Comparison of periodontal pathogens between cats and their owners

Abstract

The periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia are strongly associated with periodontal disease and are highly prevalent in humans with periodontitis. Porphyromonas and Tannerella spp. have also been isolated from the oral cavity of cats. The oral microflora in animals was compared with those in humans in earlier studies, but no studies are available on the comparison of the oral microflora from pets and their respective owners. The aim of this study was to determine the presence of these bacteria in the oral microflora of cats and their owners, since animal to human transmission, or vice versa, of oral pathogens could have public health implications. This study investigated the prevalence of Porphyromonas gulae, P. gingivalis, and T. forsythia in the oral microflora of cats and their owners, using culture and polymerase chain reaction (PCR). All Porphyromonas isolates from cats (n=64) were catalase positive, whereas the Porphyromonas isolates from owners (n=7) were catalase negative, suggesting that the isolates from cats were P. gulae whereas those from the owners were P. gingivalis. T. forsythia was recovered from both cats (n=63) and owners (n=31); the proportion of T. forsythia relative to the total CFU was higher in cats with periodontitis than in cats without periodontal disease. Genotyping of T. forsythia isolates (n=54) in six cat/owner couples showed that in one cat/owner couple the T. forsythia isolates (n=6) were identical. These T. forsythia isolates were all catalase positive, which led us to hypothesize that transmission from cats to owners had occurred and that cats may be a reservoir of T. forsythia.

Full text

Comparison of periodontal pathogens between cats and their owners
H.E. Booij-Vrieling
a,
*, W.A. van der Reijden
b
, D.J. Houwers
c
, W.E.A.J. de Wit
b
,
C.J. Bosch-Tijhof
b
, L.C. Penning
a
, A.J. van Winkelhoff
d
, H.A.W. Hazewinkel
a
a
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, P.O. Box 80154, 3508 TD Utrecht,
The Netherlands
b
Department of Oral Microbiology, Academic Center for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam and Vrije Universiteit,
Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
c
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80165, 3508 TD Utrecht, The Netherlands
d
Center for Dentistry and Oral Hygiene, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
1. Introduction
Periodontal diseases are bacterial infections of the oral
cavity. The capnophilic bacterium Aggregatibacter actino-
mycetemcomitans and the obligate anaerobic bacteria
Porphyromonas gingivalis and Tannerella forsythia are
established periodontal pathogens, with the latter two
being the strongest bacterial markers for destructive
periodontal disease in humans (Socransky et al., 1998;
van Winkelhoff et al., 2002). The prevalence of P. gingivalis
varies between 10% and 25% in persons with healthy
periodontal tissue and between 59% and 79% in patients
with periodontitis (Boutaga et al., 2003; Griffen et al., 1998;
van Winkelhoff et al., 2002); the prevalence of T. forsythia is
48% and 91%, respectively (van Winkelhoff et al., 2002).
Periodontal pathogens can be transmitted between family
members (Asikainen et al., 1997; Petit et al., 1993; van
Winkelhoff et al., 2007; von Troil-Linden et al., 1996).
Veterinary Microbiology xxx (2010) xxx–xxx
ARTICLE INFO
Article history:
Received 28 October 2009
Received in revised form 23 December 2009
Accepted 24 December 2009
Keywords:
Porphyromonas gulae
Porphyromonas gingivalis
Tannerella forsythia
Transmission
Cat
Man
Genotyping
ABSTRACT
The periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia are strongly
associated with periodontal disease and are highly prevalent in humans with period-
ontitis.
Porphyromonas and Tannerella spp. have also been isolated from the oral cavity of cats.
The oral microflora in animals was compared with those in humans in earlier studies, but
no studies are available on the comparison of the oral microflora from pets and their
respective owners. The aim of this study was to determine the presence of these bacteria in
the oral microflora of cats and their owners, since animal to human transmission, or vice
versa, of oral pathogens could have public health implications.
