{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T11:58:12Z","timestamp":1753876692045,"version":"3.41.2"},"reference-count":46,"publisher":"Oxford University Press (OUP)","license":[{"start":{"date-parts":[[2023,1,1]],"date-time":"2023-01-01T00:00:00Z","timestamp":1672531200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2023,1,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Two primer set\/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as Chlamydia pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brain swabs, and ultimately specifically identify and quantify chlamydiae, specifically C. pneumoniae in these samples.<\/jats:p>","DOI":"10.1093\/femsle\/fnad067","type":"journal-article","created":{"date-parts":[[2023,7,7]],"date-time":"2023-07-07T13:19:39Z","timestamp":1688735979000},"source":"Crossref","is-referenced-by-count":0,"title":["Quantification of chlamydiae in the endangered Houston toad (<i>Anaxyrus houstonensis<\/i>)"],"prefix":"10.1093","volume":"370","author":[{"given":"Andrea","family":"Villamizar","sequence":"first","affiliation":[{"name":"Department of Biology, Texas State University , 601 University Drive, San Marcos, TX 78666 , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Trina","family":"Guerra","sequence":"additional","affiliation":[{"name":"Department of Biology, Texas State University , 601 University Drive, San Marcos, TX 78666 , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maryanne E","family":"Tocidlowski","sequence":"additional","affiliation":[{"name":"Houston Zoo, 1513 Cambridge St. , Houston, TX 77030 , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael R J","family":"Forstner","sequence":"additional","affiliation":[{"name":"Department of Biology, Texas State University , 601 University Drive, San Marcos, TX 78666 , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8640-5938","authenticated-orcid":false,"given":"Dittmar","family":"Hahn","sequence":"additional","affiliation":[{"name":"Department of Biology, Texas State University , 601 University Drive, San Marcos, TX 78666 , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2023,7,8]]},"reference":[{"key":"2023071815525146400_bib1","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1097\/QCO.0b013e3282f3e6a5","article-title":"Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes","volume":"21","author":"Baud","year":"2008","journal-title":"Curr Opin Infect Dis"},{"key":"2023071815525146400_bib2","doi-asserted-by":"crossref","first-page":"564","DOI":"10.1638\/2013-0250R.1","article-title":"Efficacy of fenbendazole and levamisole treatments in captive Houston toads (Bufo [Anaxyrus] houstonensis)","volume":"45","author":"Bianchi","year":"2014","journal-title":"J Zoo Wildlife Med"},{"key":"2023071815525146400_bib3","doi-asserted-by":"crossref","first-page":"755426","DOI":"10.3389\/fvets.2022.755426","article-title":"Ranavirus amplification in low-diversity amphibian communities","volume":"9","author":"Bienentreu","year":"2022","journal-title":"Front Vet Sci"},{"key":"2023071815525146400_bib4","doi-asserted-by":"crossref","first-page":"81","DOI":"10.3390\/d10030081","article-title":"Effects of emerging infectious diseases on amphibians: a review of experimental studies","volume":"10","author":"Blaustein","year":"2018","journal-title":"Diversity"},{"key":"2023071815525146400_bib5","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1354\/vp.44-2-144","article-title":"Chlamydiae in free-ranging and captive frogs in Switzerland","volume":"44","author":"Blumer","year":"2007","journal-title":"Vet Pathol"},{"key":"2023071815525146400_bib6","doi-asserted-by":"crossref","first-page":"e67","DOI":"10.1017\/S0022149X21000614","article-title":"Research on helminths from Mexican amphibians: gaps, trends, and biases","volume":"95","author":"Cabrera-Guzman","year":"2021","journal-title":"J Helminthol"},{"key":"2023071815525146400_bib7","doi-asserted-by":"crossref","first-page":"860","DOI":"10.1177\/03009858221095269","article-title":"An outbreak of systemic chlamydiosis in farmed American alligators (Alligator mississippiensis)","volume":"59","author":"Carossino","year":"2022","journal-title":"Vet