Polymerase Chain Reaction Detection of Capnocytophaga canimorsus and Capnocytophaga cynodegmi as the Emerging Zoonosis

The members of genus Capnocytophaga are fastidious, Gram-negative, rod-shaped bacteria which belong to the family of Flavobacteriaceae (1) and reside in the oral cavities of humans and domestic animals. Eight species of bacteria including C. ochracea, C. sputigena, C. gingivalis, C. granulosa, C. leadbetteri, C. haemolytica, C. canimorsus, and C. cynodegmi (2) are the members of this genus. All species except for C. canimorsus and C. cynodegmi reside in the human oral cavity (3,4) and may cause periodontal diseases and severe infections in immunocompromised patients (3,5). According to several studies (1,2,6-8), these two bacteria reside in the oral cavities of dogs and cats and may be transmitted to humans and infect local open wounds causing systemic infection in humans sometimes through bites (54% of cases), scratches (8.5% of cases), or even by licks (27% of cases). Recently, Butler isolated a new species, named C. canis, from the oral cavity of healthy dogs and found that this genus has low pathogenicity for humans (6). C. canimorsus is the causative agent of wound infections and may cause disseminated infections, followed by sepsis, meningitis, and endocarditis (3-5,7). On the other hand, systemic infection is rarely caused by C. cynodegmi while it is mainly detected in wound infections (2,5,7,9). According to different reports, this organism is susceptible to penicillin. However, the prognosis of C. canimorsus infection is poor in chronic alcohol users, as well as asplenic and immunocompromised patients and thus can cause an overall mortality of 30% (4-7,10). For this reason, rapid identification to species level with the accurate and rapid initiation of treatment with appropriate antimicrobial agents is very important (3). However, except for a few cases that occur following the cat bites and scratches, most of the infections in humans caused by C. canimorsus are related to dog bites, dogs licking pre-existing wounds, or other contacts with dogs (9,11,12). Nevertheless, it is possible that close animal contact or a superficial abrasion may provoke the event. The history of a dog bite is elicited in only 43% to 57% of the cases and exposure to dogs without bites or scratches are reported in Avicenna Journal of Clinical Microbiology and Infection


Background
The members of genus Capnocytophaga are fastidious, Gram-negative, rod-shaped bacteria which belong to the family of Flavobacteriaceae (1) and reside in the oral cavities of humans and domestic animals.Eight species of bacteria including C. ochracea, C. sputigena, C. gingivalis, C. granulosa, C. leadbetteri, C. haemolytica, C. canimorsus, and C. cynodegmi (2) are the members of this genus.All species except for C. canimorsus and C. cynodegmi reside in the human oral cavity (3,4) and may cause periodontal diseases and severe infections in immunocompromised patients (3,5).According to several studies (1,2,(6)(7)(8), these two bacteria reside in the oral cavities of dogs and cats and may be transmitted to humans and infect local open wounds causing systemic infection in humans sometimes through bites (54% of cases), scratches (8.5% of cases), or even by licks (27% of cases).Recently, Butler isolated a new species, named C. canis, from the oral cavity of healthy dogs and found that this genus has low pathogenicity for humans (6).C. canimorsus is the causative agent of wound infections and may cause disseminated infections, followed by sepsis, meningitis, and endocarditis (3)(4)(5)7).On the other hand, systemic infection is rarely caused by C. cynodegmi while it is mainly detected in wound infections (2,5,7,9).According to different reports, this organism is susceptible to penicillin.However, the prognosis of C. canimorsus infection is poor in chronic alcohol users, as well as asplenic and immunocompromised patients and thus can cause an overall mortality of 30% (4)(5)(6)(7)10).For this reason, rapid identification to species level with the accurate and rapid initiation of treatment with appropriate antimicrobial agents is very important (3).
However, except for a few cases that occur following the cat bites and scratches, most of the infections in humans caused by C. canimorsus are related to dog bites, dogs licking pre-existing wounds, or other contacts with dogs (9,11,12).Nevertheless, it is possible that close animal contact or a superficial abrasion may provoke the event.The history of a dog bite is elicited in only 43% to 57% of the cases and exposure to dogs without bites or scratches are reported in 12% to 27% of the cases (9,10).Local wound infections rapidly lead to life-threatening septicemias correlated with intravascular coagulation, generalized purpura, and severe hemorrhages in adrenal glands, and finally, failure in internal organs (13).
After prolonged incubation (48 to 72 hours), colonies appear and rapidly growing bacteria are probably overgrown because the organisms are relatively fastidious and of slow-growing type.Meanwhile, many strains produce a yellow-brown pigment (14)(15)(16).The results of oxidase, catalase, ONPG (O-nitrophenyl-b-D-galactoside), and arginine dihydrolase are positive for Capnocytophaga spp.while they are negative for urease, nitrate, indole, DNase, gelatin, lysine, and ornithine (9,17).On the other hand, biochemical tests are difficult to perform due to the slow growth of bacteria.
Likewise, the identification of C. canimorsus and C. cynodegmi is very difficult owing to the similarities in genetic properties and physiological activity between these two species.Therefore, there is a need for the development of more acceptable and specific molecular techniques for identifying the Capnocytophaga spp.(18).In the current study, a polymerase chain reaction (PCR) system, described by Suzuki et al, was used to differentiate between the above-mentioned species (19).
Considering the importance of canine and feline reservoir hypothesis, obtaining more information about the epidemiology and genetics of Capnocytophaga spp.from the dogs and cats help to control the transmission and treatment of Capnocytophaga infections and to improve the overall public health.Thus, the present study aimed to investigate the role of the traditional method and PCR in identifying Capnocytophaga spp.The results (backed up by 16S rRNA gene sequencing) showed that this method reliably identifies C. canimorsus and C. cynodegmi to species level.