This study investigated the prevalence of Porphyromonas gulae,P. gingivalis, and T.
forsythia in the oral microflora of cats and their owners, using culture and polymerase
chain reaction (PCR). All Porphyromonas isolates from cats (n= 64) were catalase positive,
whereas the Porphyromonas isolates from owners (n= 7) were catalase negative,
suggesting that the isolates from cats were P. gulae whereas those from the owners
were P. gingivalis.T. forsythia was recovered from both cats (n= 63) and owners (n= 31);
the proportion of T. forsythia relative to the total CFU was higher in cats with periodontitis
than in cats without periodontal disease. Genotyping of T. forsythia isolates (n= 54) in six
cat/owner couples showed that in one cat/owner couple the T. forsythia isolates (n= 6)
were identical. These T. forsythia isolates were all catalase positive, which led us to
hypothesize that transmission from cats to owners had occurred and that cats may be a
reservoir of T. forsythia.
!2010 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +31 30 253 9228; fax: +31 30 251 8126.
E-mail address: h.e.booij-vrieling@uu.nl (H.E. Booij-Vrieling).
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doi:10.1016/j.vetmic.2009.12.046

Periodontal disease is the most common disease in cats
and dogs (Reichart et al., 1984), with a prevalence of 70%
and 80%, respectively, in 2-year-old animals (Wiggs and
Lobprise, 1997). The normal oral microflora of cats is
dominated by anaerobic bacteria, mainly of the genera
Bacteroides and Fusobacterium (Love et al., 1990). Porphyr-
omonas spp. have been isolated from dental plaque in
periodontal diseased cats (Harvey et al., 1995; Mallonee
et al., 1988; Norris and Love, 1999, 2000). Although
Tannerella spp. have also been identified in the oral
microflora of cats, on the basis of bite wound infections
(Hudspeth et al., 1999; Love et al., 2000; Stefanopoulos and
Tarantzopoulou, 2005; Talan et al., 1999; Weber et al.,
1984). Several studies have compared the composition of
the oral microflora in animals with those in humans
(Fournier and Mouton, 1993; Hudspeth et al., 1999) but not
from the perspective of the owner–pet relationship. The
aim of this study was to determine the prevalence of P.
gulae,P. gingivalis, and T. forsythia in cats and their owners.
Since owners are in close contact with their cats (Holbrook,
2008; Vitulli, 2006), we furthermore wanted to determine
whether transmission of these periodontal pathogens may
have occurred.
2. Materials and methods
2.1. Recruitment of subjects and sampling of dental plaque
The study was approved by the medical ethics
committee of the Vrije Universiteit of Amsterdam and
by the animal ethics committee of the Utrecht Uni-
versity. Approval was granted on the basis that the least
invasive sampling methods would be used and that
sampling would be anonymous. Therefore, we had no
demographic or dental information (periodontal status)
for the owners. Cats and their owners were sampled at an
international cat show (group 1, healthy group) and at
two private veterinary clinics (group 2, patient group).
Cats were included in group 1 if there were no visible
signs of gingival recession, no visible furcations, no
redness/edema, and no hyperplasia with spontaneous
bleeding of gingival tissues. Cats in group 2 were selected
if one or more of the aforementioned visible signs of
periodontal disease were present. Owners were informed
about the aim of the study and gave permission for
sampling of their cat(s). Supragingival plaque samples
from cats were taken from the buccal side of 108/208
(upper fourth premolars) using a cotton–wool stick and
transferred to a tube filled with 1.5 mL reduced transport
fluid (RTF). Oral lavage samples from the owners were
obtained by rinsing the mouth for 30 s with phosphate-
buffered saline (in group 1 and group 2). In addition, in
group 2 subgingival paper point samples were obtained
from the owners (.02/025, Dentsply, Maillefer, Montigny
le Bretonneux, France). The paper point was inserted for
10 s in the subgingival space of a first molar and
subsequently transferred to 1.5 mL RTF. After collection,
the samples were transferred immediately to the
laboratory of Oral Microbiology of the Academic Centre
for Dentistry Amsterdam (ACTA), The Netherlands, for
further processing.
