Rare ospc allele L of Borrelia burgdorferi sensu stricto is commonly found among samples

AEM Accepts, published online ahead of print on 7 December 2012 Appl. Environ. Microbiol. doi:10.1128/aem.03362-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 3 Rare ospc allele L of Borrelia burgdorferi sensu stricto is commonly found among samples collected in a coastal plain area from southeastern United States and is associated with tick Ixodes affinis and local rodent hosts Peromyscus gossypinus and Sigmodon hispidus. 4 5 6 7 8 9 10 11 12 13 14 15 16 Running title: Rare Borrelia burgdorferi ospc allele L Nataliia Rudenko 1, 3#, Maryna Golovchenko 1, 3, Libor Grubhoffer 1, 2 and James H. Oliver Jr 3. 1 Biology Centre, Institute of Parasitology, 37005, České Budějovice, Czech Republic 2 Faculty of Science, University of South Bohemia, 37005, České Budějovice, Czech Republic 3 The James H. Oliver Jr. Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA 30460-8056, U.S.A. Keywords: Borrelia burgdorferi, Lyme disease, ospc, invasive strain, rodent hosts, tick vector 17 18 Address correspondence to Nataliia Rudenko, natasharudenko@hotmail.com 1

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Abstract Globally rare ospc allele L was detected in 30% of Borrelia burgdorferi sensu stricto strains cultured from hard ticks Ixodes affinis and two rodent host species, Peromyscus gossypinus and Sigmodon hispidus, collected in a coastal plain area of Georgia and South Carolina, southeastern United States. Association of Borrelia burgdorferi sensu stricto (B. burgdorferi) ospc alleles with strains invasiveness and probable severity of Lyme disease (LD) was recently confirmed by multiple publications (2, 3, 26, 29, 30). It is believed that major ospc alleles are geographically distinct and host-specific (3, 5, 18, 26, 30). A wide variety of ospc is identified in different populations of B. burgdorferi. The dominant, common or rare ospc alleles are defined (1, 8, 18, 26, 29, 30). OspC allele L is recognized as a very rare one worldwide (all above cited). Cultivation of B. burgdorferi sensu lato from tick vectors and diverse host specimens collected in southeastern part of the United States was initiated in 1991 by Prof. J. H. Oliver Jr. and resulted in collection of more than 300 isolates from Texas, Missouri, Rhode Island, South Carolina, Georgia and Florida. It is housed today in the James H. Oliver Jr. Institute of Arthropodology and Parasitology, Georgia Southern University (Statesboro, Georgia). The collection includes 152 strains from 6 hard tick species (Ixodes affinis, I. minor, I. scapularis, I. dentatus, Dermacentor variabilis and Amblyomma americanum), 131 from 3 rodent host species (cotton mouse Peromyscus gossypinus, eastern wood rat Neotoma floridana and hispid cotton rat Sigmodon hispidus), 13 from 8 bird species (Carolina Wren Thryothorus ludovicianus, Downy Woodpecker Picoides pubescens, White-eyed Vireo Vireo griseus, Swainson s thrush Catharus ustulatus, American Redstart Setophaga ruticilla, Northern Water thrush Parkesia 2

42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 noveboracensis, Pine Warbler Setophaga pinus, and Northern Cardinal Cardinalis cardinalis) and a group of isolates with confirmed multiple spirochete species originated from different sources (James H. Oliver Jr., presented at the Annual meeting of Institute of Medicine, Washington, DC, October 10-12, 2010). Spirochetes were cultured in BSK-H medium supplemented with 6% rabbit serum, 0.15% of agarose, antibiotics (rifampicin, phosphomycin) and fungicide (amphotericin B) in 5% CO 2 at 34 o C and were stored at -80 o C after the cultures cell density reached 2 10 6 spirochetes/ml (21, 24, 25). Protocols for Borrelia DNA purification, amplification, sequence and data analyses were described earlier (27, 28). Briefly, total B. burgdorferi DNA was purified using DNeasy Blood and Tissue kit (Qiagen, U.S.A.). Partial 16S rrna gene, 5S-23S intergenic spacer (IGS), 16S-23S internal transcribed region (ITR) and ospc gene were amplified using previously described primers (27, 28). Direct sequencing of all amplicons was done on both strains by the HTGU, University of Washington (Seattle, USA). Sequences were assembled and edited using DNAStar (DNASTAR, United Kingdom). The BlastN algorithm was used to confirm identity against GenBank. All sequences obtained in this study were submitted to the GenBank (Table 1). The previous analyses of Borrelia collection were directed mostly on isolates from Missouri (12, 13, 14), Rhode Island (22) and Texas (11, 12, 13, 14). The goal of our study was to analyze Borrelia isolates cultured from hosts and vectors collected in selected coastal regions of southeastern United States, i.e., Georgia and Florida. The group of 112 spirochete isolates was of our big interest as it was originated from the regions where spirochete enzootic maintenance is supported by wide spectrum of hosts and ticks species which rarely bite human (23). Recently, this type of spirochete maintenance in nature is referred to as cryptic maintenance (6). From the other side, the regions of samples origin are the part of the Atlantic 3

