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Short communication: Distribution of Porphyromonas gulae fimA genotypes in oral specimens from dogs with mitral regurgitation
Research in Veterinary Science 102 (2015) 49–52
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/yrvsc
Short communication: Distribution of Porphyromonas gulae fimA genotypes in oral specimens from dogs with mitral regurgitation Mitsuyuki Shirai a,1, Ryota Nomura b,⁎,1, Yukio Kato c, Masaru Murakami d, Chihiro Kondo a, Soraaki Takahashi a, Yoshie Yamasaki e, Michiyo Matsumoto-Nakano e, Nobuaki Arai f, Hidemi Yasuda f, Kazuhiko Nakano b, Fumitoshi Asai a a
Department of Pharmacology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japan Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka 565–0871, Japan c Department of Veterinary Public Health II, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japan d Department of Molecular Biology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japan e Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700–8556, Japan f Yasuda Veterinary Clinic, Meguro, Tokyo 152–0034, Japan b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 8 July 2015 Accepted 13 July 2015 Available online xxxx Keywords: fimbriae mitral regurgitation periodontal disease Porphyromonas gulae
a b s t r a c t Porphyromonas gulae, a suspected pathogen for periodontal disease in dogs, possesses approximately 41-kDa fimbriae (FimA) that are encoded by the fimA gene. In the present study, the association of fimA genotypes with mitral regurgitation (MR) was investigated. Twenty-five dogs diagnosed with MR (age range 6–13 years old, average 10.8 years) and 32 healthy dogs (8–15 years old, average 10.8 years) were selected at the participating clinics in a consecutive manner during the same time period. Oral swab specimens were collected from the dogs and bacterial DNA was extracted, then polymerase chain reaction analysis was performed using primers specific for each fimA genotype, with the dominant genotype determined. The rate for genotype C dominant specimens was 48.0% in the MR group, which was significantly higher than that in the control group (18.8%) (P b0.05). These results suggest that P. gulae fimA genotype C is associated with MR. © 2015 Elsevier Ltd. All rights reserved.
Periodontal disease, including gingivitis and periodontitis, is commonly found in dogs, with prevalence rates ranging from 5070% (Harvey and Emily, 1993). Gingivitis involves inflammation of the gingiva, whereas periodontitis is a more progressive form of periodontal disease that involves the loss of tooth supporting tissues (i.e., periodontal ligament, cementum, alveolar bone). Proper periodontal therapy can reduce gingival bleeding, tooth mobility, and, in some cases, sophisticated methods can be utilized to regenerate lost tissues. The distribution of bacterial species related to periodontal disease has been investigated using oral swab specimens taken from dogs, with Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, one of the species frequently detected (Kato et al., 2011). The cell surface component Fimbrillin (FimA), a 41-kDa fimbriae subunit, of P. gulae has also been characterized and shown to be a possible factor related to periodontal disease in dogs ⁎ Corresponding author at: Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, 1–8 Yamada-oka, Suita, Osaka 565–0871, Japan. E-mail addresses: [email protected] (M. Shirai), [email protected] (R. Nomura), [email protected] (Y. Kato), [email protected] (M. Murakami), [email protected] (C. Kondo), [email protected] (S. Takahashi), [email protected] (Y. Yamasaki), [email protected] (M. Matsumoto-Nakano), [email protected] (N. Arai), [email protected] (H. Yasuda), [email protected] (K. Nakano), [email protected] (F. Asai). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.rvsc.2015.07.009 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
(Hamada et al., 2008). In our recent study, FimA of P. gulae was classified into 3 major genotypes; A, B, and C, with type C shown to have a high virulence for periodontal disease, followed by B and A (Nomura et al., 2012; Yamasaki et al., 2012). The association of periodontal disease with systemic diseases has recently been gaining attention (Pavlica et al., 2008; Rawlinson et al., 2011). Mitral regurgitation (MR) caused by myxomatous mitral valve disease is one of the most common types of cardiovascular diseases occurring in dogs (Serfass et al., 2006; Atkins et al., 2009) and characterized by chronic progression, with the condition worsening from mild to severe over time (Borgarelli et al., 2008). A few reports have shown an association of periodontal disease with MR in dogs (Glickman et al., 2009). In the present study, oral specimens from dogs diagnosed with MR were analyzed to investigate the association of the disease with the presence of P. gulae and genotypes of fimA encoding FimA. Twenty-five dogs diagnosed with MR and treated at veterinary clinics in Tokyo in 2012 and 2013 were consecutively enrolled (MR group). In addition, 36 dogs older than 6 years old and without periodontitis, MR, diabetes mellitus, or other relevant clinical history, were selected as Control group. Among them, we removed the 4 larger breed dogs that weighed more than 20 kilograms and used the remaining 32 as controls. The study protocol was approved by the Animal Research Committee of Azabu University (No. 130307–3). Prior to
50
M. Shirai et al. / Research in Veterinary Science 102 (2015) 49–52