Specimens Collection
Oral swabs were obtained from 125 dogs and 35 cats of the same age, sex, and breed during October 2014-June 2015.The animals were routinely admitted to the veterinary teaching hospital or animals were maintained for teaching at the Faculty of Veterinary Medicine, Shiraz University.In an attempt to sample a representative portion of the population, specimens were collected at irregular intervals.After getting the informed consent from the owners, swabs were taken from the animals attending this teaching hospital for various reasons.Cotton-tip sterile swabs were rubbed on the gums and tongue and then suspended in nutrient broth and transferred to the laboratory of microbiology under cold condition.Next, the cottontipped applicators were suspended in brain heart infusion broth (Merck, Germany) as enriched cultures for 24-48 hours at 35 °C in the atmosphere containing 5% CO 2 .

DNA Extractions
A 100 µL of enriched cultured samples were used for DNA extraction and the total DNA was prepared from enrichment cultures using Gram-negative DNA extraction kit (Cinnagen, Iran) according to the instructions of the kit.The extracted DNA were determined to be of good quality and DNA concentration was measured using Nanodrop (10000V 3.52).In addition, DNA concentrations were adjusted to 21.8 ng/µL before PCR amplification.Finally, extracted DNA samples were stored at -20 °C for further use.
It should be noted that the primers targeting the 16S rRNA gene of C. canimorsus and C. cynodegmi were obtained from the study by Suzuki et al (19).
The purified DNA of C. canimorsus (ATCC35979) and C. cynodegmi (ATCC49044) strains, kindly donated by Dr. M. Suzuki, and sterile nuclease-free distilled water were used as appropriate positive controls and negative control, respectively.The positive control DNA sample was used to test PCR validity, consistently leading to the expected PCR products.The employed samples were as follows.
resulted image was captured by a computer software program (AlphaEase, Alpha Innotech).

Results
The extracted DNAs from all enrichment cultures were used as the template for 16S rRNA specific primer pairs.Further, three primer sets were used, which amplify the fragments of 16S rDNA gene of Capnocytophaga genus, as well as C. canimorsus and C. cynodegmi species and should yield the PCR fragments of 124, 427, and 427 bp, respectively (Figures 1-3).Then, the primer set was tested on the purified DNA of C. canimorsus  1).
Additionally, primers in different combinations were utilized for determining the specificity of PCR to amplify the 16S rRNA gene of C. canimorsus and C. cynodegmi.Only the target sequence in the DNA of Capnocytophaga was amplified by using the CAL2 and AS1 primers while no PCR products were obtained from the DNA of other bacterial strains included in this experiment (data not   available).Similarly, only amplicon was achieved from the DNA of C. canimorsus when using CAL2 and CaR primer pair.On the other hand, primers CAL2-CyR produced a single specific band of about 427 bp from the DNA of C. cynodegmi (Table 1, Figures 2 and 3).

