ABSTRACTThe zoonotic bacteriaCapnocytophaga canimorsusandC. cynodegmi, the predominantCapnocytophagaspecies in the canine oral biota, can cause human local wound infections or lethal sepsis, usually transmitted through dog bites. Molecular surveying of theseCapnocytophagaspecies using conventional 16S rRNA-based PCR is not always accurate due to their high genetic homogeneity. In this study, we isolatedCapnocytophagaspp. from the canine oral cavity and identified them using 16S rRNA and phylogenetic analysis. A novel 16S rRNA PCR-restriction fragment length polymorphism (RFLP) method was designed based on our isolates and validated using publishedC. canimorsusandC. cynodegmi16S rRNA sequences. The results showed that 51% of dogs carriedCapnocytophagaspp. Among these,C. cynodegmi(47/98, 48%) was the predominant isolated species along with one strain ofC. canimorsus(1/98, 1%). Alignment analysis of 16S rRNA sequences revealed specific site nucleotide diversity in 23% (11/47) of theC. cynodegmiisolates, which were misidentified asC. canimorsususing previously reported species-specific PCR. Four RFLP types could be classified from all the isolatedCapnocytophagastrains. The proposed method demonstrates superior resolution in distinguishingC. cynodegmi(with site-specific polymorphism) fromC. canimorsusand especially in distinguishingC. canimorsusfrom otherCapnocytophagaspecies. Afterin silicovalidation, this method was revealed to have an overall detection accuracy of 84%; notably, accuracy reached 100% inC. canimorsusstrains isolated from human patients. Overall, the proposed method is a useful molecular tool for the epidemiological study ofCapnocytophagain small animals and for the rapid diagnosis of humanC. canimorsusinfections.IMPORTANCEWith the increased number of small animal breeding populations, zoonotic infections associated with small animals need to be taken more seriously.Capnocytophaga canimorsusandC. cynodegmiare part of common biota in the mouths of small animals and can cause human infections through bites or scratches. In this study,C. cynodegmiwith site-specific 16S rRNA sequence polymorphisms was erroneously identified asC. canimorsusduring the investigation of canineCapnocytophagaby conventional PCR. Consequently, the prevalence ofC. canimorsusis incorrectly overestimated in epidemiological studies in small animals. We designed a new 16S rRNA PCR-RFLP method to accurately distinguish zoonoticC. canimorsusfromC. cynodegmi. After validation against publishedCapnocytophagastrains, this novel molecular method had high accuracy and could detect 100% ofC. canimorsus-strain infections in humans. This novel method can be used for epidemiological studies and the diagnosis of humanCapnocytophagainfection following exposure to small animals.