East African Relapsing Fever Borrelia spp. | CDC EID

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Volume 16, Number 7–July 2010
Research
Population Structure of East African Relapsing Fever Borrelia spp.
Sally J. Cutler, E. Margarita Bonilla, and Rajbir J. Singh
Author affiliation: University of East London, London, UK

Suggested citation for this article

Abstract
Differentiation of endemic East African tick-borne relapsing fever Borrelia duttonii spirochetes from epidemic louse-borne relapsing fever (LBRF) B. recurrentis spirochetes into different species has been questioned. We assessed a noncoding intragenic spacer (IGS) region to compare genotypes found in clinical samples from relapsing fever patients. Although IGS typing was highly discriminatory and resolved 4 East African tick-borne relapsing fever groups from a disease-endemic region in Tanzania, 2 IGS clades were found among LBRF patients in Ethiopia. The 2 IGS sequence types for B. recurrentis overlapped with 2 of the 4 groups found among B. duttonii. All cultivable isolates of B. duttonii fell into a single IGS cluster, which suggests their analysis might introduce selective bias. We provide further support that B. recurrentis is a subset of B. duttonii and represents an ecotype rather than a species. These observations have disease control implications and suggest LBRF Borrelia spp. could reemerge from its tick-borne reservoirs where vectors coexist.
Relapsing fever is a recurrent febrile infection caused by various Borrelia spirochetes transmitted either by lice (epidemic relapsing fever) or by ticks (endemic relapsing fever). Relapsing fever was once a disease of global epidemic importance. However, largely as a result of the demise of the clothing louse Pediculus humanus, it is now restricted to areas where clothing lice are still commonplace, such as Ethiopia (1). In these regions it still has a major impact and was documented in a recent Ethiopian Department of Health report as being the seventh most common cause of hospital admission (2.5% of total; 3,777 cases) and fifth most common cause of death (0.9%, 42 cases) (2004) (1). The epidemic tendency of this infection is likely to reside in its vector transmission, with waves of clothing lice fleeing febrile patients and thus facilitating epidemic transmission. The endemic tick-borne relapsing fever spirochetes are transmitted by Ornithodoros ticks; O. sonrai serve as the principle vector for Borrelia crocidurae in West Africa, and O. moubata complex ticks effectively maintain these spirochetes in East Africa (1). Because soft ticks are only associated with hosts for their typically rapid nocturnal feeding, this limits the spread of infection beyond the confines of the areas where tick vectors reside.

Although these differing life styles of the vectors can account for the epidemiologic differences between these infections, recent studies have highlighted the similarity between B. recurrentis, the cause of louse-borne relapsing fever, and B. duttonii, the agent of East African of tick-borne relapsing fever (2–4). It has been postulated that B. recurrentis is a louse-adapted variant of B. duttonii (5). In these disease-endemic regions, diagnosis is typically achieved through demonstration of spirochetes in stained blood films from patients. However, this technique is unable to discriminate between the different Borrelia spp. that cause relapsing fever.

To provide a method able to reliably identify these spirochetes, we validated the use of sequence analysis of an intragenic spacer (IGS) region for typing these spirochetes (3). This method was used to analyze a noncoding spacer region and proved to be highly discriminatory; it resolved 4 groups among B. duttonii found among isolates and directly in tick vectors. Two groups were found among B. recurrentis isolates and louse vectors (3). Furthermore, a novel Borrelia species detected previously was found, and some sequence types resembled B. crocidurae, previously believed to be only in West Africa (3,6). The surprising finding was that 1 phylogenetic group of B. duttonii overlapped with a group of B. recurrentis (3). This colinearity was further supported by complete genomic sequencing of 1 isolate of B. recurrentis and 1 of B. duttonii, which suggests that B. recurrentis is a decaying genome that evolved from either B. duttonii or a common ancestral strain (5).

To further explore this apparent overlap between B. duttonii and B. recurrentis, we sequenced and compared additional gene targets. However, this study focused upon coding genes that were under different selective pressure and thus were not necessarily comparable to the noncoding IGS previously used. Furthermore, these investigations used only cultivable strains, and thus could represent bias toward those able to grow under axenic conditions. These investigations disclosed a clear demarcation between B. duttonii and B. recurrentis (4).

To resolve this apparent dichotomy, we undertook additional IGS typing directly on serum samples collected from patients with clinical cases, thus removing the selective pressure of cultivation. We report the results of those investigations.

Materials and Methods
Clinical Samples
Eighty-eight serum samples from patients in Ethiopia with relapsing fever that were blood film–positive for spirochetes were collected and stored frozen at –20°C before testing. Similarly, 23 samples collected from patients in Tanzania were collected and stored frozen. An additional series of 45 samples from family members accompanying patients to hospital as potential blood donors were available for analysis. Ethical approval for their collection and testing for relapsing fever had been granted for earlier studies (Ethiopia [7] and Commission for Science and Technology [COSTECH] 2001–330-NA-2001–25 for Tanzania).

DNA Extraction
Serum samples were centrifuged at 13,000 rpm in a microfuge for 30 min and 100 μL of the pellet was used for DNA extraction. After an initial proteinase K digestion in a waterbath at 56°C for 1 h, DNA extraction was conducted by using DNeasy reagents (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol for the QIAcube robotic platform with a final elution volume of 200 μL.

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Suggested Citation for this Article
Cutler SJ, Bonilla EM, Singh RJ. Population structure of East African relapsing fever Borrelia spp. Emerg Infect Dis [serial on the Internet]. 2010 Jul [date cited]. http://www.cdc.gov/EID/16/7/1076.htm

DOI: 10.3201/eid1607.091085