sample collection, the owners were informed of the purpose of the study and gave approval for their participation. None of the dogs or their owners received antibiotic treatment within the previous 3 months prior to collection. Oral specimens were obtained from supraand sub-gingival areas of the left and/or right upper fourth premolar and/or lower first molar using Seed-SwabR g-1 or − 2 swabs (Eiken Chemical, Tokyo, Japan), then bacterial DNA was extracted using a method previously described (Kato et al., 2011). According to the ACVIM (American College of Veterinary Internal Medicine) functional classification (Atkins et al., 2009), the 25 dogs in the MR group (18 males, 7 females; 10.8 ± 0.3 year old) were diagnosed based on physical examination, thoracic radiography, electrocardiography, and echocardiography findings, as follows; Stage A (n = 0), Stage B1 (n = 4), Stage B2 (n = 10), Stage C (n = 7), and Stage D (n = 4). In addition, the severity of MR has been reported to be associated with murmur intensity and audibility (Ljungvall et al., 2009). Thus, these dogs were also evaluated using the Levine classification, which is a scoring system used to classify the intensity or loudness of a heart murmur (Freeman and Levine, 1933), as follows; I/VI (n = 0), II/VI (n = 8), II-III/VI (n = 2), III/VI (n = 7), IV/VI (n = 7), V/VI (n = 0), and VI/VI (n = 1). Breeds represented in the MR group are listed in Supplementary Table 1. The 32 healthy dogs in the control group (17 males, 15 females, 10.8 ± 0.4 years old) had no abnormal cardiac conditions shown in physical examination, thoracic radiography, electrocardiography, and echocardiography findings. Breeds represented in the control group are listed in Supplementary Table 2. Statistical analyses were performed using the computational software packages StatView 5.0e and Prism 4f. Fisher’s protected leastsignificant difference test was utilized to compare the detection frequencies of each fimA genotype for each gingival condition. Odds ratio (OR) and 95% confidence interval (CI95) values were calculated to determine the significance of the association of each fimA genotype in the dogs with each periodontal condition. A P value of b 0.05 was considered to be statistically significant. We evaluated calculus coverage, and gingival and periodontal scores based on methods previously described, with some modifications (Warrick and Gorrel, 1997; Harvey and Emily, 1993; Löe, 1967; Wolf et al., 2005). For each dog, 22 teeth were scored according to a previously described technique (Kate and Cecilia, 1999). Dental calculus
accumulation was scored visually, as follows; (3) more than half of the area of the portion of the tooth from the gingival margin to the tip of the crown, (2) less than half of that area, (1) slight, only at gingival margin, and (0) none. Gingival scores were evaluated visually as follows; (3) prominent inflammation (intensive redness with gingival swelling) identified throughout entire area, (2) prominent inflammation (intensive redness with gingival swelling) identified in limited area, (1) slight inflammation (slight redness without gingival swelling) identified only in gingival margin, and (0) no significant findings. Periodontal scores were evaluated based on visual examination and palpation, as follows; (3) tooth mobility with severe attachment loss, (2) attachment loss identified in more than one quarter of the portion of the tooth but no tooth mobility, (1) attachment loss identified in less than one quarter of that portion, and (0) no significant findings. Table 1 summarizes the oral conditions in the MR and Control groups. There were no significant differences in regard to dental calculus and gingival scores. As for periodontal scoring, the MR group had higher scores than the control group, though there were no significant differences between the groups. Detection of P. gulae and fimA genotypes in addition to Tannerella forsythia and Campylobacter rectus were performed by PCR using speciesspecific sets of primers (Kato et al., 2011; Nomura et al., 2012; Yamasaki et al., 2012). PCR amplification was performed in a total volume of 20 μl with 1 μl of bacterial DNA using Ex Taq polymerase (Takara Bio, Shiga, Japan) with the following cycling parameters: initial denaturation at 95 °C for 4 minutes and then 30 cycles consisting of 95 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, with a final extension at 72 °C for 7 minutes. Specificity with this method was shown in our previous study, with the minimum detection level reported to be 10–100 cells (Nomura et al., 2012; Yamasaki et al., 2012). The dominant genotypes were determined using samples with multiple fimA genotypes detected. PCR was performed with titrated DNA samples (1/10, 1/100, 1/1000) from each specimen. The dominant genotype was determined as that with the lowest detection limit. When the detection limits were similar for at least two genotypes, the results were described as no dominancy. Fig. 1A shows the results of detection using the PCR system with genomic DNA extracted from titrated cultures of representative strains of each type. The lowest numbers of tested strains for identification were the same for all 3 fimA genotypes. Fig. 1B, C, and D show
Table 1 Oral conditions and distribution frequency of P. gulae and its fimA genotypes in oral specimens in MR and Control groups. MR (n = 25)
Control (n = 32)
Statistical analysis
10.8 ± 0.3 (6–13)
10.8 ± 0.4 (8–15)
NS b
5.7 ± 0.8 (1.6-14.6)
5.7 ± 0.7 (1.6-18.8)
NS b
Oral condition Dental calculus score (mean ± SE a) Gingival score (mean ± SE a) Periodontal score(mean ± SE a)
1.7 ± 0.1 0.8 ± 0.2 0.6 ± 0.2
1.8 ± 0.2 0.8 ± 0.1 0.3 ± 0.1
NS b NS b NS b
PCR detection P. gulae-positive A-dominant
24 (96.0%) 5 (20.0%)
30 (93.8%) 17 (53.1%)
B-dominant C-dominant
3 (12.0%) 12 (48.0%)
4 (12.5%) 6 (18.8%)
A=C A=B=C Untypeable
2 (8.0%) 0 (0%) 2 (8.0%)
1 (3.1%) 1 (3.1%) 1 (3.1%)
NS b P = 0.0143 OR: 0.22 (CI95: 0.07-0.73) NS b P = 0.0240 OR c: 4.00 (CI95 d: 1.22-13.08) NS b NS b NS b
Age in years Mean ± SE a (range) Body weight in kg Mean ± SE a (range)
a b c d
SE, standard error. NS, not significant. OR, odds ratio. CI95, 95% confidence interval.
