PLoS One. 2015 Apr 15;10(4):e0121274.

Identification of a novel Afipia species isolated from an Indian flying fox.


Pickering BS1, Tyler S2, Smith G3, Burton L3, Li M3, Dallaire A4, Weingartl H1.
  • 1National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, Canada; Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.
  • 2National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada.
  • 3National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, Canada.
  • 4Département de pathologie et microbiologie, faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada.



An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.

PMID: 25874801



Afipia felis is the type species of the genus Afipia and originally believed to be the agent causing cat scratch disease (CSD). In 1986, an estimated 6000 cases of CSD were reported annually with about 1% of these leading to encephalitis1. However, it was later determined the primary cause of CSD was Bartonella henselae, with only rare cases attributed to A. felis.

Alphaproteobacteria are an incredibly diverse class of bacteria with little in common aside from an ancient ancestor. Among its members, the Bradyrhizobiaceae are well-known for their autotrophic ability surviving in diverse environments. The Afipia are members of the genus Bradyrhizobium, and unlike many of its members, Afipia are considered to be intracellular pathogens.

Initially, in this work necrotic tissue was received to help identify an unknown pathogen causing disease to an old world bat. To help distinguish the cause of disease, we employed the use of electron microscopy for initial characterization of the cytopathic effect of the pathogen non Vero cells. Electron microscopy suggested particles in similar shape to filoviruses, pathogens known to associate with old world bats (figure 1). In consideration of this, we performed all subsequent work with live material in biosafety level four (BSL4) conditions.

Upon ascertaining the presence of an Afipia bacterial pathogen, 16S rRNA analysis and whole genome sequencing was performed to further characterize the organism. Impressively, the genome appears well equipped for survival in many environments in addition to genes synonymous with pathogens. The phylogenetic relationship between Afipia and the other members of the Bradyrhizobium is an interesting component of this work. Bradyrhizobium are often referred to as symbionts of host plants, suggesting a beneficial association whereby the bacteria supplies much needed nitrogen to the plant in exchange for nutrients. An alternative view of this association describes Bradyrhizobium as pathogens well suited to infect plants and transverse infection threads leading to nodule formation. In either definition, part of their specialty is to induce internalization and generate an infection of legumes. In the same way, Afipia spp. have been shown to act as intracellular pathogens2,3. The amount of bacteria and diversity of bacteria is extremely high, further stressing the need for increased identification and surveillance for new bacteria. Increased metagenomics studies and genome sequencing will be essential to providing more information about the origin and classification of bacterial species.





  1. Brenner, D. J., Hollis, D. G., Moss, C. W., English, C. K., Hall, G. S., Vincent, J., et al (1991). Proposal of Afipia gen. nov., with Afipia felis sp. nov. (formerly the cat scratch disease bacillus), Afipia clevelandensis sp. nov. (formerly the Cleveland Clinic Foundation strain), Afipia broomeae sp. nov., and three unnamed genospecies Journal Of Clinical Microbiology, 29(11), 2450-60.
  2. Scola, B. L., & Raoult, D (1999). Afipia felis in hospital water supply in association with free-living amoebae. The Lancet, 353(9161), 1330. doi:10.1016/S0140-6736(99)00906-X.
  3. Lo, S., Hung, G., Li, B., Lei, H., Li, T., Nagamine, K., et al (2013). Isolation of novel Afipia septicemium and identification of previously unknown bacteria Bradyrhizobium sp. OHSU_III from blood of patients with poorly defined illnesses Plos ONE, 8(10), e76142. doi:10.1371/journal.pone.0076142.





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