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Ureaplasma diversum in pneumonic lungs of

swine

To the editor

Members of the genus Ureaplasmaare commensals or opportu-nistic pathogens inhabiting urogenital and respiratory tracts ofvertebrate hosts, primarily birds and mammals (Brown, 2010). Thisgenus currently has seven recognized species i.e. Ureaplasmaurealyticum and Ureaplasma parvum, both isolated from humans,Ureaplasma felinum and Ureaplasma cati, both isolated fromcats, Ureaplasma canigenitalium, isolated from dogs, Ureaplasma

gallorale, isolated from birds and Ureaplasma diversum, isolatedfrom cattle (Brown, 2010).

In the literature there are few reports concerning the isolationofUreaplasmaspecies from animals, presumably because ureaplas-mas are largely avirulent or, at least, not an economic threat.Isolations have been reported from non human primates, dogs,cats, mink, cattle, sheep, goats, camels, chickens and other fowl.Identification and isolation of ureaplasmas from swine is not welldocumented or needs confirmation (Brown, 2010).

Recently,Ureaplasma spp. was detected in the lungs of swinewith pneumonia in Cuba, by amplification and nucleotide sequenc-ing of a fragment of the rRNA 16S gene (Lobo et al., 2013). To ourknowledge, there are no earlier reports of detection of ureaplasmasin the lungs of swine. To confirm the presence of Ureaplasma

species in Cuban swine, and particularly to determine whetherthese microorganisms could be found in animals with respiratorysymptoms, as well as in the healthy ones, respiratory samples col-lected from pigs at slaughterhouse from different Cuban regionsduring the period 20092012 were studied. A total of 106 samples,including 68 lung swabs, 9 bronchial mucus and one tracheobron-chiolar lavage from pneumonic lungs, 15 nasal swabs from swinewith respiratory symptoms and 13 tracheobronchiolar lavages ta-ken from healthy lungs were processed. DNA was extracted di-rectly from 500 ll of each sample as described previously(Fernndez and Chvez, 1996), except for tracheobronchiolarlavages, where the DNA extraction was carried out from 1 mL ofsample and for bronchial mucus where DNA was extracted from500ll of sample as described previously (Mayor et al., 2007). Afragment of 644 bp of the rRNA 16S was amplified by PCR using

primers UGPF (50

-GGATGAGGGTGCGACGTATC-30

) and UGPR (50

-GCGTTAGCTACAACACCGAC-3 0) designed to detect microorganismsof Ureaplasma genus (Lauerman, 1998). Ureaplasma spp. positivesamples were tested using specie-specific PCR assays that targetsthe 50 ends of the multiple-banded antigen (MBA) genes describedpreviously for U. urealyticum and U. parvum identification (DeFrancesco et al., 2009). A PCR assay that targets another fragmentin the rRNA 16S gene, previously described (Vasconcellos Cardosoet al., 2000) was used for U. diversum identification. In addition,PCR products obtained with UGPF and UGPR primers were purified

using ExoSAP-IT (Affymetrix, USA), following manufacturersinstructions and bi-directionally sequenced on an ABI PRISM 310Genetic Analyzer according to the manufacturers protocol (AppliedBiosystems, USA). All sequences determined in this study have beendeposited in GenBank with accession numbers KC686352KC686355. The phylogenetic signal of the sequence dataset wasinvestigated by means of the likelihood mapping analysis of10,000 random quartets generated using TreePuzzle (Strimmerand von Haeseler, 1997). Phylogenetic analyses were performedusing Bayesian analysis in MrBayes v3.1.2 (Huelsenbeck andRonquist, 2001), with a minimum of 10 million generations and aburn-in of 10%. Stationary was assessed at effective samplesize (ESS > 400) using Tracer v1.4.1 (part of the BEAST package)(Drummond and Rambaut, 2007). All Bioinformatic analyseswere carried out on the freely available Bioportal: http://www.bioportal.uio.no.

From 106 samples analyzed, 7 pneumonic lung samples (6.6%),including one bronchial mucus and six lung swabs, were positiveby PCR detectingUreaplasmaspp. All these samples were identifiedas U. diversum by the species-specific PCR. Only four samples hadenough yield to obtain good quality sequences. The rRNA 16S par-tial sequences obtained in the present study showed high nucleo-tide similarity (99.8%). All sequences were aligned using ClustalXtogether with relevant sequences retrieved from GenBank (avail-able from the authors on request) representative of sequencesfrom the seven recognized Ureaplasma species. The likelihood

mapping analysis indicated that the rRNA 16S gene fragment uti-lized in the present study contains sufficient phylogenetic signalsince the noise observed was less than 20% (Zehender et al.,2011). The Bayesian phylogeny obtained for the data set (Fig. 1)confirms preceding results obtained by PCR showing the geneticrelatedness of the CubanUreaplasmaisolates withU. diversumspe-cies. In general, high posterior probabilities values were observedfor all major nodes, except for the cluster including U. parvumandU. urealyticum serovars which are not properly resolved evenusing the complete rRNA 16S gene (Kong et al., 1999). Particularly,the CubanUreaplasmastrains were grouped within theU. diversumcluster with 100% of posterior probability.