M. Shirai et al. / Research in Veterinary Science 102 (2015) 49–52
A
M ATCC 51700 (Type A)
D077 (Type B)
D049 (Type C)
M
105 104 103 102 10 105 104 103 102 10 105 104 103 102 10
B
M
Primer for Type A 1
C
M
10-1 10-2 10-3
Primer for Type A 1
10-1 10-2 10-3
Primer for Type B 1
10-1 10-2 10-3
Primer for Type B 1
10-1 10-2 10-3
Primer for Type C 1
Primer for Type C 1
M
10-1 10-2 10-3
M
10-1 10-2 10-3
51
such as type II and IV, indicated that they are highly virulent for periodontitis (Nakano et al., 2008). In addition, it is interesting that analysis of the evolutionary relationship revealed that type C fimA of P. gulae is close to type IV fimA of P. gingivalis (Yamasaki et al., 2012). Taken together, these results indicate the possibility of an association of periodontal disease with MR. In summary, the present results indicate a possible association between the presence of a specific genotype of fimA of P. gulae with MR. In addition, our novel method used to specify the dominant genotype of P. gulae in the specimens obtained may enable identification of subjects at risk and in need of preventive approaches. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.rvsc.2015.07.009. Conflicts of interest The authors declare that they have no competing interests. Acknowledgements
D
M
Primer for Type A 1
10-1
10-2
10-3
Primer for Type B 1
10-1
10-2
10-3
Primer for Type C 1
10-1
10-2
M
10-3
This study was supported by Grants-in-Aid for Scientific Research for Challenging Exploratory Research (No. 23658256 and 25670873) from the Japan Society for Promotion of Science, the Promotion and Mutual Aid Corporation of Private Schools of Japan, and a Grant-in-Aid for Matching Fund Subsidy for Private Universities. We also thank Prof. Howard K. Kuramitsu (State University of New York at Buffalo) for editing the manuscript. References
Fig. 1. Methodology designed to identify the dominant genotype fimA of P. gulae, using polymerase chain reaction (PCR). A. Comparisons of PCR products using diluted genomic DNA extracted from P. gulae strains with each fimA genotype. M; molecular size marker (100-bp DNA ladder). B, C, D. Representative results obtained with clinical specimens using PCR system constructed for the present study.
representative results obtained using titrated bacterial DNA samples from the original concentration to 10−3 in the PCR system constructed for the present study. Fig. 1B presents a representative specimen, in which positive bands were observed in the original and 10−1 dilution samples for type B and the original dilution for type C, while positive bands were observed when applying the original, 10−1, 10−2, and even 10−3 dilution samples for type A detection. Thus, this specimen was classified as “type A-dominant.” Fig. 1C and D present representative results of other “type B-dominant” and “type C-dominant” specimens, respectively. The detection rates for both T. forsythia and C. rectus were not significantly between the Control and MR groups (Supplementary Tables 1 and 2). Table 1 summarizes the distribution frequencies of P. gulae and its fimA genotypes in oral specimens obtained from the MR and Control groups. P. gulae was detected in nearly all specimens from both groups. On the other hand, there was a significantly larger number of type A-dominant specimens in the Control group (P b0.05) and a significantly larger number of type C-dominant specimens in the MR group (P b 0.05). The odds ratios for type-A and type-C detection in the MR group were 0.22 and 4.00, respectively. This is the first known study to analyze oral specimens obtained from dogs with MR in comparison to those from healthy dogs, though the number of subjects, variety of breeds, and clinical assessment parameters were limited. We previously reported that genomic DNA specific for P. gingivalis was detected in approximately 10% of heart valves and 20-30% of atheromatous plaque specimens, indicating a possible association between periodontal disease and cardiovascular diseases (Nakano et al., 2009). That notion is also supported by previous evidence showing that frequent detection of fimA genotype organisms,
