Adult A. intermedius bats (n = 38) were captured in the municipality of Cocoyoc in the state of Morelos, Mexico, in May 2012. Animals were kept in captivity by following the Guidelines of the American Society of Mammalogists for the Use of Wild Mammals in Research (7). Bats were observed for 2 months to ensure that existing infectious diseases did not develop. No animals had rabies antibodies detectable through rapid fluorescent focus inhibition test. Animals were inoculated intramuscularly with rabies virus (vampire bat variant 5020, 1×10 ^5.34Fluorescent Focus). After 5 days, an adult male exhibited emaciation, restlessness, and depression. On day 20, the animal could not fly and remained on the floor of the cage. Areas of pallor appeared on its wings (Technical Appendix Figure 1). The animal died on day 28. Testing showed negative results for rabies virus by direct immunofluorescence of brain tissue smears and by PCR of nervous tissue. Skin biopsies were taken from the wing lesions for histopathologic analyses and isolation.

Figure 1

Figure 1. Bacterial cytoplasmic inclusions (arrow) in Vero cell cultures 72 h postinoculation with supernatant from homogenates of white spot lesion biopsies of adult A. intermedius bats in Mexico by using Diff–Quick stain...

Vero cells inoculated with supernatant from homogenates of white spot lesion biopsies showed cytopathic effect (CPE) within 72 to 96 h postinoculation. CPE consisted of lytic plaque formation. Acidophilic inclusions visible by using Diff-Quick (VWR International, Briare, France) staining were detected within 48 and 72 h postinoculation (Figure 1). Similar inclusions could be seen after inoculation of BHK21 cells. The microorganism could not be cultured on blood or chocolate agar, aerobically or anaerobically, when incubated for up to 7 days.

Experimental inoculation was then established. Three bats that were seronegative for the isolated microorganism were inoculated intraperitoneally. The 3 animals were euthanized on days 5, 10, and 15. The bats euthanized on days 5 and 10 postinoculation showed signs of severe multifocal interstitial pneumonia (Technical Appendix Figure 2) and severe diffuse lymphoid hyperplasia in the spleen. On euthanization, the third bat showed signs of mild multifocal interstitial pneumonia and mild diffuse lymphoid hyperplasia in the spleen.

Two additional bats were inoculated subcutaneously; areas of pallor developed in the wing skin similar to those observed in the originally infected bat (Technical Appendix Figure 3). Mononuclear cells infected with bacteria were localized in skin (Technical Appendix Figure 4) and lung lesions of experimentally inoculated animals by immunofluorescence.

Histopathological findings in the areas of pallor through hematoxylin and eosin staining revealed the presence of mononuclear cell infiltrates in all subjects. Because of the resemblance of the wing lesions to those typically seen in white nose syndrome infection, which is caused by the fungus Pseudogymnoascus destructans, we applied periodic acid–Schiff staining to rule out fungal infection. No hyphae were identified.

Hyperimmune serum samples raised against the isolated bacteria neutralized the CPE of the bacteria up to a 1:719 dilution. Five (13%) of the 38 serum samples taken during captivity neutralized the CPE of the bacteria in dilutions ranging from 1:9 to 1:81. This result suggested circulation of this bacterium within the sampled population. All experimentally inoculated animals seroconverted. Serum samples from both animals that were inoculated subcutaneously neutralized the CPE of the bacteria up to a 1:27 dilution at day 28 postinoculation. Animals inoculated intraperitoneally and euthanized at 5, 10, and 15 days postinoculation neutralized CPE up to dilutions of 1:27, 1:27, and 1:81, respectively. No serum samples from the researchers who handled the bats showed seroneutralization activity.

Figure 2

Figure 2. Phylogenetic relationships of bacterium newly identified in Artibeus intermedius fruit bats in Mexico (Waddlia cocoyoc, bold text), to other Chlamydiales. 16S sequences were used to infer relationships. X. westbladi, Xenoturbella westbladi.

DNA from skin biopsy samples and Vero and BHK 21 cells experimentally infected by using primers directed against domain I of the 23S gene of the family Waddliaceae yielded PCR products of the expected size (627 bp) (8). Vero cell culture was used to amplify the infection of the bacterial agent and DNA extracts were subjected to high throughput sequencing (SRA: PRJNA268154). Analysis of assembled contigs by using blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that sequences close to Waddlia spp. were abundant (43%) and were only surpassed by 2 sequences of primate origin (Technical Appendix Figure 5). A neighbor-joining phylogenetic tree with 10,000 replicates was built with 16S sequences from assorted members of the order Chlamydiales and the cultured microorganism. The Chlamydiales have evolved from a single genus to a diverse order including new families such as Candidatus Parichlamydiaceae and Rhabdochlamiaceae (9). Phylogenetic analyses revealed that the newly identified Waddlia sp. segregates with known Waddlia spp. Although the new Waddlia sp. fell in the same taxonomic unit, it is found in its own branch (Figure 2). This finding was confirmed by a maximum-likelihood phylogeny with approximate likelihood ratio test (Technical Appendix Figure 6).

We report the isolation of a newly identified bacterial pathogen of A. intermedius bats and propose naming it Waddlia cocoyoc. The isolated bacterium was successfully grown in cell culture but not in inert bacterial growth media, suggesting dependence on host cells. Staining of inoculated cells revealed lysis and large intracytoplasmic vacuoles. Infected bats showed areas of pallor on the wings and had severe lesions in the lungs and the spleen. Histopathological analyses on the areas of pallor revealed mononuclear cell infiltrates in infected bats. Detection of the bacterium in lesion sites by immunofluorescence and PCR strongly suggests that it caused the observed pathogenesis. Phylogenetic analyses indicate that the pathogen is closely related to organisms in the family Waddliaceae.

Diversity in Waddliaceae increases as reports of new species surface. Waddlia spp. have been previously associated with Malaysian fruit bats (10). W. chondrophila has been isolated from aborted cattle fetuses in the United States (11), and was detected in a potoroo (Potorous spp.), a threatened marsupial native to Australia (12). Serologic evidence showed a substantive association between high titers of W. chondrophila antibodies and bovine abortion (13). In addition, W. chondrophila seroprevalence was found to be high in women who have had recurrent and sporadic miscarriages (14). W. chondrophila was also found in patients with community-acquired pneumonia (15). The host range and zoonotic potential of Waddlia spp. open multiple research avenues for this newly identified organism.