Human Exposure to Novel Bartonella Species From Contact With Fruit Bats

Ying Bai; Modupe O.V. Osinubi; Lynn Osikowicz; Clifton McKee; Neil M. Vora; Maria Rosales Rizzo; Sergio Recuenco; Lora Davis; Mike Niezgoda; Ajoke M. Ehimiyein; Grace S.N. Kia; Akin Oyemakinde; Olufunmilayo Sanni Adeniyi; Yemi H. Gbadegesin; Olugbon A. Saliman; Abiodun Ogunniyi; Albert B. Ogunkoya; Michael Y. Kosoy; Idanre Bat Festival Investigation Team


Emerging Infectious Diseases. 2018;24(12):2317-2323. 

In This Article

Materials and Methods

Human subjects work was approved by the Centers for Disease Control and Prevention (CDC) Institutional Review Board, the Ahmadu Bello University Human Ethics Board, and the National Health Research Ethics Committee of Nigeria. All animal procedures were conducted in compliance with a field protocol approved by the CDC Animal Institutional Care and Use Committee.

Field Sites, bat Capture, and Sample Collection

We captured bats by nets in 2 caves in Idanre Hills, Ondo State, southwestern Nigeria, in September 2010 (n = 106) and February 2013 (n = 71). We identified all bats by morphologic characteristics as Egyptian fruit bats (R. aegyptiacus). Captured bats were anesthetized by intramuscular injection of ketamine hydrochloride (0.05–0.1 mg/g bat weight) and exsanguinated via cardiac puncture after surface sterilization with 75% alcohol. Serum and blood clots were separated by centrifugation. Clots were stored at −80°C except while still in the field or being shipped, during which time they were stored on dry ice.

Bat Blood Culture and Characterization of Bartonella Strain

We plated bat blood clots on heart infusion agar containing 10% rabbit blood and incubated in an aerobic atmosphere with 5% carbon dioxide at 35°C for up to 4 weeks. Bacterial colonies morphologically identified as Bartonella were subcultured to obtain pure cultures.

We prepared crude genomic DNA by heating a heavy suspension of pure culture for 10 minutes at 95°C, followed by centrifugation of the lysed cells for 1 minute at 3,000 rpm. The supernatant was then transferred to a clean centrifuge tube to be used as the template DNA. We first verified all isolates obtained from the blood clots as Bartonella spp. by PCR amplification targeting a fragment of the citrate synthase gene (gltA).[15] Positive (B. doshiae) and negative (deionized water) controls were included to ensure that the PCR worked properly.

We purified and sequenced all PCR products of gltA in both directions by using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). We used the Lasergene software package (DNASTAR, Madison, WI, USA) to compare the generated gltA sequences with all available Bartonella species/genotypes. Once the sequences were identified, we selected 1 representative strain (R-191) for further characterization with multilocus sequence typing on the basis of sequence analysis of 8 molecular markers (ftsZ, gltA, nuoG, ribC, rpoB, ssrA, 16S rRNA, and internal transcribed spacers [ITS]).[16] For phylogenetic analyses, we used the neighbor-joining method by the Kimura 2-parameter distance method and bootstrap calculations with 1,000 replicates.

Bat Ectoparasite Collection and Detection of Bartonella DNA

We collected ectoparasites from the skin and pelage of bats and stored them in microcentrifuge tubes with 70% ethanol. Ectoparasite species were identified by using available morphologic keys,[17] and identifications were later confirmed by sequencing of the mitochondrial 16S rRNA and cytochrome oxidase I (COI) genes.[18,19]

Using a Bullet Blender Gold homogenizer (Next Advance, Averill Park, NY, USA), we homogenized whole ectoparasites in Navy Eppendorf bead tubes (Next Advance) containing 400 μL brain–heart infusion broth (CDC, Atlanta, GA, USA). We extracted DNA from the homogenates by using the KingFisher Flex Purification System and the associated MagMAX Pathogen RNA/DNA Kit (both ThermoFisher, Waltham, MA, USA) according to the manufacturer's protocols. Detection of Bartonella DNA in ectoparasite samples was performed by nested PCR for gltA[20] because of low concentrations of DNA and by conventional PCR for ITS,[21] followed by sequencing and sequence analysis of amplicons.

Preparation of Antigen From the Bartonella Strain Obtained From Bats

We produced a whole-cell antigen by co-cultivating Vero E6 cells with the pure culture (≈106 agar-grown organisms) of the Bartonella strain (R-191) obtained from Egyptian fruit bats. Both were put into T-150 flasks that contained minimum essential medium supplemented with 10% fetal calf serum, 10 mmol HEPES buffer solution, 10 mmol nonessential amino acids, and 2 mmol L-glutamine. The flasks were incubated at 35°C and harvested on postinoculation day 4. At harvest, all but 2 mL of the medium was removed from the flask, sterile glass beads were added, and the flask was gently rocked to remove the Vero E6 cell monolayer. Drops (≈15 μL) of the cell suspension were mounted on each well of 12-well glass slides, which were then air dried, fixed in acetone for 15 minutes, and stored at −70°C until use.

Human Serum Collection and Testing for Antibodies

Persons in communities surrounding the caves who gave consent were enrolled in the study 11–15 days after the first bat festival of 2013 (February 19, 2013); not all of these persons had participated in all bat festival activities. Participants were asked about their contact with bats and their role in the festival, and some provided a blood sample (considered an acute-phase specimen). About 69–78 days later, a follow-up survey was conducted and a second blood sample (considered a convalescent-phase specimen) was collected (the second bat festival of 2013 did not take place between collection of the acute- and convalescent-phase samples). Serum and blood clots were separated by centrifugation; serum was stored at −80°C except while in the field or being shipped, during which time it was stored on dry ice.

To screen human serum, we used an indirect immunofluorescence assay at an initial dilution of 1:32 for IgG against the specific Bartonella antigen from the bat-associated isolate. Antigen-covered wells of the slide were overlaid with dilutions of human serum. Separate slides were included with positive and negative controls. The positive control against the Egyptian fruit bat–associated Bartonella species was produced in laboratory mice via mouse immunization with heat-inactivated bacterium (ProSci Incorporated, Poway, CA, USA). All slides were incubated at 35°C for 30 minutes and then washed in phosphate-buffered saline for 15 minutes. We used anti-human and anti-mouse conjugates (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA) for human and control serum samples, respectively. Each human serum sample reactive at the initial dilution was further titrated in 2-fold dilutions to endpoint; to check for cross-reactivity, we tested the final positive samples (defined as a titer >1:64) for 3 other Bartonella antigens (B. elizabethae, B. henselae, and B. quintana) previously reported in Africa.[22–24]