Atkins, C., Bonagura, J., Ettinger, S., Fox, P., Gordon, S., Haggstrom, J., Hamlin, R., Keene, B., Luis-Fuentes, V., Stepien, R., 2009. Guidelines for the diagnosis and treatment of canine chronic valvular heart disease. J. Vet. Intern. Med. 23, 1142–1150. Borgarelli, M., Savarino, P., Crosara, S., Santilli, R.A., Chiavegato, D., Poggi, M., Bellino, C., La Rosa, G., Zanatta, R., Haggstrom, J., Tarducci, A., 2008. Survival characteristics and prognostic variables of dogs with mitral regurgitation attributable to myxomatous valve disease. J. Vet. Intern. Med. 22, 120–128. Freeman, A.R., Levine, S.A., 1933. Clinical significance of systolic murmurs: Study of 1000 consecutive “noncardiac” cases. Ann. Intern. Med. 6, 1371–1379. Glickman, L.T., Glickman, N.W., Moore, G.E., Goldstein, G.S., Lewis, H.B., 2009. Evaluation of the risk of endocarditis and other cardiovascular events on the basis of the severity of periodontal disease in dogs. J. Am. Vet. Med. Assoc. 234, 486–494. Hamada, N., Takahashi, Y., Watanabe, K., Kumada, H., Oishi, Y., Umemoto, T., 2008. Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis. Vet. Microbiol. 128, 108–117. Harvey, C.E., Emily, P.P., 1993. Small Animal Dentistry. 1st ed. Mosby – Year Book Inc, St. Loius, p. 413. Kate, E.I., Cecilia, Gorrel, 1999. Assessing oral health and hygiene in dogs. Waltham Focus 9, 32–33. Kato, Y., Shirai, M., Murakami, M., Mizusawa, T., Hagimoto, A., Wada, K., Nomura, R., Nakano, K., Ooshima, T., Asai, F., 2011. Molecular detection of human periodontal pathogens in oral swab specimens from dogs in Japan. J. Vet. Dent. 28, 84–89. Ljungvall, I., Ahlstrom, C., Höglund, K., Hult, P., Kvart, C., Borgarelli, M., Ask, P., Häggström, J., 2009. Use of signal analysis of heart sounds and murmurs to assess severity of mitral valve regurgitation attribute to myxomatous mitral valve disease in dogs. Am. J. Vet. Res. 70, 604–613. Löe, H., 1967. The gingival index, the plaque index and the retention index systems. J. Periodontol. 38, 610–616. Nakano, K., Inaba, H., Nomura, R., Nemoto, H., Takeuchi, H., Yoshioka, H., Toda, K., Taniguchi, K., Amano, A., Ooshima, T., 2008. Distribution of Porphyromonas gingivalis fimA genotypes in cardiovascular specimens from Japanese patients. Oral Microbiol. Immunol. 23, 170–172. Nakano, K., Nemoto, H., Nomura, R., Inaba, H., Yoshioka, H., Taniguchi, K., Amano, A., Ooshima, T., 2009. Detection of oral bacteria in cardiovascular specimens. Oral Microbiol. Immunol. 24, 64–68. Nomura, R., Shirai, M., Kato, Y., Murakami, M., Nakano, K., Hirai, N., Mizusawa, T., Naka, S., Yamasaki, Y., Matsumoto-Nakano, M., Ooshima, T., Asai, F., 2012. Diversity of Fimbrillin among Porphyromonas gulae clinical isolates from Japanese dogs. J. Vet. Med. Sci. 74, 885–891. Pavlica, Z., Petelin, M., Juntes, P., Erzen, D., Crossley, D.A., Skaleric, U., 2008. Periodontal disease burden and pathological changes in organs of dogs. J. Vet. Dent. 25, 97–105. Rawlinson, J.E., Goldstein, R.E., Reiter, A.M., Attwater, D.Z., Harvey, C.E., 2011. Association of periodontal disease with systemic health indices in dogs and the systemic response to treatment of periodontal disease. J. Am. Vet. Med. Assoc. 238, 601–609. Serfass, P., Chetboul, V., Sampedrano, C.C., Nicolle, A., Benalloul, T., Laforge, H., Gau, C., Hébert, C., Pouchelon, J.L., Tissier, R., 2006. Retrospective study of 942 small-sized
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dogs: Prevalence of left apical systolic heart murmur and left-sided heart failure, critical effects of breed and sex. J. Vet. Cardiol. 8, 11–18. Warrick, J., Gorrel, C.A., 1997. More sensitive method of scoring calculus. Proceedings of the 11th Annual Veterinary Dental Forum, Denver, USA, 30 Oct-2 Nov, pp. 134–136. Wolf, H.F., Rateitschak, E.M., Rateitschak, K.H., Hassel, T.M., 2005. Color atlas of dental medicine: periodontology. 3rd ed. Georg Thieme Verlag, Stuttgart.
