volunteers who rehabilitate wild reptiles (Lewbart et al. 2005). Between May and July Identification of vector-borne diseases in 2014, samples of 53 spleens were collected reptiles has largely been unexplored. Our from turtles admitted to the TRT that either project was inspired by the documentation of died or were euthanized, mostly due to Bartonella spp. in sea turtles in North trauma (mainly vehicular). The 53 specimens Carolina (Valentine et al. 2007), because included 23 eastern box turtles (Terrapene subsequent investigations of reptiles, includ- carolina carolina), 14 yellow-bellied sliders ing terrestrial turtles, as a possible reservoir (Trachemys scripta scripta), six river cooters host remained lacking. Various Bartonella spp. (Pseudemys concinna), five eastern painted have been identified in North Carolina turtles (Chrysemys picta), three common cetaceans (Harms et al. 2008; Maggi et al. snapping turtles (Chelydra serpentina), one 2008) and river otters (Chinnadurai et al., common mud turtle (Kinosternon subru- 2010). Also, known and novel Mycoplasma brum), and one common musk turtle (Sterno- spp. have been documented in reptiles, the therus odoratus). Turtles originated from majority of which are associated with clinical central North Carolina. After death, speci- signs involving the respiratory tract (Brown et mens were stored at 4 C for up to 2 d until al. 2001; Feldman et al. 2006; Ossiboff et al. tissue harvest. We performed necropsies with 2015). Hemotropic mycoplasmas (hemoplas- the guidance of a sea turtle necropsy manual mas) are small, epicellular, -deficient (Work 2000) using appropriate modifications bacteria encountered in the blood of a variety made for each species. If DNA extraction of animal species that can contribute to could not be performed immediately, spleen pathogenic outcomes such as anemia, ill-thrift, specimens (up to 5 g, depending on the size of

1 2 JOURNAL OF WILDLIFE DISEASES, VOL. 54, NO. 2, APRIL 2018 turtle) were harvested and stored frozen at 0 fications were performed in an Eppendorf C in 5 mL screw-cap centrifuge tubes. Mastercycler EPgradient St (Eppendorf, Spleen specimens were thawed or cut Hauppauge, New York, USA). directly after necropsy into small pieces using We used 2% agarose gel electrophoresis to a scalpel blade. Pathogen DNA extraction was analyze PCR products, with detection using performed with a standardized kit (DNeasy ethidium bromide under ultraviolet light. Blood & Tissue Kit; Qiagen, Valencia, Cal- Bartonella spp. DNA was not amplified from ifornia, USA), following the manufacturer’s any turtle spleen. Two turtle spleens con- instructions for DNA extraction from tissues. tained Mycoplasma spp. DNA. Amplicon Pathogen DNA concentrations and absor- products were sequenced by Genewiz (RTP, bance ratios at 260 nm and 280 nm were North Carolina, USA) to establish species determined using the Nanodropt ND-1000 strain identification using chromatogram and (Thermo, Wilmington, Delaware, USA). to alignment analysis (ContigExpresst and assess the concentration and purity of the AlignX software, Vector NTIt v10, Invitrogen, DNA samples collected. Repeated DNA Carlsbad, California, USA). After sequencing, extractions were performed in eight cases to phylogenetic and taxonomic comparisons ensure that yields were .15 ng/lL. were made using GenBank. The DNA se- We used pathogen DNA amplification, quence from the eastern box turtle number 24 through PCR, to detect the presence of (GenBank accession number MG649987) was Bartonella spp. and hemotropic Mycoplasma most similar to the following Mycoplasma spp. DNA in each sample. We amplified sequences: 87% (490/564 base pairs [bp]) hemotropic Mycoplasma by targeting a con- similar to bacterium served region of the 16SrRNA using oligonu- T2110, (GenBank: DQ318957.1), an unclassi- cleotides HemMycop16S-322s: 50 GCC CAT fied mycoplasma found in Atlantic Cod ATT CCT ACG GGA AGC AGC AGT 30 and (Gadus morhua); 87% (484/557 bp) similar HemMyco16S-938as: 50 CTC CAC CAC TTG to Candidatus Mycoplasma haemohominis, TTC AGG TCC CCG TC 30 as previously found in humans; and 92% (483/564 bp) described (Maggi et al. 2013). Detection of similar to Candidatus Mycoplasma haemomi- Bartonella DNA was performed by targeting nutum, found in cats (Fig. 1). The DNA the 16S–23S intergenic spacer region (ITS) sequence from yellow-bellied slider number using oligonucleotides 325s (50 CCTCAGAT 47 (GenBank