2.2. Isolation and detection of P. gulae/P. gingivalis and T.
forsythia
After sampling, 1.3 mL of the dental plaque samples
was stored at !80 8C until polymerase chain reaction (PCR)
was performed; two 100-
m
L samples were used for the
culture and isolation procedures. The latter samples were
tenfold serially diluted in RTF and aliquots of 100
m
L were
used to inoculate 5% sheep blood agar plates (Oxoid no. 2,
Basingstoke, UK) supplemented with hemin (5 mg/L) and
menadione (1 mg/L). Plates were incubated anaerobically
(BBL Gaspack Anaerobic System, Beckton, Dickinson and
Co., Sparks, MD, USA) at 37 8C up to 14 days. Two to four
colonies of suspected Porphyromonas spp. (average of 3.9)
and Tannerella spp. (average of 3.2) per plate were streaked
to purity and identified to species level. Identification of
the isolates was performed based on Gram-staining,
typical colony morphology, and the biochemical and
enzymatic profile in the API 32A system (Biomerieux, La
Balme Les Grottes, France). Additional tests included
detection of trypsin-like activity based on the degradation
of benzoyl-
DL
-arginine-2-naphthylamide (BANA) (Sigma,
St Louis, MO, USA) (Slots, 1981; van Winkelhoff et al.,
1988). Differentiation between P. gulae and P. gingivalis
was based on catalase activity, detected with 3% H
2
O
2
(Fournier et al., 2001). After primary culturing, pure
cultures of T. forsythia were prepared on Trypticase Soy
agar plates (Beckton Dickinson Microbiology Systems,
Cockeysville, MD, USA) supplemented with 5% horse blood,
hemin (5 mg/L), menadione (1 mg/L), and N-acetyl-mur-
aminic acid (10 mg/L) (TSNAM plates) (van der Reijden
et al., 2006). Identical T. forsythia isolates, based on AFLP
analysis, were tested for catalase activity. Isolates were
stored on glass beads (Protect Bacterial Preservers, TSC
Ltd., Lancashire, UK) at !80 8C until further processing.
2.3. DNA extraction
Pure bacterial colonies from TSNAM agar plates were
resuspended in 100
m
L Tris/EDTA buffer (10 mmol/L Tris–
HCl, 1 mmol/L EDTA, pH 8.0) buffer and adjusted to match
a turbidity of 0.5 McFarland. The suspension was then
incubated with 10 mg/mL lysozyme at 37 8C for at least 1 h.
Thereafter DNA was isolated with the MagNA Pure DNA
Isolation kit III (Roche, Molecular Diagnostics, Almere, The
Netherlands). Bacteria were lysed by incubation with
proteinase K (20 mg/mL) for 1 h at 56 8C, and DNA was
eluted in 100
m
L elution buffer (Roche) and stored at
!20 8C until needed. DNA extraction for determination of
P. gingivalis and T. forsythia by real-time PCR was
performed similarly with 100
m
L of the dental plaque
suspension.
2.4. Real-time PCR
Amplification of species-specific 16S rDNA sequences
was performed in a 20-
m
L reaction mixture containing
10
m
L of 2x LightCycler
1
480 Probes Master (Roche),
300 nM of species-specific primers, 100 nM of a species-
specific probe (both from TIB MolBiol GmbH, Berlin,
Germany; modified by a FAM reporter and a BHQ-2
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quencher), and 5
m
L of DNA purified from the plaque
samples. The sequences of species-specific primers and
probes are described by Boutaga et al. (2003, 2005). The
target sequences of the PCR could not distinguish between
P. gulae and P. gingivalis by BLAST comparison. Five
milliliters of the DNA extracted from P. gingivalis strain
HG66 (W83) or T. forsythia (ATCC 43037) was used to
prepare a standard curve as positive control; 5
m
L of sterile
H
2
O was used as a non-template control.
The samples were subjected to an initial single
incubation at 95 8C for 10 min, followed by 45 cycles at
95 8C for 10 s and 60 8C for 20 s. DNA amplification was
monitored by quantitatively analyzing the fluorescence
emission (LightCycler 480, software version 1.5, Roche)
during each annealing-extension step.