Yamasaki, Y., Nomura, R., Nakano, K., Inaba, H., Kuboniwa, M., Hirai, N., Shirai, M., Kato, Y., Murakami, M., Naka, S., Iwai, S., Matsumoto-Nakano, M., Ooshima, T., Amano, A., Asai, F., 2012. Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype. Vet. Microbiol. 28, 196–205.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/yrvsc
Short communication: Distribution of Porphyromonas gulae fimA genotypes in oral specimens from dogs with mitral regurgitation Mitsuyuki Shirai a,1, Ryota Nomura b,⁎,1, Yukio Kato c, Masaru Murakami d, Chihiro Kondo a, Soraaki Takahashi a, Yoshie Yamasaki e, Michiyo Matsumoto-Nakano e, Nobuaki Arai f, Hidemi Yasuda f, Kazuhiko Nakano b, Fumitoshi Asai a a
Department of Pharmacology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japan Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka 565–0871, Japan c Department of Veterinary Public Health II, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japan d Department of Molecular Biology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japan e Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700–8556, Japan f Yasuda Veterinary Clinic, Meguro, Tokyo 152–0034, Japan b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 8 July 2015 Accepted 13 July 2015 Available online xxxx Keywords: fimbriae mitral regurgitation periodontal disease Porphyromonas gulae
a b s t r a c t Porphyromonas gulae, a suspected pathogen for periodontal disease in dogs, possesses approximately 41-kDa fimbriae (FimA) that are encoded by the fimA gene. In the present study, the association of fimA genotypes with mitral regurgitation (MR) was investigated. Twenty-five dogs diagnosed with MR (age range 6–13 years old, average 10.8 years) and 32 healthy dogs (8–15 years old, average 10.8 years) were selected at the participating clinics in a consecutive manner during the same time period. Oral swab specimens were collected from the dogs and bacterial DNA was extracted, then polymerase chain reaction analysis was performed using primers specific for each fimA genotype, with the dominant genotype determined. The rate for genotype C dominant specimens was 48.0% in the MR group, which was significantly higher than that in the control group (18.8%) (P b0.05). These results suggest that P. gulae fimA genotype C is associated with MR. © 2015 Elsevier Ltd. All rights reserved.
Periodontal disease, including gingivitis and periodontitis, is commonly found in dogs, with prevalence rates ranging from 5070% (Harvey and Emily, 1993). Gingivitis involves inflammation of the gingiva, whereas periodontitis is a more progressive form of periodontal disease that involves the loss of tooth supporting tissues (i.e., periodontal ligament, cementum, alveolar bone). Proper periodontal therapy can reduce gingival bleeding, tooth mobility, and, in some cases, sophisticated methods can be utilized to regenerate lost tissues. The distribution of bacterial species related to periodontal disease has been investigated using oral swab specimens taken from dogs, with Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, one of the species frequently detected (Kato et al., 2011). The cell surface component Fimbrillin (FimA), a 41-kDa fimbriae subunit, of P. gulae has also been characterized and shown to be a possible factor related to periodontal disease in dogs ⁎ Corresponding author at: Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, 1–8 Yamada-oka, Suita, Osaka 565–0871, Japan. E-mail addresses: [email protected] (M. Shirai), [email protected] (R. Nomura), [email protected] (Y. Kato), [email protected] (M. Murakami), [email protected] (C. Kondo), [email protected] (S. Takahashi), [email protected] (Y. Yamasaki), [email protected] (M. Matsumoto-Nakano), [email protected] (N. Arai), [email protected] (H. Yasuda), [email protected] (K. Nakano), [email protected] (F. Asai). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.rvsc.2015.07.009 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
(Hamada et al., 2008). In our recent study, FimA of P. gulae was classified into 3 major genotypes; A, B, and C, with type C shown to have a high virulence for periodontal disease, followed by B and A (Nomura et al., 2012; Yamasaki et al., 2012). The association of periodontal disease with systemic diseases has recently been gaining attention (Pavlica et al., 2008; Rawlinson et al., 2011). Mitral regurgitation (MR) caused by myxomatous mitral valve disease is one of the most common types of cardiovascular diseases occurring in dogs (Serfass et al., 2006; Atkins et al., 2009) and characterized by chronic progression, with the condition worsening from mild to severe over time (Borgarelli et al., 2008). A few reports have shown an association of periodontal disease with MR in dogs (Glickman et al., 2009). In the present study, oral specimens from dogs diagnosed with MR were analyzed to investigate the association of the disease with the presence of P. gulae and genotypes of fimA encoding FimA. Twenty-five dogs diagnosed with MR and treated at veterinary clinics in Tokyo in 2012 and 2013 were consecutively enrolled (MR group). In addition, 36 dogs older than 6 years old and without periodontitis, MR, diabetes mellitus, or other relevant clinical history, were selected as Control group. Among them, we removed the 4 larger breed dogs that weighed more than 20 kilograms and used the remaining 32 as controls. The study protocol was approved by the Animal Research Committee of Azabu University (No. 130307–3). Prior to
50
M. Shirai et al. / Research in Veterinary Science 102 (2015) 49–52
