Andrade Senhorinho

Chengxu Liu

Gerusa Neyla

Mario J Avila-Campos

Sydney M Finegold

Viviane Nakano

Yuli Song

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Anaerobe 17 (2011) 257e258Contents lists avaiAnaerobe
journal homepage: www.elsevier .com/locate/anaerobeNote
Detection of Porphyromonas gulae from subgingival biofilms of dogs with and
without periodontitis
Gerusa Neyla Andrade Senhorinho a, Viviane Nakano a, Chengxu Liu b, Yuli Song b,
Sydney M. Finegold b,c,d, Mario J. Avila-Campos a,*
aAnaerobe Laboratory, Department of Microbiology, Institute of Biomedical Science, University of São Paulo, Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil
bVA Medical Center West Los Angeles, Los Angeles, CA, USA
cDepartment of Medicine, UCLA School of Medicine, Los Angeles, CA, USA
dDepartment of Microbiology, Immunology and Molecular Genetics, UCLA School of Medicine, Los Angeles, CA, USAa r t i c l e i n f o
Article history:
Received 1 February 2011
Received in revised form
29 April 2011
Accepted 2 June 2011
Available online 23 June 2011
Keywords:
Porphyromonas gulae
Dog
Periodontal disease
PCR approach* Corresponding author. Tel.: þ55 11 30917344.
E-mail address: mariojac@usp.br (M.J. Avila-Camp
1075-9964/$ e see front matter  2011 Elsevier Ltd.
doi:10.1016/j.anaerobe.2011.06.002a b s t r a c t
A rapid PCR approach was developed to detect Porphyromonas gulae strains from subgingival samples of
dogs with and with periodontitis. The presence of P. gulae was observed in 92% and 56%, respectively, in
dogs with and without periodontitis. The new primer pair was specific to detect this microorganism, and
this technique could be used to evaluate a correlation between periodontitis and P. gulae in companion
animals.
 2011 Elsevier Ltd. All rights reserved.Periodontal disease is a chronic and multi-factorial disease that
leads to inflammation of the gingival destruction of periodontal
tissue and loss of alveolar bone in humans and animals [1]. Gingi-
vitis and periodontitis are common in dogs; however, their oral
microbiota has been poorly characterized [2].
In animals, particularly dogs, periodontitis occurs spontane-
ously increasing in prevalence and severity with age, and different
species of Porphyromonas appear to be associated with periodontal
disease [3], but their pathogenicity is still unclear [4]. Differences
between human and companion animal Porphyromonas isolates
have been reported. Strains of animal origin, closely resembling
P. gingivalis of humans, and both can be distinguished phenotypic
and genotypically. Catalase-producing P. gingivalis strains are
referred as Porphyromonas gulae, and this microorganism has been
recovered from subgingival samples of different companion
animals displaying periodontitis [3e5]. Because the bacterial
dispersion from dog bites to humans is observed, the study of its
oral microbiota is important [6,7].
Molecular methods are currently available for detecting perio-
dontopathogens and polymerase chain reaction has been used toos).
All rights reserved.identify these pathogens from subgingival microbiota [8,9]. In this
study, a rapid detection of P. gulae from subgingival biofilm of dogs
with and without periodontitis was performed.
Subgingival samples were taken from 50 dogs with and 50
without periodontitis undergoing routine dental treatment at the
Private Dental Veterinary Clinic (Odontovet, São Paulo, SP, Brazil)
and at the School of Veterinary Medicine and Zootechny of the
University of São Paulo. Dogs (62) and bitches (38) with and
without periodontitis from 7-months to 10-years of age were
selected and represented 12 different breeds. Periodontitis was
verified by different clinical indices (degree of gingival inflamma-
tion, amount of supragingival plaque, probing pocket depths
(5 mm), bleeding on probing, tooth mobility and alveolar bone
loss. None animal had received antibiotic treatment within the
previous three months of the collection. The Ethics Committee for
Animal Experimentation at the Institute of Biomedical Science/USP
(116/CEEA) approved this study.
Animals were anesthetized with propofol (2 mg/kg) and diaz-
epam (5.5 mg/kg) by an intramuscular injection, and received iso-
flurane and oxygen by an endotracheal way. Supragingival biofilm
was removed by using a sterile gauze, and subgingival samples
from only one gingival site (healthy dogs) or periodontal pocket
(dogs with periodontitis) were collected by using two fine sterile
paper points (N. 30, Tanariman Ind Ltd, AM, Brazil), placed for 60 s.
G.N.A. Senhorinho et al. / Anaerobe 17 (2011) 257e258258Points were then placed into VMGA III transport medium and
processed within 4 h of collection. All samples 10-fold diluted were
plated (0.1 ml) onto Brucella blood agar (Difco Laboratories) con-
taining 5% defibrinated horse blood, 0.0005 mg/ml hemin and
0.0001 mg/ml menadione, and incubated in atmosphere of 10%
CO2 þ 90% N2, at 37 C for 5 days.
Black-pigmented hemolytic colonies were selected and identi-
fied as P. gulae by using a Rapid ID 32A kit (bioMérieux), by
a b-hemolitic activity and producing catalase and indole. P. gulae
ATCC 51700 was used as control. Then, bacterial genomic DNA was
extracted and purified from cells in the mid-logarithmic growth
phase using a QIAamp DNA mini kit (Qiagen Inc, Chatsworth, CA).
