<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article  PUBLIC "-//NLM//DTD Journal Publishing DTD v3.0 20080202//EN" "http://dtd.nlm.nih.gov/publishing/3.0/journalpublishing3.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" dtd-version="3.0" xml:lang="en" article-type="research article"><front><journal-meta><journal-id journal-id-type="publisher-id">OALibJ</journal-id><journal-title-group><journal-title>Open Access Library Journal</journal-title></journal-title-group><issn pub-type="epub">2333-9705</issn><publisher><publisher-name>Scientific Research Publishing</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.4236/oalib.1102790</article-id><article-id pub-id-type="publisher-id">OALibJ-69505</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biomedical&amp;Life Sciences</subject><subject> Business&amp;Economics</subject><subject> Chemistry&amp;Materials Science</subject><subject> Computer Science&amp;Communications</subject><subject> Earth&amp;Environmental Sciences</subject><subject> Engineering</subject><subject> Medicine&amp;Healthcare</subject><subject> Physics&amp;Mathematics</subject><subject> Social Sciences&amp;Humanities</subject></subj-group></article-categories><title-group><article-title>
 
 
  Report of Mollicutes in the Ear Canal of Domestic Dogs in Brazil
 
</article-title></title-group><contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Sandra</surname><given-names>Batista dos Santos</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Ma&amp;#237;na</surname><given-names>de Souza Almeida</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Luana</surname><given-names>Thamires Rap&amp;#244;so da Silva</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Júnior</surname><given-names>Mário Baltazar de Oliveira</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Carlos</surname><given-names>Adriano de Santana Leal</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>José</surname><given-names>Wilton Pinheiro Júnior</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Rinaldo</surname><given-names>Aparecido Mota</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><addr-line>Laboratory of Infectious Disease of Animals, Universidade Federal Rural de Pernambuco, Recife, Brazil</addr-line></aff><author-notes><corresp id="cor1">* E-mail:<email>sanbsantos@gmail.com(SBDS)</email>;<email>mainaalmeida@gmail.com(MDSA)</email>;<email>luana.raposo.vet@gmail.com(LTRDS)</email>;<email>juniormariobaltazar@gmail.com(JMBDO)</email>;<email>c_adrianosl@hotmail.com(CADSL)</email>;<email>wiltonjrufrpe@gmail.com(JWPJ)</email>;<email>rinaldo.mota@hotmail.com(RAM)</email>;</corresp></author-notes><pub-date pub-type="epub"><day>31</day><month>07</month><year>2016</year></pub-date><volume>03</volume><issue>07</issue><fpage>1</fpage><lpage>5</lpage><history><date date-type="received"><day>2</day>	<month>June</month>	<year>2016</year></date><date date-type="rev-recd"><day>accepted</day>	<month>8</month>	<year>July</year>	</date><date date-type="accepted"><day>11</day>	<month>July</month>	<year>2016</year></date></history><permissions><copyright-statement>&#169; Copyright  2014 by authors and Scientific Research Publishing Inc. </copyright-statement><copyright-year>2014</copyright-year><license><license-p>This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/</license-p></license></permissions><abstract><p>
 
 
   
   Several microorganisms as bacteria, fungi, yeast and ectoparasites compose canine ear canal microbiota. These agents can be native or invasive pathogens that cause clinical otitis. The aim of this study was to associate Mollicutes in external ear canal with clinical otitis. 41 domestic dogs were examined, and a total of 82 otologic samples were collected using sterile swabs, placed in a plastic storage tube containing 2 milliliters of sterile phosphate buffer saline (PBS, pH 7.2) and appropriately labeled. The samples were processed for isolation of Mollicutes using Hayflick’s medium. The frequency of Mollicutes class in the isolation was 32.9% (27/82). 14.8% (4/27) of the dogs, which were positive for Mollicutes, had external otitis and 85.2% (23/27) were healthy dogs. The genus 
   Mycoplasma 
   spp. was detected in 7.4% (2/27) by digitonin test. It was the first isolation of Mollicutes from the external ear canal in dogs. Although there have been important findings in this study, the presence of these bacteria cannot indicate the onset and perpetua
   tion of external otitis in dogs. 