sample collection, the owners were informed of the purpose of the study and gave approval for their participation. None of the dogs or their owners received antibiotic treatment within the previous 3 months prior to collection. Oral specimens were obtained from supraand sub-gingival areas of the left and/or right upper fourth premolar and/or lower first molar using Seed-SwabR g-1 or − 2 swabs (Eiken Chemical, Tokyo, Japan), then bacterial DNA was extracted using a method previously described (Kato et al., 2011). According to the ACVIM (American College of Veterinary Internal Medicine) functional classification (Atkins et al., 2009), the 25 dogs in the MR group (18 males, 7 females; 10.8 ± 0.3 year old) were diagnosed based on physical examination, thoracic radiography, electrocardiography, and echocardiography findings, as follows; Stage A (n = 0), Stage B1 (n = 4), Stage B2 (n = 10), Stage C (n = 7), and Stage D (n = 4). In addition, the severity of MR has been reported to be associated with murmur intensity and audibility (Ljungvall et al., 2009). Thus, these dogs were also evaluated using the Levine classification, which is a scoring system used to classify the intensity or loudness of a heart murmur (Freeman and Levine, 1933), as follows; I/VI (n = 0), II/VI (n = 8), II-III/VI (n = 2), III/VI (n = 7), IV/VI (n = 7), V/VI (n = 0), and VI/VI (n = 1). Breeds represented in the MR group are listed in Supplementary Table 1. The 32 healthy dogs in the control group (17 males, 15 females, 10.8 ± 0.4 years old) had no abnormal cardiac conditions shown in physical examination, thoracic radiography, electrocardiography, and echocardiography findings. Breeds represented in the control group are listed in Supplementary Table 2. Statistical analyses were performed using the computational software packages StatView 5.0e and Prism 4f. Fisher’s protected leastsignificant difference test was utilized to compare the detection frequencies of each fimA genotype for each gingival condition. Odds ratio (OR) and 95% confidence interval (CI95) values were calculated to determine the significance of the association of each fimA genotype in the dogs with each periodontal condition. A P value of b 0.05 was considered to be statistically significant. We evaluated calculus coverage, and gingival and periodontal scores based on methods previously described, with some modifications (Warrick and Gorrel, 1997; Harvey and Emily, 1993; Löe, 1967; Wolf et al., 2005). For each dog, 22 teeth were scored according to a previously described technique (Kate and Cecilia, 1999). Dental calculus
accumulation was scored visually, as follows; (3) more than half of the area of the portion of the tooth from the gingival margin to the tip of the crown, (2) less than half of that area, (1) slight, only at gingival margin, and (0) none. Gingival scores were evaluated visually as follows; (3) prominent inflammation (intensive redness with gingival swelling) identified throughout entire area, (2) prominent inflammation (intensive redness with gingival swelling) identified in limited area, (1) slight inflammation (slight redness without gingival swelling) identified only in gingival margin, and (0) no significant findings. Periodontal scores were evaluated based on visual examination and palpation, as follows; (3) tooth mobility with severe attachment loss, (2) attachment loss identified in more than one quarter of the portion of the tooth but no tooth mobility, (1) attachment loss identified in less than one quarter of that portion, and (0) no significant findings. Table 1 summarizes the oral conditions in the MR and Control groups. There were no significant differences in regard to dental calculus and gingival scores. As for periodontal scoring, the MR group had higher scores than the control group, though there were no significant differences between the groups. Detection of P. gulae and fimA genotypes in addition to Tannerella forsythia and Campylobacter rectus were performed by PCR using speciesspecific sets of primers (Kato et al., 2011; Nomura et al., 2012; Yamasaki et al., 2012). PCR amplification was performed in a total volume of 20 μl with 1 μl of bacterial DNA using Ex Taq polymerase (Takara Bio, Shiga, Japan) with the following cycling parameters: initial denaturation at 95 °C for 4 minutes and then 30 cycles consisting of 95 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, with a final extension at 72 °C for 7 minutes. Specificity with this method was shown in our previous study, with the minimum detection level reported to be 10–100 cells (Nomura et al., 2012; Yamasaki et al., 2012). The dominant genotypes were determined using samples with multiple fimA genotypes detected. PCR was performed with titrated DNA samples (1/10, 1/100, 1/1000) from each specimen. The dominant genotype was determined as that with the lowest detection limit. When the detection limits were similar for at least two genotypes, the results were described as no dominancy. Fig. 1A shows the results of detection using the PCR system with genomic DNA extracted from titrated cultures of representative strains of each type. The lowest numbers of tested strains for identification were the same for all 3 fimA genotypes. Fig. 1B, C, and D show
Table 1 Oral conditions and distribution frequency of P. gulae and its fimA genotypes in oral specimens in MR and Control groups. MR (n = 25)
Control (n = 32)
Statistical analysis
10.8 ± 0.3 (6–13)
10.8 ± 0.4 (8–15)
NS b
5.7 ± 0.8 (1.6-14.6)
5.7 ± 0.7 (1.6-18.8)
NS b
Oral condition Dental calculus score (mean ± SE a) Gingival score (mean ± SE a) Periodontal score(mean ± SE a)
1.7 ± 0.1 0.8 ± 0.2 0.6 ± 0.2
1.8 ± 0.2 0.8 ± 0.1 0.3 ± 0.1
NS b NS b NS b
PCR detection P. gulae-positive A-dominant
24 (96.0%) 5 (20.0%)
30 (93.8%) 17 (53.1%)
B-dominant C-dominant
3 (12.0%) 12 (48.0%)
4 (12.5%) 6 (18.8%)
A=C A=B=C Untypeable
2 (8.0%) 0 (0%) 2 (8.0%)
1 (3.1%) 1 (3.1%) 1 (3.1%)
NS b P = 0.0143 OR: 0.22 (CI95: 0.07-0.73) NS b P = 0.0240 OR c: 4.00 (CI95 d: 1.22-13.08) NS b NS b NS b
Age in years Mean ± SE a (range) Body weight in kg Mean ± SE a (range)
a b c d
SE, standard error. NS, not significant. OR, odds ratio. CI95, 95% confidence interval.