Pathol"},{"key":"2023071815525146400_bib8","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1007\/978-1-4939-0820-2_12","article-title":"Amphibian declines in the twenty-first century: why we need assisted reproductive technologies","volume":"753","author":"Clulow","year":"2014","journal-title":"Adv Exp Med Biol"},{"key":"2023071815525146400_bib9","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/ilar.48.3.235","article-title":"Diseases of amphibians","volume":"48","author":"Densmore","year":"2007","journal-title":"Ilar J"},{"key":"2023071815525146400_bib10","doi-asserted-by":"crossref","first-page":"363","DOI":"10.3996\/112013-JFWM-071","article-title":"Documenting extinction in real time: decline of the Houston toad on a primary recovery site","volume":"5","author":"Duarte","year":"2014","journal-title":"J Fish Wildl Manag"},{"key":"2023071815525146400_bib11","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.mcp.2005.09.003","article-title":"Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies","volume":"20","author":"Ehricht","year":"2006","journal-title":"Mol Cell Probe"},{"key":"2023071815525146400_bib12","doi-asserted-by":"crossref","first-page":"2139","DOI":"10.1007\/s10482-020-01483-6","article-title":"Chronic wasting associated with Chlamydia pneumoniae in three ex situ breeding facilities for tropical frogs","volume":"113","author":"Eisenberg","year":"2020","journal-title":"Antonie Van Leeuwenhoek"},{"key":"2023071815525146400_bib13","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1038\/s41564-020-00834-3","article-title":"Community-led, integrated, reproducible multi-omics with anvi'o","volume":"6","author":"Eren","year":"2021","journal-title":"Nat Microbiol"},{"key":"2023071815525146400_bib14","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1128\/JCM.37.3.575-580.1999","article-title":"Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests","volume":"37","author":"Everett","year":"1999","journal-title":"J Clin Microbiol"},{"key":"2023071815525146400_bib15","doi-asserted-by":"crossref","first-page":"783","DOI":"10.2307\/2408678","article-title":"Confidence limits of phylogenies: an approach using the bootstrap","volume":"39","author":"Felsenstein","year":"1985","journal-title":"Evolution"},{"key":"2023071815525146400_bib16","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1038\/s41579-020-0335-x","article-title":"Chytrid fungi and global amphibian declines","volume":"18","author":"Fisher","year":"2020","journal-title":"Nat Rev Microbiol"},{"key":"2023071815525146400_bib17","first-page":"71","article-title":"Houston toad population supplementation in Texas, USA","volume-title":"Global Re-Introduction Perspectives: 2011","author":"Forstner","year":"2011"},{"key":"2023071815525146400_bib18","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1177\/0300985819844818","article-title":"Chlamydia pneumoniae polioencephalomyelitis and ganglionitis in captive Houston toads (Anaxyrus houstonensis)","volume":"56","author":"Fratzke","year":"2019","journal-title":"Vet Pathol"},{"key":"2023071815525146400_bib19","doi-asserted-by":"crossref","first-page":"1425","DOI":"10.1136\/bjo.2006.096420","article-title":"Detection by broad-range real-time PCR assay of Chlamydia species infecting human and animals","volume":"90","author":"Goldschmidt","year":"2006","journal-title":"Brit J Ophthalmol"},{"key":"2023071815525146400_bib20","doi-asserted-by":"crossref","first-page":"243","DOI":"10.3354\/dao02138","article-title":"Ecology and pathology of amphibian ranaviruses","volume":"87","author":"Gray","year":"2009","journal-title":"Dis Aquat Organ"},{"key":"2023071815525146400_bib21","first-page":"215","article-title":"Diagnosis of Aeromonas hydrophila, Mycobacterium species, and batrachochytrium dendrobatidis in an African Clawed Frog (Xenopus laevis)","volume":"49","author":"Hill","year":"2010","journal-title":"J Am Assoc Lab Anim"},{"key":"2023071815525146400_bib22","doi-asserted-by":"publisher","first-page":"e1","DOI":"10.1093\/nar\/gks808","article-title":"Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies","volume":"41","author":"Klindworth","year":"2013","journal-title":"Nucleic Acids