2.5. Amplified fragment length polymorphism (AFLP) typing
T. forsythia strains were genotyped using ALFP. DNA was
restricted with two enzymes, and two adapters were ligated
to the restriction sites simultaneously. The reaction
mixtures consisted of 10ng DNA, of 1"T4 DNA ligase
buffer, 0.05 mol/L NaCl, 1 mg/mL bovine serum albumin,
5 pmol of the PST-I adapter (Eurogentec, Seraing, Belgium),
20 pmol of the MSE-I adapter (Eurogentec), and 80 U of T4
DNA ligase, 0.5 U of PstI, 2 U of MseI. All restriction enzymes
and ligase are fromNew England Biolabs (Beverly, MA, USA).
After incubation at 37 8C for 3 h, the mixtures were diluted
1:20 in 0.1"Tris/EDTAbuffer pH 8.0. For amplification of the
restrictionfragments, 5
m
L of the dilutedmixture was added
to 5
m
L of PCR mixture, which consisted of 1"PCR buffer
(Applied Biosystems, Foster City, CA, USA), 2mmol/L dNTPs
(Promega Benelux, Leiden, The Netherlands), 15 mmol/L
MgCl
2
(Applied Biosystems), 1 U AmpliTaq DNApolymerase
(Applied Biosystems), 20 ng of PST-0 primer (FAM-5
0
-
GACTGCGTACATGCAG-3
0
), and 60 ng of MSE-C primer (5
0
-
GATGAGTCCTGAGTAAC-3
0
). PST-0 was fluorescently labeled
with carboxyfluorescein (Eurogentec, Maastricht, The Neth-
erlands). Amplification was carried out in a GeneAmp PCR
System 9700 (Applied Biosystems) under the following
conditions: 2 min at 72 8C, followed by 12 cycles of 30s at
94 8C, 30 s at 65 8C with a decreasing gradient of 0.7 8C per
cycle, and 1 min at 72 8C followed by 23 cycles of 30 s at
94 8C, 30 s at 56 8C and 1 min at 72 8C, ended by a single
extension at 72 8C for 1 min.
Before analysis on an ABI Prism 3100 capillary
electrophoresis sequencing system (Applied Biosystems),
2.5
m
L of each PCR product was added to 22
m
L deionized
formamide and 0.5
m
L GeneScan-500 ROX standard
(Applied Biosystems). Data were analyzed with the
BioNumerics software package, version 3.0 (Applied
Maths, Sint-Martens-Latem, Belgium). Similarity coeffi-
cients were calculated with Pearson’s correlation, and
dendrograms were obtained by the unweighted pair group
method using arithmetic averages (upgma) clustering.
2.6. 16S rRNA sequencing
Confirmation of the identification of identical T. forsythia
strains from cat and owners was done by sequence analysis
of a 502-bp region of the 16S rRNA gene with the primers of
the MicroSeq Microbial Identification system (Hall et al.,
2003). Double-ended sequencing reactions of the 16S rRNA
sequence were carried out with BigDye 3.1 terminator
chemistry (Applied Biosystems, Nieuwerkerk a/d IJssel, The
Netherlands) and resolved with an ABI Prism 3130 Genetic
Analyzer (Applied Biosystems).
2.7. Statistical analysis
The differences in the ages of the cats, CFU, and total
amounts of P. gulae,P. gingivalis, and T. forsythiabetween the
two groups of cats and owners as well as differences in CFU
and total amounts of P. gingivalis and T. forsythia between
oral lavage samples and subgingival samples were com-
pared by a Mann–Whitney Test (SPSS 15.0 for Windows
(SPSS Inc., Chicago, IL)). Significance was defined as p#0.05.
3. Results
Group 1 (control group) consisted of 62 cats (51
owners) with a mean age of 1.7 years (SEM 0.2, range 0.3–
6.8). Group 2 (patient group) consisted of 8 cats (7 owners)
with a mean age of 8.8 years (SEM 2.3, range 1.9–20.3). The
age difference between the two groups was statistically
significant (p<0.001).