M. Shirai et al. / Research in Veterinary Science 102 (2015) 49–52
A
M ATCC 51700 (Type A)
D077 (Type B)
D049 (Type C)
M
105 104 103 102 10 105 104 103 102 10 105 104 103 102 10
B
M
Primer for Type A 1
C
M
10-1 10-2 10-3
Primer for Type A 1
10-1 10-2 10-3
Primer for Type B 1
10-1 10-2 10-3
Primer for Type B 1
10-1 10-2 10-3
Primer for Type C 1
Primer for Type C 1
M
10-1 10-2 10-3
M
10-1 10-2 10-3
51
such as type II and IV, indicated that they are highly virulent for periodontitis (Nakano et al., 2008). In addition, it is interesting that analysis of the evolutionary relationship revealed that type C fimA of P. gulae is close to type IV fimA of P. gingivalis (Yamasaki et al., 2012). Taken together, these results indicate the possibility of an association of periodontal disease with MR. In summary, the present results indicate a possible association between the presence of a specific genotype of fimA of P. gulae with MR. In addition, our novel method used to specify the dominant genotype of P. gulae in the specimens obtained may enable identification of subjects at risk and in need of preventive approaches. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.rvsc.2015.07.009. Conflicts of interest The authors declare that they have no competing interests. Acknowledgements
D
M
Primer for Type A 1
10-1
10-2
10-3
Primer for Type B 1
10-1
10-2
10-3
Primer for Type C 1
10-1
10-2
M
10-3
This study was supported by Grants-in-Aid for Scientific Research for Challenging Exploratory Research (No. 23658256 and 25670873) from the Japan Society for Promotion of Science, the Promotion and Mutual Aid Corporation of Private Schools of Japan, and a Grant-in-Aid for Matching Fund Subsidy for Private Universities. We also thank Prof. Howard K. Kuramitsu (State University of New York at Buffalo) for editing the manuscript. References
Fig. 1. Methodology designed to identify the dominant genotype fimA of P. gulae, using polymerase chain reaction (PCR). A. Comparisons of PCR products using diluted genomic DNA extracted from P. gulae strains with each fimA genotype. M; molecular size marker (100-bp DNA ladder). B, C, D. Representative results obtained with clinical specimens using PCR system constructed for the present study.
representative results obtained using titrated bacterial DNA samples from the original concentration to 10−3 in the PCR system constructed for the present study. Fig. 1B presents a representative specimen, in which positive bands were observed in the original and 10−1 dilution samples for type B and the original dilution for type C, while positive bands were observed when applying the original, 10−1, 10−2, and even 10−3 dilution samples for type A detection. Thus, this specimen was classified as “type A-dominant.” Fig. 1C and D present representative results of other “type B-dominant” and “type C-dominant” specimens, respectively. The detection rates for both T. forsythia and C. rectus were not significantly between the Control and MR groups (Supplementary Tables 1 and 2). Table 1 summarizes the distribution frequencies of P. gulae and its fimA genotypes in oral specimens obtained from the MR and Control groups. P. gulae was detected in nearly all specimens from both groups. On the other hand, there was a significantly larger number of type A-dominant specimens in the Control group (P b0.05) and a significantly larger number of type C-dominant specimens in the MR group (P b 0.05). The odds ratios for type-A and type-C detection in the MR group were 0.22 and 4.00, respectively. This is the first known study to analyze oral specimens obtained from dogs with MR in comparison to those from healthy dogs, though the number of subjects, variety of breeds, and clinical assessment parameters were limited. We previously reported that genomic DNA specific for P. gingivalis was detected in approximately 10% of heart valves and 20-30% of atheromatous plaque specimens, indicating a possible association between periodontal disease and cardiovascular diseases (Nakano et al., 2009). That notion is also supported by previous evidence showing that frequent detection of fimA genotype organisms,