  
 
</p></abstract><kwd-group><kwd>Mollicutes</kwd><kwd> Otites</kwd><kwd> Canines</kwd><kwd> Microbiota</kwd><kwd> Brazil</kwd></kwd-group></article-meta></front><body><sec id="s1"><title>1. Introduction</title><p>Microbiota of the ear canal in dogs is composed up of commensals and opportunists microorganisms. Bacteria of the genus Staphylococcus spp., Streptococcus spp. Corynebacterium spp., Proteus spp. are often found as a component of the normal flora of the ear canal, however unstable conditions may cause clinical otitis [<xref ref-type="bibr" rid="scirp.69505-ref1">1</xref>] . Identified Staphylococcus intermedius, Staphylococcus aureus subsp. aureus, Pseudomonas spp., Proteus mirabillis and Klebsiella pneumoniae in dogs with severe otitis [<xref ref-type="bibr" rid="scirp.69505-ref2">2</xref>] . Many authors have described Staphylococcus spp. as the most common bacteria in otitis cases [<xref ref-type="bibr" rid="scirp.69505-ref2">2</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref6">6</xref>] . However, up to 35% of the dog’s clinical otitis isolation are Pseudomonas spp. and Gram-negative bacteria that not constitute the normal microbiota [<xref ref-type="bibr" rid="scirp.69505-ref7">7</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref9">9</xref>] .</p><p>Mollicutes class is the smallest prokaryotes reported with replication capacity, and it is the most fastidious bacteria found in nature, that requires complex molecules such as cholesterol, sterol and urea for growth. These microorganisms are found in avian, human, insect, mammalian, plant and reptilian hosts. Some species are pathogenic and have been associated with different infections such as anemia, arthritis, otitis, infertility and respiratory disease [<xref ref-type="bibr" rid="scirp.69505-ref10">10</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref12">12</xref>] . Mycoplasmas in dogs has been described [<xref ref-type="bibr" rid="scirp.69505-ref13">13</xref>] . Nowadays, 15 species are known and two species with incompletely described. Species of mycoplasmas detected or isolated in dogs are: Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma canis, Mycoplasma cynos, Mycoplasma edwardii, Mycoplasma feliminutum, Mycoplasma felis, Mycoplasma gateae, Mycoplasma haemocanis, Mycoplasma maculosum, Mycoplasma molare, Mycoplasma opalescens, Mycoplasma sp. HRC689, Mycoplasma sp. VJC358, Mycoplasma spumans and Ureaplasma canigenitalium [<xref ref-type="bibr" rid="scirp.69505-ref14">14</xref>] [<xref ref-type="bibr" rid="scirp.69505-ref15">15</xref>] .</p><p>Some species detected in canine have been isolated in others hosts, though the canines’ mycoplasma flora were not be fully described. This report refers to species that occur in the respiratory and reproductive tract, oral and nasal cavity, conjunctiva, pericardial sac, kidney, liver, lymph node, spleen, bladder, cerebral spinal fluid, blood, synovial membrane [<xref ref-type="bibr" rid="scirp.69505-ref16">16</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref19">19</xref>] . Several authors described canines’ mycoplasmas species in different parts of the body [<xref ref-type="bibr" rid="scirp.69505-ref14">14</xref>] [<xref ref-type="bibr" rid="scirp.69505-ref19">19</xref>] . No previous studies of Mycoplasmas found the bacteria from the ear canal of dogs. The present study was conducted to investigate members of the Mollicutes class in the ear canal of canines with and without otitis.</p></sec><sec id="s2"><title>2. Material and Methods</title><sec id="s2_1"><title>2.1. Animals and Samples</title><p>Domestic dogs from different localities attended veterinary hospital of the Federal Rural University-UFRPE, Recife-PE. 41 dogs at different ages and breeds, females and males were used in this study. Anamnesis, clinical examination and otoscopy were performed in the right and left sides the ear canal of each dog, in a total 82 samples collected. The otologic samples were collected with sterile swabs placed in a plastic storage tube containing 2 milliliters of sterile phosphate buffer saline (PBS, pH 7.2) and appropriately labeled. Furthermore, otologic swabs were collected for direct yeast examination. All samples were maintained at 4˚C in a cooler with ice and forwarded to the Laboratory of Infectious Diseases of Animals (LDIC/DMV/UFRPE).