Capnocytophaga and Neisseria species in the Blood of Stray Dogs from Northern Mexico

INTRODUCTION

Understanding the bacteria that inhabit the mouths of dogs is crucial for developing effective prevention and treatment strategies. This includes education on the importance of proper wound cleaning after a bite, as well as the appropriate prescription of antibiotics. In addition, the study of these bacteria allows for the development of vaccines or preventive treatments, which could be administered to people at greater risk of being bitten or to dogs to reduce the bacterial load in their oral cavities. Recent studies have employed advanced molecular techniques to characterize oral bacterial communities in healthy dogs, revealing a more complex picture than was previously known.^[1] The genus Capnocytophaga consists of gram-negative, facultative anaerobic bacteria known to be part of the oral microbiome of dogs, often colonizing the gingival sulcus and other mucosal surfaces.^[2] However, the potential impact of these bacteria on the well-being of companion canines has been the subject of increasing research and clinical interest.^[3] Human Capnocytophaga infections caused by dog bites have been reported in various parts of the world, primarily in regions where close contact with dogs is common and where health services allow for accurate diagnosis of bacterial infections. In the United States, the Centers for Disease Control and Prevention has documented cases across the country, especially in people with compromised immune systems, who are more susceptible to infection after dog bites.^[4] In Europe, countries such as Germany, France, and the United Kingdom have reported Capnocytophaga infections following dog licking or bites.^[5,6] In France, for example, a study conducted in several hospitals identified the presence of this bacterium in cases of serious infections in humans.^[7] Infections have also been reported in Australia, where serious cases have been documented in people who have been bitten by dogs.^[8] Therefore, this genus represents a significant public health concern due to its potential to cause severe infections, particularly in older and immunocompromised individuals.^[9]

The genus Neisseria is a group of Gram-negative, aerobic, oxidase-positive bacteria that are known for their importance in human health and disease, particularly the well-known N. gonorrhoeae and N. meningitidis, which cause the sexually transmitted disease gonorrhea and severe, often fatal meningitis, respectively.^[10,11] While the majority of Neisseria infections in humans are well documented, the presence and significance of this genus in animal populations have received less attention. Nonetheless, studies have reported the isolation of Neisseria from various animal species, including cattle, sheep, goats, and wildlife.^[12-15] These findings suggest that Neisseria has a broader host range, raising concerns about its potential zoonotic implications. Dog bites, while generally not considered a significant risk factor for Neisseria infections, have been reported as a potential mode of transmission in a small number of cases. Therefore, the study of Neisseria in dogs may provide valuable insights into the epidemiology and transmission dynamics of these bacteria, as these animals often live in close proximity to humans and can act as reservoirs for zoonotic diseases.

The identification and characterization of Capnocytophaga and Neisseria species can be challenging. Conventional biochemical methods, as well as more advanced techniques such as real-time polymerase chain reaction (PCR), 16S rRNA gene sequencing, and whole-genome sequencing, have been employed to differentiate between the various species within these genera.^[16,17] Blood is considered an enriched sample that can provide insight into an animal’s oral, intestinal, and dermal microbiome because bacteria can translocate from organs into the bloodstream or enter through wounds;^[18] therefore, this type of sample is preferable. In the present study, massive sequencing of the V3-V4 16S rRNA gene was used in blood samples from stray dogs in northern Mexico to identify Capnocytophaga and Neisseria species carried by these animals. This information will complement existing knowledge about the Capnocytophaga and Neisseria species that inhabit dogs, benefiting public health.

MATERIAL AND METHODS

In October 2023, blood was collected aseptically using 3 mL syringes (23 G) from the cephalic vein of 12 randomly selected and apparently healthy adult stray dogs in Comarca Lagunera, Mexico (25°34’45”N 103°26’49”W). Ten drops (50 mg of blood) were collected from each sampled dog and placed in BashingBead lysis tubes containing 750 µL of lysing/stabilizing solution (Zymo Research, Irvine, CA USA); then, the samples were processed in a Terralyzer (Zymo Research, Irvine, CA USA) cell disruptor. Deoxyribonucleic acid (DNA) extraction was performed through the ZymoBiomics DNA Miniprep Kit (Zymo Research, Irvine, CA USA) following the established protocol.

The samples were processed at Novogene Corporation, Inc. (Davis, CA USA), and V3‒V4 16S rRNA amplicons were obtained through the 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) primers. PCR reactions were carried out with 15 µL of Phusion^® High-Fidelity PCR Master Mix (New England Biolabs), 2 µM forward and 2 µM reverse primers, and approximately 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s. Finally, the samples were heated at 72°C for 5 min. The same volume of ×1 loading buffer [containing Synthetic, yellow, bromine (Sybr^®) green] and PCR products were mixed for electrophoresis on a 2% agarose gel for detection. The PCR products were mixed in equal ratios. The mixed PCR products were subsequently purified with a Qiagen Gel Extraction Kit (Qiagen, Germany). The sequencing libraries were generated through a TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations, and index codes were added. Library quality was assessed on a Qubit@ 2.0 fluorometer (Thermo Scientific, Waltham, MA USA) and an Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform, and 250 base pairs (bp) paired-end reads were generated.