3.1. Prevalence of periodontal pathogens
In the healthy cats (group 1), Porphyromonas was
detected by cell culture in 90% of samples (56 of 62 cats)
(Fig. 1, (1)),and all these typical Porphyromonas-like colonies
(n= 219) tested positive for catalase reactivity (mean 3.9
colonies per cat). The prevalence of Porphyromonas
gingivalis/gulae was 97% (60 of 62 cats) when analyzed
by PCR. The prevalence of T. forsythia in cats was 89% (55 of
62 cats) when analyzed by culture(Fig. 1, (1)) and 98% (61 of
62 cats) when analyzed by PCR. A total of 174 colonies of
T. forsythia (mean 3.2 isolates per cat) were streaked to
purity. In the cat owners, Porphyromonas spp. were not
detected in oral lavage samples analyzed by cell culture but
were detectedin 96% of samples (49 of 51 samples) analyzed
by PCR (Fig. 1, (1)). The prevalence of T. forsythia by culture
was 49% (25/51) and 80% (41/51) by PCR detection.
In the cats with periodontal disease and their owners
(group 2), catalase positive Porphyromonas spp. and T.
forsythia were detected by cell culture in all samples taken
from the cats (8 of 8 cats) (Fig. 1, (2)). Of the seven oral
lavage and seven subgingival samples of plaque collected
from the owners, Porphyromonas was detected in 57% (4 of
7 owners) of the oral lavage samples and in 100% (7 of 7
owners) of the subgingival samples (Fig. 1, (2)). All
Porphyromonas isolates were catalase negative. T. forsythia
was detected by culture in 86% (6 of 7 owners) of the oral
lavage and subgingival samples (Fig. 1, (2)). The positive
culture results for Porphyromonas and T. forsythia in cats
and owners were confirmed by PCR.
3.2. Relative frequencies
The mean total CFU in the plaque samples from cats
with periodontal disease (group 2) was significantly higher
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than that in the plaque samples from the healthy cats
(1.08E+08 vs 3.06E+07, p= 0.01). P. gulae accounted for
35.8% and 19.8% of the total CFU in cats with periodontal
disease (group 2) and healthy cats, respectively (p= 0.03),
and T. forsythia for 3.7% and 1.9%, respectively (p= 0.01)
(Table 1).
The mean total CFU was higher in oral lavage samples
from the owners of healthy cats than in similar samples
from the owners of cats with periodontal disease
(1.26E+07 vs 4.09E+06, p= 0.003). Moreover, the mean
total CFU for subgingival plaque samples was higher than
that for oral lavage samples taken from the owners of cats
with periodontal disease (2.48E+07 vs 4.09E+06, p= 0.006).
P. gingivalis was not recovered from the owners of healthy
cats but was detected in 4.6% of the oral lavage samples
and in 1.5% of the subgingival plaque samples taken from
the owners of cats with periodontal disease (the difference
between the two types of sample was not significantly
different). The relative frequency of T. forsythia in the oral
lavage samples from the owners of cats without or with
periodontal disease was 0.1% and 0.5%, respectively (not
significant). In the owners of cats with periodontal disease,
the relative frequency of T. forsythia was 2.4% and 0.5% in
the subgingival plaque samples and oral lavage samples,
respectively (p= 0.01) (Table 2). T.forsythia accounted for a
greater proportion of the mean number of CFU in cats with
that in cats without periodontal disease (Fig. 1).
Genotyping of T. forsythia isolated from samples from
cats with periodontal disease and their owners (six
combinations cat/owner) identified six identical isolates
of T. forsythia in one cat/owner combination (Fig. 2).
Sequence analyses of a maximal 502-bp 16S rDNA
fragment confirmed the T. forsythia identity of the isolates
from the cat and its owner by comparison to known T.
forsythia gene sequences from the sequence database of
the National Center of Biotechnology Information (NCBI,
Bethesda, MD). The 16S rDNA sequences of strains T.
forsythia 75 mucosa 3, T. forsythia 75 mucosa 1 and T.
forsythia 75 cat 1, are available at the NCBI sequence
database by accession numbers: GU350448, GU350449,
and GU350450 respectively. The T. forsythia isolates that
were identical in cat and owner were all catalase positive.
4. Discussion
We found the prevalence of P. gulae to be 90% in healthy
cats and 100% in cats with visible signs of periodontal
disease. For comparison, the prevalence of P. gingivalis in
healthy persons is about 10%, compared with 43–69% in
persons with periodontitis (Boutaga et al., 2003; van
Winkelhoff et al., 2002). The much lower prevalence of
Porphyromonas spp.in healthy humans might reflect a
Fig. 1. Prevalence of Porphyromonas gulae,Porphyromonas gingivalis, and
Tannerella forsythia in healthy cats and their owners (group 1, (1)) and in
cats with periodontal disease and their owners (group 2, (2)) (by culture).