</p></sec><sec id="s2_2"><title>2.2. Reagents Used for Isolation</title><p>For isolation of Mollicutes the samples collected in phosphate buffer saline (PBS, pH 7.2) were cultivated in modified Hayflick’s medium. Mollicutes isolates were confirmed by Dienes probe and Mycoplasma genus through a digitonin sensitivity test, according to [<xref ref-type="bibr" rid="scirp.69505-ref20">20</xref>] .</p></sec><sec id="s2_3"><title>2.3. Procedures</title><p>The Mollicutes isolation procedures consisted in the swabs were homogenized in the transport solution, and 100 uL was relocated to tubes containing 900 uL of modified Hayflick’s medium, and diluted up to 10<sup>−1</sup> at 10<sup>−5</sup> and spread in 2 mL and agar modified Hayflick’s medium. All culture samples sowed were incubated at 37˚C in CO<sub>2</sub> incubator for at least 30 days, and blind peals were made for up to 60 days, before disposal of samples. The cultures were checked daily by using a stereomicroscopy (40&#215;) for the presence colonies.</p></sec><sec id="s2_4"><title>2.4. Statistical Analysis</title><p>Associations between Mollicutes and external otitis were analysed by Fisher test, in the Software [<xref ref-type="bibr" rid="scirp.69505-ref21">21</xref>] .</p></sec></sec><sec id="s3"><title>3. Results</title><p>A total frequency of Mollicutes in the isolation was 32.9% (27/82). 14.8% (4/27) of the dogs positive for Mollicutes were also external otitis and 85.2% (23/27) in dogs without clinical otitis (<xref ref-type="table" rid="table1">Table 1</xref>). The presence of</p><table-wrap id="table1" ><label><xref ref-type="table" rid="table1">Table 1</xref></label><caption><title> Mollicutes in domestic dogs in the metropolitan region from Recife-PE, Brazil</title></caption><table><tbody><thead><tr><th align="center" valign="middle"  rowspan="2"  >Mollicutes</th><th align="center" valign="middle"  colspan="2"  >Positive otitis</th><th align="center" valign="middle"  colspan="2"  >Negative otitis</th><th align="center" valign="middle"  colspan="2"  >Total</th></tr></thead><tr><td align="center" valign="middle" >(AF)<sup> </sup></td><td align="center" valign="middle" >(RF%)</td><td align="center" valign="middle" >(AF)</td><td align="center" valign="middle" >(RF%)</td><td align="center" valign="middle" >(AF)</td><td align="center" valign="middle" >(RF%)</td></tr><tr><td align="center" valign="middle" >Positive</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >14.8</td><td align="center" valign="middle" >23</td><td align="center" valign="middle" >85.2</td><td align="center" valign="middle" >27</td><td align="center" valign="middle" >100</td></tr><tr><td align="center" valign="middle" >Negative</td><td align="center" valign="middle" >2</td><td align="center" valign="middle" >100</td><td align="center" valign="middle" >0</td><td align="center" valign="middle" >0</td><td align="center" valign="middle" >2</td><td align="center" valign="middle" >100</td></tr><tr><td align="center" valign="middle" >Total</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" ></td><td align="center" valign="middle" >23</td><td align="center" valign="middle" ></td><td align="center" valign="middle" >29</td><td align="center" valign="middle" >100</td></tr></tbody></table></table-wrap><p>P = 0.036. Absolut frequency (AF), Relative frequency (RF).</p><p>fried-egg colonies on solid medium and other appearance variations as raindrops were observed. In the digitonin senbility test 7.4% (2/27) were positives, confirming presence Mycoplasma spp. genus. In the analysis of association of Mollicutes with otitis, this disease influenced negatively the isolation of Mollicutes in the ear canal of dogs, this association was significant (P = 0.036).