Res"},{"key":"2023071815525146400_bib23","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1016\/j.cvex.2013.01.005","article-title":"Selected emerging diseases of amphibia","volume":"16","author":"Latney","year":"2013","journal-title":"Vet Clin North Am Exot Anim Pract"},{"key":"2023071815525146400_bib24","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1670\/0022-1511(2003)037[0215:CIWFFS]2.0.CO;2","article-title":"Chytridiomycosis in wild frogs from Southern Costa Rica","volume":"37","author":"Lips","year":"2003","journal-title":"J Herpetol"},{"key":"2023071815525146400_bib25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3354\/dao03039","article-title":"Patterns of amphibian infection prevalence across wetlands on the Savannah River Site, South Carolina, USA","volume":"121","author":"Love","year":"2016","journal-title":"Dis Aquat Organ"},{"key":"2023071815525146400_bib26","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1111\/j.1574-6976.1994.tb00132.x","article-title":"Bacterial phylogeny based on 16S and 23S ribosomal-RNA sequence analysis","volume":"15","author":"Ludwig","year":"1994","journal-title":"FEMS Microbiol Rev"},{"key":"2023071815525146400_bib27","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1099\/jmm.0.46335-0","article-title":"Development of a real-time PCR for the detection of Chlamydia psittaci","volume":"55","author":"Menard","year":"2006","journal-title":"J Med Microbiol"},{"key":"2023071815525146400_bib28","first-page":"187","article-title":"Detection of chlamydia psittaci in amphibians using an immunofluorescence test (IFT)","volume":"111","author":"Mutschmann","year":"1998","journal-title":"Berl Munch Tierarztl"},{"key":"2023071815525146400_bib29","doi-asserted-by":"crossref","first-page":"1187","DOI":"10.1099\/00207713-50-3-1187","article-title":"Sequence analysis of domains III and IV of the 23S rRNA gene of verticillate streptomycetes","volume":"50","author":"Palmano","year":"2000","journal-title":"Int J Syst Evol Microbiol"},{"key":"2023071815525146400_bib30","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/j.tvjl.2008.02.025","article-title":"New real-time PCR tests for species-specific detection of chlamydophila psittaci and chlamydophila abortus from tissue samples","volume":"181","author":"Pantchev","year":"2009","journal-title":"Vet J"},{"key":"2023071815525146400_bib31","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1002\/zoo.21166","article-title":"Causes of mortality in anuran amphibians from an ex situ survival assurance colony in Panama","volume":"33","author":"Pessier","year":"2014","journal-title":"Zoo Biol"},{"key":"2023071815525146400_bib32","doi-asserted-by":"crossref","first-page":"D590","DOI":"10.1093\/nar\/gks1219","article-title":"The SILVA ribosomal RNA gene database project: improved data processing and web-based tools","volume":"41","author":"Quast","year":"2012","journal-title":"Nucleic Acids Res"},{"key":"2023071815525146400_bib33","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1099\/00221287-138-6-1167","article-title":"Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes","volume":"138","author":"Roller","year":"1992","journal-title":"J Gen Microbiol"},{"key":"2023071815525146400_bib34","doi-asserted-by":"crossref","first-page":"106318","DOI":"10.1016\/j.mimet.2021.106318","article-title":"Development and comparative evaluation of droplet digital PCR and quantitative PCR for the detection and quantification of Chlamydia psittaci","volume":"190","author":"Sahu","year":"2021","journal-title":"J Microbiol Meth"},{"key":"2023071815525146400_bib35","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1146\/annurev-animal-020518-115056","article-title":"The role of reproductive technologies in amphibian conservation breeding programs","volume":"7","author":"Silla","year":"2019","journal-title":"Annu Rev Anim Biosci"},{"key":"2023071815525146400_bib36","doi-asserted-by":"crossref","first-page":"817","DOI":"10.7589\/2015-04-104","article-title":"Monitoring ranavirus-associated mortality in a Dutch heathland in the aftermath of a ranavirus disease outbreak","volume":"52","author":"Sluijs","year":"2016","journal-title":"J