The 16S rRNA gene fragments were amplified [10]. Briefly, two
subregions of 16S rRNA gene were amplified by using two primer
pairs. Subregion A was defined as 899-bp sequences between
primers 8UA and 907B, and subregion B was defined as 711-bp
sequences between primers 774A and 1485B. PCR was performed
for 35 cycles of 30 s at 95 C, 30 s at 45 C, and 1 min at 72 C, with
a final extension at 72 C for 5 min. PCR products were excised from
a 1% agarose gel after electrophoresis and purified using a QIA quick
gel extraction kit (Qiagen). An ABI 3100 genetic analyzer (Applied
Biosystems, Foster City, CA) was used for DNA sequencing.
Analysis of P. gulae isolateswas performedbycomparing the type
strains sequences determined in this study, as well as related
sequences retrieved from GenBank, as follow: P. gulae ATCC 51700
[AY546476]; P. gulae [AF285873]; P. gulae [AF285872]; P. gulae
[AF285874] and P. gulae [AF285871] which were analyzed by mul-
tisequence alignment with CLUSTAL-W (http://genome.kribb.re.kr).
Based on the multi-alignment analysis data, two primers were
designed as follows: F-primer: 50- TTG GTT GCA TGA TCG GG -30 and
R-primer: 50- GCT TAT TCT TAC GGT ACA TTC AYA -30. Sequences
were analyzed for secondary structure formation, Gþ C content, and
primeredimer formation with the NetPrimer analysis software
(http://www.premierbiosft.com/netprimer). The specificities of the
primerswere compared toall available sequencesbyusing theBLAST
database search program (www.ncbi.nlm.nih.gov/BLAST). Primers
were tested by running PCRwith DNA from nine different reference
strains (P. gingivalis ATCC 33277, P. macacae ATCC 33141, Prevotella
intermedia ATCC 25611, P. nigrescens ATCC 33563, Fusobacterium
nucleatum ATCC 25586, Treponema denticola ATCC 35308, Tannerella
forsythia ATCC 25540, Bacteroides fragilis ATCC 25285 and Para-
bacteroides distasonisATCC 8503). A colony from Brucella blood agar
was resuspended in distilledwater (200 ml), boiled (20min), and 2 ml
of supernatant (DNA) was used for subsequent amplifications.
PCR amplification was carried out in a 30-mL reaction mix con-
taining 1  PCR buffer, 50 mMMgCl2, 0.2 mM of each dNTP, 0.4 mM
of each primer pair, 0.5 U Taq Platinum DNA polymerase (Invi-
trogen) and 1 ng of DNA. Amplificationwas carried out in a thermal
cycle (GeneAmp PCR System 9700), programmed for 1 cycle of
94 C (5min) followed by 35 cycles at 94 C (30 s), 58 C (1min) and
72 C (30 s), and 1 cycle of 72 C (5 min). A negative control without
DNA was included in each PCR run, and PCR products were
analyzed by electrophoresis in 1% agarose gel. A 300 bp amplicon
was expected (Fig. 1).Fig. 1. DNA from P. gulae isolates and another non-related organism amplified with the
specific primer pair. Lanes 1 to 4, P. gulae isolates; lane 5, P. gulae ATCC 51700; lane 6,
negative control; lane 7, 100-bp DNA ladder.Species of Porphyromonas were recovered from dogs with (50)
and without (50) periodontitis, respectively, in 46 (92%) and 28
(56%) dogs. Of the 46 periodontal and of the 28 healthy dogs, 38 and
15 P. gulae, respectively, were isolated. The presence of P. gulae in
dogs without periodontitis did not show any statistically significant
difference; however, the presence of this microorganism in
dogs with periodontitis showed statistically significant values
(P ¼ 0.008).
Because periodontal disease is considered as the most common
disease in small animals, previous companion animal studies
determining the oral microbiota using biochemical assays have
been performed; however, these procedures are limited by the
quality of the biochemical databases. In this study, we detected the
presence of P. gulae in canine periodontal lesions. Paper point
samples from the crevicular space that are diluted and plated on
growth media have proven to be the most reliable method for
isolating bacteria from crevicular fluid. The traditional culture and
identification methods may limit the bacterial recovery due to the
absence of viable cells and growth requirements [3]. The identifi-
cation of canine periodontal pathogens, such as P. gulae allows for
a more directed approach to companion animal periodontitis
prevention and treatment [3,11].
Hardham et al. [7] using the 16S rRNA gene sequences identified
26 different species of black-pigmented anaerobic bacteria,
including P. gulae in the most evaluated dogs. The new synthesized
primers were specific to all isolated P. gulae. In addition, all the PCR
products belonging to P. gulae were confirmed by sequencing. No
amplification with the other bacterial DNA was observed (data not
shown). These primers distinguished P. gulae from P. gingivalis-like
organisms and they proved to be reliable and applicable for a rapid
identification of P. gulae. In addition, these primersmight be used to
a rapid diagnosis of periodontitis in animals.Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (Grant 04/09077-7).References
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Detection of Porphyromonas gulae from subgingival biofilms of dogs with and without periodontitis

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Detection of Porphyromonas gulae from subgingival biofilms of dogs with and without periodontitis

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