This Ureaplasma specie can inhabit all mucosal membranes ofcattle causing urogenital infections as well as subclinical respira-tory infections in young calves, which occasionally develop bron-

chopneumonia (Brown, 2010). The presence ofUreaplasma in therespiratory tract of swine has not been described in the literature.To our knowledge, this is the first report ofU. diversumin the lungof swine, being remarkable that it was found in pneumonic lungsof swine but not in the healthy ones. Our research has limitationsbecause of the small number of positive samples; no rRNA 16S full-length sequences were obtained nor isolation of this Ureaplasmafrom clinical samples. Nonetheless, we consider it relevant to com-municate these results to the veterinary research community toframe this important finding.

1567-1348/$ - see front matter 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.meegid.2013.07.003

Infection, Genetics and Evolution 21 (2014) 486488

Contents lists available at ScienceDirect

Infection, Genetics and Evolution

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m e e g i d

http://www.bioportal.uio.no/http://www.bioportal.uio.no/http://dx.doi.org/10.1016/j.meegid.2013.07.003http://dx.doi.org/10.1016/j.meegid.2013.07.003http://dx.doi.org/10.1016/j.meegid.2013.07.003http://www.elsevier.com/locate/meegidhttp://www.sciencedirect.com/science/journal/15671348http://dx.doi.org/10.1016/j.meegid.2013.07.003http://dx.doi.org/10.1016/j.meegid.2013.07.003http://www.bioportal.uio.no/http://www.bioportal.uio.no/http://crossmark.crossref.org/dialog/?doi=10.1016/j.meegid.2013.07.003&domain=pdf

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Mollicutes usually exhibit a strict host and tissue specificity.However, some Mycoplasma species have been found in unusualhosts (Razin et al., 1998). Koromyslov et al. reported the presenceof the avian mycoplasma, Mycoplasma gallisepticum in plants(Koromyslov et al., 1987), and Chernov et al, proved thephytopathogenic potential of this avian mycoplasma (Chernovet al., 2010). Another example is the bovine mycoplasma, Myco-

plasma bovis, which was identified in pigs by Spergser et al.(2013). In that case, the fact that a single M. bovis strain crossedthe host species barrier by infecting pigs was demonstrated.

Our findings constitute another example ofMycoplasmaspeciescrossingfrom bovine to swine. Further studies will be necessary todetermine whether U. diversum has some pathogenic potential inpigs or not. In addition, the implications of its presence as part ofthe porcine respiratory disease complex deserve more studies.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

This study was supported by CAPES (Brazil). We thank AricelmaP.Frana for invaluable technical assistance.

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Fig. 1. Bayesian phylogeny ofureaplasmas species based on 16S rRNA partial nucleotide sequences. For clarity, posterior probabilities obtained via Bayesian methods are

shown for the main clades only. All horizontal branch lengths are drawn to scale; bar, 0.02 substitutions per site. The tree is rooted using the sequence of the strain

Mycoplasm penetrans HF2 (Accession Number BA000026.2). The asterisks () indicate those sequences that are published for the first time in the current study.

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Yaima Burgher a,a National Center for Animal and Plant Health, Cuba

Corresponding author. Address: Laboratory for Mycoplasmas

Diagnosis, MYCOLAB, National Center for Animal and Plant Health,

CENSA, San Jos de las Lajas, Apartado Postal 10, Mayabeque, Cuba.

Tel.:+53 047849153; fax:+53 047861104.

E-mail addresses:yaima@censa.edu.cu,joan@censa.edu.cu

Lucas Miranda b,c

b Instituto Multidisciplinar em Sade, Universidade Federal da Bahia,

Brazilc Instituto de Cincias Biomdicas-II, Universidade de So Paulo, Brazil

Rosmari Rodriguez-Roche d

d Pedro Kouri Tropical Medicine Institute, Cuba

Anglica C. de Almeida Campos c

c Instituto de Cincias Biomdicas-II, Universidade de So Paulo, Brazil

Evelyn Lobo a

a National Center for Animal and Plant Health, Cuba

Tssia Neves c

c

Instituto de Cincias Biomdicas-II, Universidade de So Paulo, BrazilOrlando Martnez e

e Universidad de las Ciencias Informticas, Cuba

Jorge Timenetsky cc Instituto de Cincias Biomdicas-II, Universidade de So Paulo, Brazil

Available online 11 July 2013

488 Yaima Burgher et al./ Infection, Genetics and Evolution 21 (2014) 486488

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