accession number MG519832) GATGATCCCAAGCCTTTTGGCG 30) and was most similar to the following Mycoplasma 1000as (50 CTGAGCTACGGCCCCTAAAT sequences: 92% (514/557 bp) similar to CAGG 30) as forward and reverse primers, Mycoplasma insons, previously found in green respectively (Valentine et al. 2007). Amplifi- iguanas; 92% (514/557 bp) similar to Myco- cation for each was performed in a 25 plasma cavipharyngis, found in guinea pigs lL final volume reaction. The 25 lL reaction (Cavia porcellus); 92% (512/557 bp) similar to mix contained 12.5 lL of MyTaqe Red Mix Mycoplasma fastidiosum, found in equines (Bioline, Taunton, Massachusetts, USA), 0.2 (Fig. 2). lLof50lM of each forward and reverse Hemotropic Mycoplasma spp. have not primer (IDTt DNA Technologies, Coralville, previously been amplified from a reptile. Iowa, USA), 7.3 lL of molecular grade water, The potential clinical impact that hemotropic and 5 lL (75–150 ng total) of template DNA. Mycoplasma spp. have on reptiles is unknown We prepared positive controls using either 5 because their pathogenicity in reptile species lL Mycoplasma hematoparvum 16SrRNA or remains undetermined. Many turtles enter a Bartonella henselae ITS plasmid vectors rehabilitation facility already in compromised (pGEM-T Easy Vector Systems, Promega, health, and stresses associated with trauma Madison, Wisconsin, USA) at a final concen- and rehabilitation can exacerbate what would tration of five copies per microliter, for both otherwise be nonclinical or minor health Mycoplasma and Bartonella detection. Ampli- issues. If hemotrophic Mycoplasma spp. are SHORT COMMUNICATIONS 3

FIGURE 1. Sequencing results highlighting the similarities between the Mycoplasma spp. identified in turtle number 24 (eastern box turtle, Terrapene carolina carolina) and turtle number 47 (yellow-bellied slider, Trachemys scripta scripta) to each other as well as to species of Mycoplasma found in canines and felines. normally nonpathogenic in chelonian species, Although we did not amplify DNA from a hemoplasmosis could have a negative impact Bartonella spp. from terrestrial turtles in this on an animal’s recovery and chance for study, a negative PCR result does not rule out ultimate release for those animals in rehabil- a potential Bartonella spp. infection. In non- itation facilities where concurrent diseases can reservoir hosts, Bartonella spp. are most often exacerbate nonpathogenic conditions. found in very low concentrations in blood and We do not know if the Mycoplasma spp. in splenic tissues, which could result in false chelonians are contagious to humans or to negative PCR amplification. The role, if any, other animal species. Hemotrophic Mycoplas- which reptiles play in transmission of Myco- plasma is unknown, and further investigation ma spp. amplified from reservoir hosts, into hemotropic Mycoplasma and other blood including bats (Mascarelli et al. 2014), deer pathogens is necessary. Our study provided (Maggi et al. 2013), raccoon (Procyon lotor; additional information supporting the pres- Frerichs and Holbrook 1971), rodents (Elko ence in reptiles of hemotropic Mycoplasmas, and Cantrell 1968), and other animals (Mes- which were genetically very similar to patho- sick 2004), might serve as sources for human genic strains found in humans and other infections. Zoonotic diseases such as Salmo- mammals. Until further investigation has nella spp. and Eastern Equine Encephalitis evaluated the pathogenicity and spread of virus have occurred following contact with hemotropic Mycoplasma in reptiles, isolation nonclinical reptiles (White 2011; Middleton of turtles with compromised health status, 2014). We used splenic extracts in our maintaining cleanliness and sterility of surgi- analyses and presumed that the Mycoplasma cal and medical rehabilitation supplies or use spp. was present in erythrocytes and not in of separate supplies for infected turtles, and splenic stroma. having personal protective equipment for

