Infection, Genetics and Evolution 21 (2014) 486488

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Infection, Genetics and Evolution

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Ureaplasma diversum in pneumonic lungs of

swine

To the editor
Members of the genus Ureaplasma are commensals or opportunistic pathogens inhabiting urogenital and respiratory tracts of
vertebrate hosts, primarily birds and mammals (Brown, 2010). This
genus currently has seven recognized species i.e. Ureaplasma
urealyticum and Ureaplasma parvum, both isolated from humans,
Ureaplasma felinum and Ureaplasma cati, both isolated from
cats, Ureaplasma canigenitalium, isolated from dogs, Ureaplasma
gallorale, isolated from birds and Ureaplasma diversum, isolated
from cattle (Brown, 2010).
In the literature there are few reports concerning the isolation
of Ureaplasma species from animals, presumably because ureaplasmas 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 well
documented or needs confirmation (Brown, 2010).
Recently, Ureaplasma spp. was detected in the lungs of swine
with pneumonia in Cuba, by amplification and nucleotide sequencing of a fragment of the rRNA 16S gene (Lobo et al., 2013). To our
knowledge, there are no earlier reports of detection of ureaplasmas
in the lungs of swine. To confirm the presence of Ureaplasma
species in Cuban swine, and particularly to determine whether
these microorganisms could be found in animals with respiratory
symptoms, as well as in the healthy ones, respiratory samples collected from pigs at slaughterhouse from different Cuban regions
during the period 20092012 were studied. A total of 106 samples,
including 68 lung swabs, 9 bronchial mucus and one tracheobronchiolar lavage from pneumonic lungs, 15 nasal swabs from swine
with respiratory symptoms and 13 tracheobronchiolar lavages taken from healthy lungs were processed. DNA was extracted directly from 500 ll of each sample as described previously
(Fernndez and Chvez, 1996), except for tracheobronchiolar
lavages, where the DNA extraction was carried out from 1 mL of
sample and for bronchial mucus where DNA was extracted from
500 ll of sample as described previously (Mayor et al., 2007). A
fragment of 644 bp of the rRNA 16S was amplified by PCR using
primers UGPF (50 -GGATGAGGGTGCGACGTATC-30 ) and UGPR (50 GCGTTAGCTACAACACCGAC-30 ) designed to detect microorganisms
of Ureaplasma genus (Lauerman, 1998). Ureaplasma spp. positive
samples were tested using specie-specific PCR assays that targets
the 50 ends of the multiple-banded antigen (MBA) genes described
previously for U. urealyticum and U. parvum identification (De
Francesco et al., 2009). A PCR assay that targets another fragment
in the rRNA 16S gene, previously described (Vasconcellos Cardoso
et al., 2000) was used for U. diversum identification. In addition,
PCR products obtained with UGPF and UGPR primers were purified
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http://dx.doi.org/10.1016/j.meegid.2013.07.003

using ExoSAP-IT (Affymetrix, USA), following manufacturers

instructions and bi-directionally sequenced on an ABI PRISM 310
Genetic Analyzer according to the manufacturers protocol (Applied
Biosystems, USA). All sequences determined in this study have been
deposited in GenBank with accession numbers KC686352
KC686355. The phylogenetic signal of the sequence dataset was
investigated by means of the likelihood mapping analysis of
10,000 random quartets generated using TreePuzzle (Strimmer
and von Haeseler, 1997). Phylogenetic analyses were performed
using Bayesian analysis in MrBayes v3.1.2 (Huelsenbeck and
Ronquist, 2001), with a minimum of 10 million generations and a
burn-in of 10%. Stationary was assessed at effective sample
size (ESS > 400) using Tracer v1.4.1 (part of the BEAST package)
(Drummond and Rambaut, 2007). All Bioinformatic analyses
were 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 positive
by PCR detecting Ureaplasma spp. All these samples were identified
as U. diversum by the species-specific PCR. Only four samples had
enough yield to obtain good quality sequences. The rRNA 16S partial sequences obtained in the present study showed high nucleotide similarity (99.8%). All sequences were aligned using ClustalX
together with relevant sequences retrieved from GenBank (available from the authors on request) representative of sequences
from the seven recognized Ureaplasma species. The likelihood
mapping analysis indicated that the rRNA 16S gene fragment utilized in the present study contains sufficient phylogenetic signal
since 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 genetic
relatedness of the Cuban Ureaplasma isolates with U. diversum species. In general, high posterior probabilities values were observed
for all major nodes, except for the cluster including U. parvum
and U. urealyticum serovars which are not properly resolved even
using the complete rRNA 16S gene (Kong et al., 1999). Particularly,
the Cuban Ureaplasma strains were grouped within the U. diversum
cluster with 100% of posterior probability.
This Ureaplasma specie can inhabit all mucosal membranes of
cattle causing urogenital infections as well as subclinical respiratory infections in young calves, which occasionally develop bronchopneumonia (Brown, 2010). The presence of Ureaplasma in the
respiratory tract of swine has not been described in the literature.
To our knowledge, this is the first report of U. diversum in the lung
of swine, being remarkable that it was found in pneumonic lungs
of swine but not in the healthy ones. Our research has limitations
because of the small number of positive samples; no rRNA 16S fulllength sequences were obtained nor isolation of this Ureaplasma
from clinical samples. Nonetheless, we consider it relevant to communicate these results to the veterinary research community to
frame this important finding.

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487

Fig. 1. Bayesian phylogeny of ureaplasmas 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.

Mollicutes usually exhibit a strict host and tissue specificity.

However, some Mycoplasma species have been found in unusual
hosts (Razin et al., 1998). Koromyslov et al. reported the presence
of the avian mycoplasma, Mycoplasma gallisepticum in plants
(Koromyslov et al., 1987), and Chernov et al, proved the
phytopathogenic potential of this avian mycoplasma (Chernov
et al., 2010). Another example is the bovine mycoplasma, Mycoplasma bovis, which was identified in pigs by Spergser et al.
(2013). In that case, the fact that a single M. bovis strain crossed
the host species barrier by infecting pigs was demonstrated.
Our findings constitute another example of Mycoplasma species
crossing from bovine to swine. Further studies will be necessary to
determine whether U. diversum has some pathogenic potential in
pigs or not. In addition, the implications of its presence as part of
the 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 Aricelma
P. Frana for invaluable technical assistance.
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Yaima Burgher 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
a

Lucas Miranda b,c

b
Instituto Multidisciplinar em Sade, Universidade Federal da Bahia,
Brazil
c
Instituto de Cincias Biomdicas-II, Universidade de So Paulo, Brazil

Anglica C. de Almeida Campos c

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

Rosmari Rodriguez-Roche d
Pedro Kouri Tropical Medicine Institute, Cuba

Evelyn Lobo a
National Center for Animal and Plant Health, Cuba

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

Orlando Martnez e
Universidad de las Ciencias Informticas, Cuba

Jorge Timenetsky c
Instituto de Cincias Biomdicas-II, Universidade de So Paulo, Brazil
Available online 11 July 2013