Cats were sampled by using a cotton wool stick (subgingival sample).
Owners were sampled by oral lavage (OL) (group 1 and 2), and subgingival
by using a paperpoint (Sub) (group 2). NR: not recovered.
Table 1
Mean age (
$SEM), mCFU (mean colony forming units) ($SEM) and % relative
to CFU of Porphyromonas gulae,Porphyromonas gingivalis and Tannerella
forsythia isolated from cats.
Cats
(subgingival plaque sample)
Group 1 (n= 62) Group 2 (n= 8)
Mean age 1.7 years (
$0.2, 0.3–6.8)
8.8 years (
$2.3, 1.9–20.3)
***
mCFU 3.06E+07 (
$7.48E+04)
1.08E+08 (
$2.07E+04)
**
%P. gulae 19.8 35.8
*
%P. gingivalis 00
%T. forsythia 1.9 3.7
**
*
p#0.05.
**
p#0.01.
***
p#0.001.
Table 2
Mean age (
$SEM), mCFU (mean colony forming units) ($SEM) and % relative
to CFU of Porphyromonas gulae,Porphyromonas gingivalis and Tannerella
forsythia isolated from owners.
Owners
(oral lavage or subgingival sample)
Group 1 (n= 51) Group 2 (n= 7)
mCFU 1.26E+07
(
$1.52E+06) (OL)**
4.09E+06
(
$7.97E+05) (OL)
2.48E+07
($1.20E+07) (Sub)**
%P. gulae NR 0
%P. gingivalis OL NR 4.6
%P. gingivalis Sub ND 1.5
%T. forsythia OL 0.1 0.5
%T. forsythia Sub ND 2.4**
OL: oral lavage sample, Sub: subgingival plaque sample, NR: not
recovered and ND: not determined. *p#0.05, **p#0.01, and ***p#0.001.
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higher virulence of P. gingivalis in humans relative to the
virulence of P. gulae in healthy cats (Fournier and Mouton,
1993). P. gulae in cats might be an opportunistic pathogen
that could initiate an endogenous infection. Although P.
gingivalis isolates were not recovered from the owners of
healthy cats, isolates were recovered from the owners of
cats with periodontal diseases. All Porphyromonas-like
isolates from plaque samples from cats were BANA positive
and catalase positive, whereas those recovered from
owners were BANA positive and catalase negative. This
is in line with earlier findings for cats and humans
(Fournier and Mouton, 1993; Fournier et al., 2001;
Hudspeth et al., 1999). On the basis of this difference in
catalase activity, we conclude that the Porphyromonas
isolates from cats and their owners were not homologous
and that Porphyromonas was not transmitted from cats to
owners.
Earlier studies reported a prevalence of subgingival T.
forsythia of up to 91% in patients with periodontitis and of
up to 48% in control subjects (van Winkelhoff et al., 2002).
We found the prevalence of T. forsythia to be high in plaque
samples from both healthy cats (89% on culture, 98% with
PCR) and cats with periodontal disease (100% by both
methods). Moreover, T. forsythia was cultured in 6 of 7
(86%) plaque samples from the owners of these cats. While
the prevalence of T. forsythia is comparable in cats with
periodontal disease and humans with periodontitis (100%
and 91%, respectively), the prevalence of T. forsythia in
healthy cats was much higher than that in humans without
periodontitis (89% vs 48%, respectively) (van Winkelhoff
et al., 2002). The relative load of T. forsythia (expressed
relative to the mean total CFU) was significantly higher in
cats with periodontitis than in cats without periodontal
disease, which suggests that T. forsythia is associated with
periodontal disease in cats as well as humans since it is
known that the relative frequency of T. forsythia is a
stronger marker of periodontal disease than the prevalence
only (Fujise et al., 2002; van Winkelhoff et al., 2002).
Though cats in group 1 had a significantly lower age
than cats in group 2, the prevalence of Porphyromonas and
T. forsythia isolates was high in both groups. We therefore
assume that age was not a confounding factor in this study.