Atkins, C., Bonagura, J., Ettinger, S., Fox, P., Gordon, S., Haggstrom, J., Hamlin, R., Keene, B., Luis-Fuentes, V., Stepien, R., 2009. Guidelines for the diagnosis and treatment of canine chronic valvular heart disease. J. Vet. Intern. Med. 23, 1142–1150. Borgarelli, M., Savarino, P., Crosara, S., Santilli, R.A., Chiavegato, D., Poggi, M., Bellino, C., La Rosa, G., Zanatta, R., Haggstrom, J., Tarducci, A., 2008. Survival characteristics and prognostic variables of dogs with mitral regurgitation attributable to myxomatous valve disease. J. Vet. Intern. Med. 22, 120–128. Freeman, A.R., Levine, S.A., 1933. Clinical significance of systolic murmurs: Study of 1000 consecutive “noncardiac” cases. Ann. Intern. Med. 6, 1371–1379. Glickman, L.T., Glickman, N.W., Moore, G.E., Goldstein, G.S., Lewis, H.B., 2009. Evaluation of the risk of endocarditis and other cardiovascular events on the basis of the severity of periodontal disease in dogs. J. Am. Vet. Med. Assoc. 234, 486–494. Hamada, N., Takahashi, Y., Watanabe, K., Kumada, H., Oishi, Y., Umemoto, T., 2008. Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis. Vet. Microbiol. 128, 108–117. Harvey, C.E., Emily, P.P., 1993. Small Animal Dentistry. 1st ed. Mosby – Year Book Inc, St. Loius, p. 413. Kate, E.I., Cecilia, Gorrel, 1999. Assessing oral health and hygiene in dogs. Waltham Focus 9, 32–33. Kato, Y., Shirai, M., Murakami, M., Mizusawa, T., Hagimoto, A., Wada, K., Nomura, R., Nakano, K., Ooshima, T., Asai, F., 2011. Molecular detection of human periodontal pathogens in oral swab specimens from dogs in Japan. J. Vet. Dent. 28, 84–89. Ljungvall, I., Ahlstrom, C., Höglund, K., Hult, P., Kvart, C., Borgarelli, M., Ask, P., Häggström, J., 2009. Use of signal analysis of heart sounds and murmurs to assess severity of mitral valve regurgitation attribute to myxomatous mitral valve disease in dogs. Am. J. Vet. Res. 70, 604–613. Löe, H., 1967. The gingival index, the plaque index and the retention index systems. J. Periodontol. 38, 610–616. Nakano, K., Inaba, H., Nomura, R., Nemoto, H., Takeuchi, H., Yoshioka, H., Toda, K., Taniguchi, K., Amano, A., Ooshima, T., 2008. Distribution of Porphyromonas gingivalis fimA genotypes in cardiovascular specimens from Japanese patients. Oral Microbiol. Immunol. 23, 170–172. Nakano, K., Nemoto, H., Nomura, R., Inaba, H., Yoshioka, H., Taniguchi, K., Amano, A., Ooshima, T., 2009. Detection of oral bacteria in cardiovascular specimens. Oral Microbiol. Immunol. 24, 64–68. Nomura, R., Shirai, M., Kato, Y., Murakami, M., Nakano, K., Hirai, N., Mizusawa, T., Naka, S., Yamasaki, Y., Matsumoto-Nakano, M., Ooshima, T., Asai, F., 2012. Diversity of Fimbrillin among Porphyromonas gulae clinical isolates from Japanese dogs. J. Vet. Med. Sci. 74, 885–891. Pavlica, Z., Petelin, M., Juntes, P., Erzen, D., Crossley, D.A., Skaleric, U., 2008. Periodontal disease burden and pathological changes in organs of dogs. J. Vet. Dent. 25, 97–105. Rawlinson, J.E., Goldstein, R.E., Reiter, A.M., Attwater, D.Z., Harvey, C.E., 2011. Association of periodontal disease with systemic health indices in dogs and the systemic response to treatment of periodontal disease. J. Am. Vet. Med. Assoc. 238, 601–609. Serfass, P., Chetboul, V., Sampedrano, C.C., Nicolle, A., Benalloul, T., Laforge, H., Gau, C., Hébert, C., Pouchelon, J.L., Tissier, R., 2006. Retrospective study of 942 small-sized
52
M. Shirai et al. / Research in Veterinary Science 102 (2015) 49–52
dogs: Prevalence of left apical systolic heart murmur and left-sided heart failure, critical effects of breed and sex. J. Vet. Cardiol. 8, 11–18. Warrick, J., Gorrel, C.A., 1997. More sensitive method of scoring calculus. Proceedings of the 11th Annual Veterinary Dental Forum, Denver, USA, 30 Oct-2 Nov, pp. 134–136. Wolf, H.F., Rateitschak, E.M., Rateitschak, K.H., Hassel, T.M., 2005. Color atlas of dental medicine: periodontology. 3rd ed. Georg Thieme Verlag, Stuttgart.
Yamasaki, Y., Nomura, R., Nakano, K., Inaba, H., Kuboniwa, M., Hirai, N., Shirai, M., Kato, Y., Murakami, M., Naka, S., Iwai, S., Matsumoto-Nakano, M., Ooshima, T., Amano, A., Asai, F., 2012. Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype. Vet. Microbiol. 28, 196–205.