Porphyromonas gulae infection in canines, pet owners and veterinarians in China: an epidemiological study and risk factor analysis

P. gulae exhibits the following characteristics: black-pigmented colonies, obligate anaerobic growth, Gram stain-negative, non-motile, non-spore-forming, and rod-shaped in appearance under a light microscope. This bacterium grows slowly (7–10 days) on modified brain-heart infusion (BHI) agar containing hemin (Solarbio, 5 μg/mL) and VK1 (Solarbio, 1 μg/mL). Smooth, uniformly-shaped colonies were observed on BHI agar medium containing 5% fetal bovine serum (FBS) after 5–10 days of culture. In liquid medium, P. gulae was more difficult to enrich. Refer to Fig. 2 for the growth of P. gulae in BHI agar medium with hemin and VK1.

SB42 and SB40 of P. gulae. The modified BHI plates were incubated anaerobically, then cells were Gram stained and examined by microscopy (100× magnification)

Gingival crevicular fluid (GCF) samples were collected from 72 canines with PD attending the Teaching Animal Hospital of China Agricultural University from February 2021 to February 2022. Anaerobic culturing was performed on all samples. A total of 22 species and 92 strains of anaerobic bacteria were isolated and purified, including P. gulae, Prevotella intermedia, Porphyromonas crevioricanis, Porphyromonas macacae and other canine oral Porphyromonas spp. The isolation and purification results were consistent with the high-throughput sequencing results. Based on these findings, P. gulae appeared to be the predominant pathogen of canine PD, with a detection rate of 31.5% (29/92). The strain information obtained by culture was confirmed by the results of first generation sequencing of cloned 16S rRNA gene.

Of the 103 canine owners tested for P. gulae, 25 were positive (24.3%), including 19 males and 6 females. The age range was from 20 to 61 years, with an average age of 46.3 (±12.7) years. Sex and age can affect the immune system and the progression of PD. We classified the degree of contact between canine owners and canines into eight levels, designated A–H (based on the research of Yamasaki et al. [19]). The highest level of contact (level A) included raising at home, kissing and other close-contact behaviors. The lowest level of contact (level H) indicated no direct contact. Among those individuals testing positive for P. gulae, 19 (76.0%) were classified as level A (raising at home, kissing, and other direct intimate contact behaviors), 6 (24.0%) were classified as level B (raising at home, licking hands, feeding by hands, and other indirect intimate contact behaviors), and no individuals were classified as level C to H. Systemic diseases are considered to increase the incidence and severity of PD directly or indirectly. In our study, 8 out of 25 individuals with a history of systemic disease tested positive for P. gulae. Smoking and drinking alcohol are directly related to PD as these behaviors alter the physical and chemical environment of the periodontium. Among the individuals that tested positive, 12 (48.0%) consumed alcohol and 16 (64.0%) were smokers. Oral health status indirectly affects the composition and abundance of microbes in the oral cavity. Among those testing positive for P. gulae, 12 had good oral health (48.0%), 9 had gingivitis (36.0%), 3 had periodontitis (12.0%) and 1 had dental caries (4.0%). Oral cleaning can destroy and remove microorganisms in the oral cavity. Typically, an individual may develop gingivitis after not brushing their teeth for 10 days. Among those who tested positive for P. gulae, 19 individuals brushed their teeth twice a day and 6 brushed their teeth once a day. Detailed statistical data are shown in Table 1.

Next, we cross-checked the canine survey information, including P. gulae test results, years of feeding, and periodontal status evaluation, with the P. gulae test results of the owners. Of the 101 canine samples tested for P. gulae, 93 (92.1%) were positive. Generally, if the owner tested negative for P. gulae, so did their dog. Four canine samples without corresponding owner samples were not included in the statistics, resulting in a total number of 97 canine samples shown in Table 2.

The owners of 28 canines tested positive for P. gulae, including 8 canines without PD, 8 canines with gingivitis, and 12 canines with periodontitis. Among all samples, 61 canines had been raised for 5 years or fewer (62.9%), and of the corresponding owners, 20 tested positive (80.0% of positive owners); 28 canines had been raised for 5 to 10 years (28.9%), and of the corresponding owners, 6 tested positive (24.0% of positive owners); and, finally, 8 canines had been raised for 10 years or longer (8.2%), and of the corresponding owners, 2 tested positive (8.0% of positive owners). Among the canines raised for 5 years or fewer, the positive rate of their corresponding owners was 32.8%. The positive rate of owners who raised canines for 5 to 10 years was 21.4%. And the positive rate of owners who raised canines for 10 years or longer was 25.0%. The results showed that the positive rate did not increase with an increase in feeding years. The detailed statistical data are shown in Table 2.

The positive detection rate of P. gulae was taken as the basic variable, and the relationship among the test variables was analyzed using a logistic regression model. The classification regression model calculation of P. gulae detection in the canine owners revealed a significant difference (P = 0.001, P