FIGURE 2. The relationship between the novel Mycoplasma species in turtle number 24 (eastern box turtle, Terrapene carolina carolina) and turtle number 47 (yellow-bellied slider, Trachemys scripta scripta) to each other and to known Mycoplasma spp. using the 550 base pair region of 16S ribosomal RNA gene. 4 JOURNAL OF WILDLIFE DISEASES, VOL. 54, NO. 2, APRIL 2018 individuals handling potentially infected tur- species in white-tailed deer (Odocoileus virginianus). tles might be indicated. Comp Immunol Microbiol Infect Dis 36:607–611. We thank the Robert J. Koller Aquatic Maggi RG, Compton SM, Trull CL, Mascarelli PE, Mozayeni BR, Breitschwerdt EB. 2013b. Infection Animal Medicine Research Endowment for with hemotropic Mycoplasma species in patients with financial support and the Turtle Rescue Team or without extensive arthropod or animal contact. J students who assisted in the procurement of Clin Microbiol 51:3237–3241. specimens and samples. Maggi RG, Raverty SA, Lester SJ, Huff DG, Haulena M, Ford SL, Nielsen O, Robinson JH, and Breitschwerdt LITERATURE CITED EB. 2008. Bartonella henselae in captive and hunter- harvested beluga (Delphinapterus leucas). J Wildl Dis Brown MB, Brown DR, Klein PA, McLaughlin GS, 44:871–877. Schumacher IM, Jacobson ER, Adams HP, Tully JG. Messick JB. 2004. Hemotrophic mycoplasmas (hemoplas- 2001. Mycoplasma agassizii sp. nov., isolated from mas): A review and new insights into pathogenic the upper respiratory tract of the potential. Vet Clin Pathol 33:2–13 (Gopherus agassizii) and the (Go- Middleton DM, La Flamme AC, Gartrell BD, Nelson NJ. pherus polyphemus). Int J Syst Evol Microbiol 51: 2014. Reptile reservoirs and seasonal variation in the 413–418. environmental presence of salmonella in an island Chinnadurai SK, Birkenheuer AJ, Blanton HL, Maggi ecosystem, Stephens Island, New Zealand. J Wildl RG, Belfiore N, Marr HS, Breitschwerdt EB, Dis 50:655–659. Stoskopf MK. 2010. Prevalence of selected vector- Ossiboff RJ, Raphael BL, Ammazzalorso AD, Seimon TA, borne organisms and identification of Bartonella Niederriter H, Zarate B, Newton AL, McAloose D. species DNA in North American river otters (Lontra canadensis). J Wildl Dis 46:947–950. 2015. A Mycoplasma species of Emydidae turtles in Elko EE, Cantrell W. 1968. Phagocytosis and anemia in the northeastern USA. J Wildl Dis 51:466–470. rats infected with Haemobartonella muris. J Infect Steer JA, Tasker S, Barker EN, Jensen J, Mitchell J, Stocki Dis 118:324–332. T, Chalker VJ, Hamon M. 2011. A novel hemotropic Feldman SH, Wimsatt J, Marchang RE, Johnson AJ, Mycoplasma (Hemoplasma) in a patient with hemo- Brown W, Mitchell JC, Sleeman JM. 2006. A novel lytic anemia and pyrexia. Clin Infect Dis 53:e147– Mycoplasma detected in association with upper e151. respiratory disease syndrome in free-ranging eastern Sykes JE, Lindsay LL, Maggi RG, Breitschwerdt EB. box turtles (Terrapene carolina carolina) in Virginia. J 2010. Human coinfection with Bartonella henselae Wildl Dis 42: 279–289. and two hemotropic mycoplasma variants resembling Frerichs WM, Holbrook AA. 1971. Haemobartonella Mycoplasma ovis. J Clin Microbiol 48:3782–3785. procyoni sp. n. in the raccoon, Procyon lotor. J Valentine KH, Harms CA, Cadenas MB, Birkenheuer AJ, Parasitol 57:1309–1310. Marr HS, Braun-McNeill J, Maggi RG, Breitsch- Guimaraes AS, Santos AP, do Nascimento NC, Time- werdt EB. 2007. Bartonella DNA in loggerhead sea netsky J, Messick JB. 2014. Comparative genomics turtles. Emerg Infect Dis 13:949–950. and phylogenomics of hemotrophic mycoplasmas. van Kuppeveld FJ, van der Logt JT, Angulo AF, van Zoest PLoS One 9:e91445. MJ, Quint WG, Niesters HG, Galama JM, Melchers Harms CA, Maggi RG, Breitschwerdt EB, Clemons- WJ. 1992. Genus- and species-specific identification Chevis CL, Solangi M, Rotstein DS, Fair PA, Hansen of mycoplasmas by 16S rRNA amplification. Appl LJ, Hohn AA, Lovewell GN, et al. 2008. Bartonella Environ Microbiol 58:2606–2615. species detection in captive, stranded and free White G, Ottendorfer C, Graham S, Unnasch TR. 2011. ranging cetaceans. Vet Res 39:59. Competency of reptiles and amphibians for eastern Lewbart GA, Kishimori J, Christian LS. 2005. The North equine encephalitis virus. Am J Trop Med Hyg 85: Carolina State University College of Veterinary 421–425. Medicine Turtle Rescue Team: A model for a Work TM. 2000. Sea turtle necropsy manual for biologists successful wild-reptile clinic. J Vet Med Educ 32: in remote refuges. US Geological Survey National 377–381. Wildlife Health Center, www.nwhc.usgs.gov/hfs/ Mascarelli PE, Keel MK, Yabsley M, Last LA, Breitsch- werdt EB, Maggi RG. 2014. Hemotropic mycoplasma Globals/Products/turtleml.pdf. Accessed November in little brown bats (Myotis lucifugus). Parasite 2017. Vectors 7:117. Maggi RG, Chitwood MC, Kennedy-Stoskopf SK, De- Submitted for publication 1 July 2017. Perno CS. 2013a. Novel hemotropic Mycoplasma Accepted 15 November 2017.