Owing to overgrowth of the primary cultures and the
subsequent loss of pure subcultures in group 1 we could
not compare the homology of isolates from healthy cats
and their owners. However, we were able to analyze the
clonality of T. forsythia isolates from six owner/cat
combinations for the cats with periodontal disease. We
selected up to four randomly chosen colonies from each
individual. Therefore it is possible that identical clones
between cat and owners were missed and the probability
of transmission could even be higher in practice. Statistical
modeling indicated that the probability of isolating a
representative part of the microflora was 50% when
isolating four colonies with a confidence level of 95%
(Loos et al., 1992). With an average number of P. gingivalis-
like isolates of 3.9, the probability of missing an identical
clone was approximately 50%. For T. forsythia the prob-
ability would have been even higher, up to 60%. Duplicate
AFLP analyses revealed that in one owner/cat couple six
identical isolates of T. forsythia could be isolated.
Identical isolates of T. forsythia have not been described
before in pets and their owners. The finding of two
identical isolates (based on AFLP typing) suggests that
transmission between cat and owner or vice versa had
occurred. Because of the high prevalence of T. forsythia in
cats in both groups (89% and 100%, respectively) we
hypothesize that transmission from cat to owner is most
likely and that cats might be a reservoir for T. forsythia. This
was confirmed by the positive catalase activity of the
identical T. forsythia isolates in the cat and its owner,
because human T. forsythia strains are catalase negative
(Hudspeth et al., 1999). However, it is possible that there
was a common source of the identical T. forsythia isolates.
We realize that we only found one owner/cat couple with
identical T. forsythia isolates. To further underpin our
suggestion of transmission from cat to owner, a larger trail
should be designed.
The transmission of bacteria depends on many factors
such as the source of infection, number of microorganisms
that are shed, route of infection, genetic factors of the
microorganisms, frequency of contact between infected
and susceptible individuals, and survival of the micro-
organism in the environment (Asikainen et al., 1997; von
Fig. 2. Amplified fragment length polymorphism (AFLP) analyzes for one owner/pet couple showing six identical isolates of T. forsythia derived from the cat
(cat) and its owner (mucosa) and five identical isolates with a different banding pattern from the mucosa and subgingival plaque from the owner.
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Please cite this article in press as: Booij-Vrieling, H.E., et al., Comparison of periodontal pathogens between cats and their
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Troil-Linden et al., 1996). While the transmission of
periodontal bacteria among humans has been described
(Asikainen et al., 1997; Petit et al., 1993; von Troil-Linden
et al., 1996), the specific transmission of P. gingivalis
between different species, i.e. humans and cats, has not
been observed. Yet the transmission of oral bacteria is
conceivable given the fact that many cat owners allow
their pets to eat from their plates, lick their faces, and sleep
on their pillows (Holbrook, 2008).
A possible limitation of our study, and one that could
explain why P. gingivalis was not detected in the owners of
healthy cats, is that our methodology was not sensitive
enough. For example, the recovery of P. gingivalis is lower
with oral lavage (Boutaga et al., 2007) than with
subgingival paper point sampling, a method used for the
owners of cats with periodontal diseases in addition to oral
lavage. While PCR can be used to detect periodontal
pathogens in patients with periodontal disease, there was a
large difference in mean recovery rate between subgingi-
val plaque samples and oral lavage samples (48% vs 10%)
(Boutaga et al., 2007). Although study approval was
dependent on the use of minimally invasive methods,
least invasive method, i.e. oral lavage samples from owners
and using cotton–wool stick in a non-anesthetized cat, it
can be questioned whether oral sampling is suitable for
sampling human subjects with an unknown periodontal
status.
A larger study with more cats and owners is needed to
establish whether oral pathogens are transmitted from pet
cats to their owners. Until then, it would be sensible to bear
in mind that cats, and perhaps other pets, might harbor
periodontal pathogens.
Acknowledgment
We thank Dr J.E.C. Sykes for revision of English
language, grammar, and style.
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owners. Vet. Microbiol. (2010), doi:10.1016/j.vetmic.2009.12.046