Subject Areas: Microbiology, Veterinary Medicine
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  . Identified Staphylococcus intermedius, Staphylococcus aureus subsp. aureus, Pseudomonas spp., Proteus mirabillis and Klebsiella pneumoniae in dogs with severe otitis  . Many authors have described Staphylococcus spp. as the most common bacteria in otitis cases  -  . However, up to 35% of the dog’s clinical otitis isolation are Pseudomonas spp. and Gram-negative bacteria that not constitute the normal microbiota  -  .
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  -  . Mycoplasmas in dogs has been described  . 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   .
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  -  . Several authors described canines’ mycoplasmas species in different parts of the body   . 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.
2. Material and Methods
2.1. Animals and Samples
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).
2.2. Reagents Used for Isolation
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  .
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 10−1 at 10−5 and spread in 2 mL and agar modified Hayflick’s medium. All culture samples sowed were incubated at 37˚C in CO2 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×) for the presence colonies.
2.4. Statistical Analysis
Associations between Mollicutes and external otitis were analysed by Fisher test, in the Software  .
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 (Table 1). The presence of
Table 1. Mollicutes in domestic dogs in the metropolitan region from Recife-PE, Brazil.
P = 0.036. Absolut frequency (AF), Relative frequency (RF).
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).
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  -   . 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   . 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.
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.
For financial support of the CNPq (Post doctoral, Process No. 158980/2014-0 and Master Science).
Conflicts of Interest
The authors declare no conflicts of interest.
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