Discussion
The members of the genus Capnocytophaga are gramnegative, fastidious, catalase, oxidase negative rods (i.e., C. canimorsus and C. cynodegmi are catalase and oxidase positive) and are considered as one of the emerging bacterial zoonotic diseases.The organism is a part of the oral microbiota of dogs and cats, which cause no disease in these animals.These agents can cause different infectious diseases when they are transmitted to a human, directly or indirectly.For example, it leads to sudden and acute septicemia with disseminated intravascular coagulation that involves many internal organs.Middle-aged and elderly persons are at a greater risk of disease contraction.In addition, individuals who spend a greater portion of their time with canines and felines such as veterinarians, breeders, pet owners, and lab workers are included in a higher risk category, especially immunocompromised individuals.Further, the chance of infection varies between 3% and 20% after dog bites and as regards the bites of the cats, it may be even more.Nevertheless, it is conceivable that close animal contact or a trivial lesion may be the inciting event.Likewise, a history of a dog bite is obtained only from 43% to 57% of the cases and exposure to dogs without bites or scratches is reported in 12% to 27% of the cases.Finally, no report is available regarding man to man transmission (20).
Although biochemical methods are still useful for identifying rapidly growing bacterial strains in clinical microbiology laboratories, molecular methods are becoming more common for detecting slowly growing strains.Several molecular methods are currently available for the identification and differentiation of bacterial species.Indeed, 16S rRNA gene sequencing is regarded as the "gold standard" for bacterial identification.
According to different studies, the prevalence of C. canimorsus in the dog samples was 8, 24, and 25%, respectively, when using the culture (21)(22)(23).On the other hand, other researchers such as Gaastra and Lipman (18), Mally et al (1), and van Dam et al (24) found that the prevalence of C. canimorsus was 41%, 57%, and 73%, respectively, in dog samples by using the PCR.These data indicate that the sensitivity of PCR for the detection of C. canimorsus is more than that of traditional microbiological methods such as culture and phenotypical traits.
Suzuki et al established a specific PCR which could identify and distinguish C. canimorsus from C. cynodegmi (19).Using this method, they determined the prevalence of Capnocytophaga spp. in dogs and cats.Based on their finding, C. canimorsus was detected in 74% of dogs and 57% of cats and C. cynodegmi was found in 86% of dogs and 84% of cats.The prevalence of Capnocytophaga spp.obtained in this study is somewhat higher than those previously reported where the bacterial isolation method was used for identification.This is probably because PCR detection is more sensitive compared to the bacterial culture concerning detecting C. canimorsus and C. cynodegmi in samples taken from dogs and cats.
Similarly, Umeda et al genetically compared C. canimorsus isolates using 16S rRNA gene sequence analysis and pulsed field gel electrophoresis and indicated that C. canimorsus was detected in 69.7% of dogs and 54.8% of cats (25).
The PCR method, established by Suzuki et al (19), can be applied to identify and distinguish C. canimorsus from C. cynodegmi since it is rapid and sufficiently sensitive.In our research, the prevalence of Capnocytophaga spp. in dogs and cats was determined using the PCR method.The 16S rRNA gene of Capnocytophaga spp. was observed in 32% of dog and 65.7% of cat samples.According to our finding, the 16S rRNA gene of C. canimorsus and/or C. cynodegmi was detected in 20% and 90% of dogs and 62.5% and 100% of cats, respectively.This finding demonstrated that 16 dog samples and 8 cat samples contained both Capnocytophaga spp.The prevalence of C. canimorsus was lower in dogs compared to the cats, but C. cynodegmi was detected in roughly a similar proportion in both animal groups.Based on some previous reports, the prevalence of Capnocytophaga spp.by bacterial isolation was shown to be 36-60% for dogs and 24% for cats (1,22).However, the detection rate for C. canimorsus was 69.7% and 54.8% in dogs and cats, respectively, when using the PCR technique (25).
In the present study, the DNA of Capnocytophaga spp. was detected in 32% and 65.7% of samples through the PCR test, respectively.Furthermore, our finding revealed that the detection of the DNA of C. canimorsus in 50% and 62.2% of dog and cat samples, respectively.Conversely, these findings were 90% and 100% in dogs and cats, respectively, respecting the DNA of C. cynodegmi.The findings of the current study are roughly in line with the findings of a study by Suzuki et al in Japan.

Conclusions
To the best of our knowledge, our finding is the first report about the presence of Capnocytophaga spp. in the swab samples of dog and cat in Iran.The risk of infection with C. canimorsus is high owing to the high percentage of Capnocytophaga in the mouth of the cats and dogs and the severe consequences of infection for humans.Accordingly, cat and dog owners should be notified about this risk, especially if they belong to specific risk groups like elderly people, pregnant women, young children, and immune (ATCC 35979) and C. cynodegmi (ATCC 49044).The presence of Capnocytophaga genus, along with C. canimorsus and C. cynodegmi species DNA in the enrichment cultures was thus indicated by positive PCRs for 16S rDNA gene.Furthermore, the DNA of Capnocytophaga genus was prespecified in 32% (n = 40) of the total 125 oral swabs of the dogs.When PCR was conducted for the presence of the DNA of C. canimorsus and C. cynodegmi, 20 (50%) and 36 samples were found positive for the DNA of C. canimorsus and C. cynodegmi, respectively.Moreover, from 35 oral swabs of the cats, 23 samples were detected positive for the presence of the DNA of Capnocytophaga genus.Based on the results, all the samples analyzed by the primer specified Capnocytophaga species were positive for DNA of C. cynodegmi although only 15 samples were detected as positive for the presence of the DNA of C. canimorsus (Figures 1-3 and Table

Table 1 .
PCR Results for the Presence of Capnocytophaga DNA in Different Samples