Stephen Balinandi

In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ~2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (~six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.

To the Editor: Marburg virus (MARV) and Ravn virus (RAVV), collectively called marburgviruses, cause Marburg hemorrhagic fever (MHF) in humans. In July 2007, 4 cases of MHF (1 fatal) occurred in miners at Kitaka Mine in southern Uganda. Later, MHF occurred in 2 tourists who visited Python Cave, ≈50 km from Kitaka Mine. One of the tourists was from the United States (December 2007) and 1 was from the Netherlands (July 2008); 1 case was fatal (1,2,3). The cave and the mine each contained 40,000–100,000 Rousettus aegyptiacus bats (Egyptian fruit bats). Longitudinal investigations of the outbreaks at both locations were initiated by the Viral Special Pathogens Branch of the Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA, and Entebbe, Uganda) in collaboration with the Uganda Wildlife Authority (UWA) and the Uganda Virus Research Institute (UVRI). During these studies, genetically diverse MARVs and RAVVs were isolated directly from bat tissues, and infection levels of the 2 viruses were found to increase in juvenile bats on a predictable bi-annual basis (4,5). However, investigations at Kitaka Mine were stopped when the miners exterminated the bat colony by restricting egress from the cave with papyrus reed barriers and then entangling the bats in fishing nets draped over the exits. The trapping continued for weeks, and the entrances were then sealed with sticks and plastic. These depopulation efforts were documented by researchers from UVRI, the CDC, the National Institute of Communicable Diseases (Sandringham, South Africa), and UWA during site visits to Kitaka Mine (Technical Appendix Figure). In August 2008, thousands of dead bats were found piled in the forest, and by November 2008, there was no evidence of bats living in the mine; whether 100% extermination was achieved is unknown. CDC, UVRI, and UWA recommended against extermination, believing that any results would be temporary and that such efforts could exacerbate the problem if bat exclusion methods were not complete and permanent (6,7). In October 2012, the most recent known marburgvirus outbreak was detected in Ibanda, a town in southwest Uganda. Ibanda is ≈20 km from the Kitaka Mine and is the urban center that serves smaller communities in the Kitaka area. This MHF outbreak was the largest in Ugandan history: 15 laboratory-confirmed cases occurred (8). In November 2012, an ecologic investigation of the greater Ibanda/Kitaka area was initiated. The investigation included interviews with local authorities to locate all known R. aegyptiacus colonies in the area. Although minor colonies of small insectivorous bats were found, the only identifiable colony of R. aegyptiacus bats was found inside the re-opened Kitaka Mine, albeit at much reduced size, perhaps 1%–5% of that found before depopulation efforts. To determine whether the R. aegyptiacus bats that had repopulated Kitaka Mine were actively infected with marburgviruses, we tested 400 bats by using previously described methods (4,5). Viral RNA was extracted from ≈100 mg of liver and spleen tissue by using the MagMAX Total Nucleic Acid Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s recommended protocol. The Fisher exact test was conducted by using IBM SPSS Statistics, version 19.0 (IBM Corp., Armonk, NY, USA). Of the 400 R. aegyptiacus bats collected, 53 (13.3%) were positive for marburgvirus RNA by quantitative reverse transcription PCR (32/233 [13.7%] adults and 21/167 [12.6%] juveniles; Technical Appendix Table); marburgvirus was isolated from tissue samples from 9 of the 400 bats. The overall level of active infection was significantly higher than that found in Kitaka Mine during 2007–2008 (5.1%) (5) (Fisher exact test, p 0.5 for both), and overall, the presence of virus-specific IgG among the bats was 16.5%, a finding consistent with that in previous studies (4,5). Figure Phylogeny of concatenated marburgvirus nucleoprotein (NP) and viral protein 35 (VP35) gene fragments as determined by using the maximum-likelihood method. Sequences from the NP (289–372 nt) and VP35 (203–213 nt) genes were amplified and ... Phylogenetic analysis of viral RNA genome fragment sequences in this study showed high marburgvirus genetic diversity, including the presence of RAVVs and MARVs. Sequences for isolates from 3 bats were nearly identical to those of the MARV isolates obtained from patients in the 2012 Ibanda outbreak (8), suggesting that bats from Kitaka Mine were a likely source of the virus. Technical Appendix: Photographs taken during August 2008–September 2009 of bat extermination efforts at Kitaka Mine, and table showing demographic characteristics of bats captured during a Marburg hemorrhagic fever outbreak investigation at the mine in November 2012, Uganda. Click here to view.(124K, pdf)

Two large outbreaks of Ebola hemorrhagic fever occurred in Uganda in 2000 and 2007. In May 2011, we identified a single case of Sudan Ebola virus disease in Luwero District. The establishment of a permanent in-country laboratory and cooperation between international public health entities facilitated rapid outbreak response and control activities.