</p></sec><sec id="s4"><title>4. Discussion<sup> </sup></title><p>The etiology complex of the canines’ otitis has been widely studied in worldwide. Although multiple factors are involved, the species richness of fungi, bacterias and ectoparasites can act as primary factors in clinical otitis in dogs [<xref ref-type="bibr" rid="scirp.69505-ref2">2</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref6">6</xref>] [<xref ref-type="bibr" rid="scirp.69505-ref22">22</xref>] . Mollicutes are fastidious for bacterial cultivation and few laboratories routinely cultivate these microrganisms, though culture is the most used method for detection of mycoplasmas in dogs [<xref ref-type="bibr" rid="scirp.69505-ref14">14</xref>] [<xref ref-type="bibr" rid="scirp.69505-ref17">17</xref>] . In the present study it was verified high frequency of Mollicutes in the ear canal of healthy dogs as compared to otitis, showing that possibly this bacteria is a component of the normal ear canal microbiota. In this study the dogs with otitis were identified other bacteria and yeasts in the ear canal.</p><p>The presence of other bacteria, fungi and yeasts in the ear canal of dogs can compete for nutrients creating undesirable conditions for mycoplasmas growth, since righ requirements and long-term isolation are needed. Several authors [<xref ref-type="bibr" rid="scirp.69505-ref16">16</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref19">19</xref>] [<xref ref-type="bibr" rid="scirp.69505-ref28">28</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref30">30</xref>] reported mycoplasmas in different sites of canine body, however in the ear canal there is no references list. The lack of studies may occur as result of greater relevance given to the association of other microorganisms as the main causes of otitis than mycoplasmas. Mycoplasmas have been found as a component of the microbiota of the ear canal in other animals species, such as cattle and goats, therefore it could be source of mycoplasmas for outbreak diseases (pneumonia, otitis, arthritis, mastitis) [<xref ref-type="bibr" rid="scirp.69505-ref23">23</xref>] - [<xref ref-type="bibr" rid="scirp.69505-ref26">26</xref>] . Mycoplasma cynos, which is frequently found in dogs respiratory disease, affects the upper respiratory tract. Mycoplasma edwardii, Mycoplasma spumans and Mycoplasma maculosum are usually found in the nasal cavity [<xref ref-type="bibr" rid="scirp.69505-ref11">11</xref>] [<xref ref-type="bibr" rid="scirp.69505-ref12">12</xref>] and Mycoplasma mucosicanis found in the oral cavity [<xref ref-type="bibr" rid="scirp.69505-ref15">15</xref>] . Furthermore clinical otitis caused by Malassezia, a common yeast found in dogs, acts as a limiting factor to the isolation of Mollicutes, by reason of Malassezia genus needs lipid and fatty acids for growth [<xref ref-type="bibr" rid="scirp.69505-ref27">27</xref>] . More studies are necessary to verify the relevance of Mollicutes in the ear canal of dogs and the role these bacteria in the etiopathogeny of otitis and other mycoplasmosis in dogs.</p></sec><sec id="s5"><title>5. Conclusion</title><p>This is the first report of members of the Mollicutes class and Mycoplasma spp. in the ear canal of dogs however their association with clinical otitis cases remains unclear, and more studies are necessary for proving this relation.</p></sec><sec id="s6"><title>Acknowledgements</title><p>For financial support of the CNPq (Post doctoral, Process No. 158980/2014-0 and Master Science).</p></sec><sec id="s7"><title>Conflicts of Interest</title><p>The authors declare no conflicts of interest.</p></sec><sec id="s8"><title>Cite this paper</title><p>Sandra Batista dos Santos,Ma&#237;na de Souza Almeida,Luana Thamires Rap&#244;so da Silva,J&#250;nior M&#225;rio Baltazar de Oliveira,Carlos Adriano de Santana Leal,Jos&#233; Wilton Pinheiro J&#250;nior,Rinaldo Aparecido Mota, (2016) Report of Mollicutes in the Ear Canal of Domestic Dogs in Brazil. 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