Wildl Dis"},{"key":"2023071815525146400_bib37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1017\/S0950268821002715","article-title":"Molecular characterization of chlamydia species in commercial and backyard poultry farms in Costa Rica","volume":"150","author":"Solorzano-Morales","year":"2022","journal-title":"Epidemiol Infect"},{"key":"2023071815525146400_bib38","doi-asserted-by":"crossref","first-page":"171","DOI":"10.2478\/s11686-018-00018-4","article-title":"Description of a new species of Rhabdias Stiles and Hassall, 1905 (Nematoda, Rhabdiasidae) in Duttaphrynus melanostictus (Schneider, 1899) from West Bengal, India","volume":"64","author":"Sou","year":"2019","journal-title":"Acta Parasitol"},{"key":"2023071815525146400_bib39","doi-asserted-by":"crossref","first-page":"2688","DOI":"10.1093\/bioinformatics\/btl446","article-title":"RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models","volume":"22","author":"Stamatakis","year":"2006","journal-title":"Bioinformatics"},{"key":"2023071815525146400_bib40","doi-asserted-by":"crossref","first-page":"1312","DOI":"10.1093\/bioinformatics\/btu033","article-title":"RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies","volume":"30","author":"Stamatakis","year":"2014","journal-title":"Bioinformatics"},{"key":"2023071815525146400_bib41","doi-asserted-by":"crossref","first-page":"D593","DOI":"10.1093\/nar\/gku1201","article-title":"rrnDB: improved tools for interpreting rRNA gene abundance in bacteria and archaea and a new foundation for future development","volume":"43","author":"Stoddard","year":"2015","journal-title":"Nucleic Acids Res"},{"key":"2023071815525146400_bib42","first-page":"57","article-title":"Some probabilistic and statistical problems in the analysis of DNA sequences","volume":"17","author":"Tavar\u00e9","year":"1986","journal-title":"Am Math Socy: Lect Math Life Sci"},{"key":"2023071815525146400_bib43","doi-asserted-by":"crossref","first-page":"503","DOI":"10.7589\/JWD-D-20-00145","article-title":"Mycobacteria in skin lesions and the habitat of the endangered Houston toad (Anaxyrus houstonensis)","volume":"57","author":"Vemulapally","year":"2021","journal-title":"J Wildl Dis"},{"key":"2023071815525146400_bib44","doi-asserted-by":"crossref","first-page":"126342","DOI":"10.1016\/j.syapm.2022.126342","article-title":"Quantification of members of the Mycobacterium chelonae-abscessus complex in lesions of the endangered Houston toad (Anaxyrus houstonensis)","volume":"45","author":"Villamizar","year":"2022","journal-title":"Syst Appl Microbiol"},{"key":"2023071815525146400_bib45","doi-asserted-by":"crossref","first-page":"2642","DOI":"10.1111\/jam.15422","article-title":"Identification of parachlamydiaceae DNA in nasal and rectal passages of healthy dairy cattle","volume":"132","author":"Wheelhouse","year":"2022","journal-title":"J Appl Microbiol"},{"key":"2023071815525146400_bib46","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.jcv.2006.01.005","article-title":"Development of a rapid real-time PCR assay for detection and quantification of four familiar species of Chlamydiaceae","volume":"36","author":"Yang","year":"2006","journal-title":"J Clin Virol"}],"container-title":["FEMS Microbiology Letters"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/femsle\/advance-article-pdf\/doi\/10.1093\/femsle\/fnad067\/50843249\/fnad067.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/femsle\/article-pdf\/doi\/10.1093\/femsle\/fnad067\/50905547\/fnad067.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/femsle\/article-pdf\/doi\/10.1093\/femsle\/fnad067\/50905547\/fnad067.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,7,18]],"date-time":"2023-07-18T16:08:25Z","timestamp":1689696505000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/femsle\/article\/doi\/10.1093\/femsle\/fnad067\/7221645"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023]]},"references-count":46,"URL":"https:\/\/doi.org\/10.1093\/femsle\/fnad067","relation":{},"ISSN":["1574-6968"],"issn-type":[{"type":"electronic","value":"1574-6968"}],"subject":[],"published-other":{"date-parts":[[2023]]},"published":{"date-parts":[[2023]]},"article-number":"fnad067"}}