Sequence analysis was performed in quantitative insights into microbial ecology 2 (QIIME2).^[19] The divisive amplicon denoising algorithm 2 was used to remove low-quality sequences, filter chimeric/singleton sequences, and generate Amplicon Sequence Variants (ASVs).^[20] The Greengenes2 database^[21] was initially used to assign taxonomy. We then searched for Capnocytophaga and Neisseria species from the output taxonomic file. The ASV sequences of both genera were confirmed in the National Center for Biotechnology Information database using the basic local alignment search tool, considering an E value of 0.0 and a minimum identity percentage of 97% to accept the species.

RESULTS

The species of Capnocytophaga that was recorded from the blood samples was Capnocytophaga cynodegmi, Capnocytophaga catalasegens, and Capnocytophaga canimorsus. A total of six species of Neisseria were identified (Neisseria canis, Neisseria perflava, Neisseria weixii, Neisseria weaveri, Neisseria dumasiana, and Neisseria zoodegmatis). The total frequency of the sequences and the mean percentages of the bacterial species are shown in Table 1.

DISCUSSION

According to Iwai,^[22] one of the main sources of enrichment of the blood microbiome is the oral microbiome; this enrichment is caused by the breakdown of epithelial cells or damage to gums. The oral cavity is then the route of entry of Capnocytophaga and Neisseria species into the blood of these dogs. Therefore, although the abundance of these bacteria is very low, it is important to consider their presence in terms of bites to the population. Two of them, C. canimorsus and C. cynodegmi, are particularly notable for their ability to infect humans through animal bites, scratches, or even licks, leading to potentially life-threatening systemic infections or death;^[23,24] meanwhile, C. catalasegens was recently described as part of the oral microbiome of cats in Japan,^[25] and their zoonotic potential has not yet been documented. On the other hand, epidemiological data on the incidence of Neisseria infections due to dog bites are limited, as these cases are relatively rare. However, the potential for these infections to occur, coupled with potentially serious consequences, underscores the importance of prompt recognition, appropriate treatment, and preventive measures. The clinical presentation and severity of these infections can vary depending on the specific Neisseria involved, the extent of the injury, and the host’s immune response. The pathogenic potential of N. canis in humans is not fully understood, but it has been associated with a range of clinical manifestations, including local skin and soft-tissue infections, as well as more severe systemic infections.^[26,27] Information on infections caused by N. perflava is scarce; it has been reported only in isolated cases of human endocarditis.^[28,29] N. weaveri is part of the normal oropharyngeal microbiome of dogs,^[30] but there are several reports of bacteremia and cellulitis in humans caused by the transmission of this species through dog bites.^[31,32] On the other hand, N. dumasiana was recently described from the sputum of elderly people and the oral cavity of dogs.^[33] N. weixii was recently described by Zhang et al.^[34] from the rectal contents of the Tibetan Plateau pika (Ochotona curzoniae); there are no records that this bacterial species has the potential to cause infection. Finally, N. zoodegmatis is a common inhabitant of the oral cavity of dogs and cats but has been reported to cause infections in wounds caused by cat bites.^[35-37]

In Mexico, as in many other countries in the world, available information on specific Capnocytophaga and Neisseria infections is limited, suggesting that cases may be infrequent or not systematically reported. Some factors that may influence the low frequency of reports are as follows: (1) limited diagnosis (identification of these infections requires specialized microbiological tests that may not be widely available in all healthcare facilities); (2) underreporting (there may be underreporting of cases due to lack of clinical suspicion, especially in patients without obvious risk factors, such as immunosuppression); and (3) epidemiological characteristics (Capnocytophaga and Neisseria infections are usually more common in people with weakened immune systems, so the prevalence of this condition in the general human population could influence the frequency of reports).

CONCLUSION

The impact of Capnocytophaga and Neisseria on public health is significant due to the morbidity they cause. Continued research is essential to address the challenges posed by these bacterial taxa. Blood offers the advantage of acting as a compiler of most of the bacteria that enter or inhabit the body of an animal; in the case of Capnocytophaga and Neisseria, the oral cavity of dogs. Hence, it is important to continue using sequencing technologies for public health concerning pathogen transmission from animals to increase information and develop better methods for prevention, diagnosis, and treatment.