65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 Flyway, one of the major bird migration routes that follow the Atlantic Coast of North America. It was shown that migratory birds represent the mechanism by which diverse Borrelia species are distributed worldwide by spreading infected ticks within and between continents and transferring the pathogen from the zones of cryptic transmission to the zones of epidemiological importance. By maintaining the spirochetes in foci where classic I. scapularis/ white-footed mouse transmission cycle is absent, birds are increasing the risk of contracting Lyme disease to humans (6). Analysis of 5S-23S IGS, 16S rrna genes and 16S-23S ITR of 112 isolates revealed a presence of 36 Borrelia bissettii strains (unpublished results), 16 strains recently described as B. carolinensis (27), 7 strains recently described as B. americana (28) and 53 B. burgdorferi strains (unpublished results). Analysis of a highly polymorphic ospc locus of 53 B. burgdorferi strains was conducted with the aim to clarify the molecular epidemiology of LD spirochete, to evaluate spirochete-vector-host associations, and possible disease risk to humans in a geographic region where the presence of LD is controversial. Clustering analysis (not shown) using the Neighbor Joining method (4) of ospc from southeastern B. burgdorferi population revealed that 16 sequences out of 53 analyzed (30.2%) clustered together with ospc allele L of European B. burgdorferi strains (X81524, EF537402 and EF537406) and sequence L42899 from the U.S.A. Although L42899 represents B. burgdorferi ospc allele L strain isolated from a rodent host in Michigan in 1995 (15), this allele was considered to be exclusively European till 2010 (2, 3, 26). Our analysis revealed that ospc allele L is one of two most prevalent ospc alleles among B. burgdorferi strains originated from southeastern part of United States, sharing the frequency of distribution with ospc allele B strains (30.2% each). Two other ospc alleles detected among 4

88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 the 53 B. burgdorferi strains were alleles G and H (28.3% and 11.3%, respectively). Ten ospc allele L strains were cultured from unfed female and male ticks that usually do not bite humans, thus playing little if any role in direct transmission of B. burgdorferi to human (Table 1). But, serves as a maintenance vector in the enzootic cycle of B. burgdorferi and is considered to be more important than human biting tick species in areas where it appears (23). I. affinis is reported from Central and South Africa and is found less often in North America (20). Its distribution in the U.S.A is restricted to coastal plain counties of Florida, Georgia, South Carolina and North Carolina (7, 16, 20). Recently, establishment of population was detected in Virginia. It is highly possible that distribution of in the U.S.A. is wider than yet recognized (17). Another 6 southeastern B. burgdorferi ospc allele L strains were cultured from 5 ear clip samples and 1 bladder of two major reservoir hosts of B. burgdorferi in southeastern U.S.A., P. gossypinus and S. hispidus (Table 1). feeds on many common species of mammals, amplifying in such a way the prevalence of B. burgdorferi in reservoir hosts and potentially increasing the number of infected hosts. Sharing infected hosts with the bridge-vector it could later transmit the spirochete to I. scapularis (6, 17, 20, 23). Twenty five years ago the major known reservoir hosts of nymphs and larvae were cotton mouse, wood rat and cotton rat. White-tailed deer served as reservoir host for adult I. affinis ticks (20, 23). Today the hosts list includes already 16 animal species whose geographical distribution is associated with southeastern U.S.A. (7). feeds on birds, thus the existence of bird - cryptic cycle is highly possible in areas where this tick species occurs. Both cycles, rodent - and bird -, might operate in parallel, or overlap (23). The detection of rather extensive group of B. burgdorferi ospc allele L strains in James H. Oliver Jr. 5

111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 collection of isolates supports the fact that this allele is associated with vector tick and rodent reservoir hosts distributed mostly in southeastern U.S.A. (23). However, B. burgdorferi ospc allele L strains were found in only 2 host seeking nymphs and 4 ticks collected from various hosts, representing the lowest frequency of any allele type found in north-eastern U.S.A. (1.1%) (3). Such low prevalence of ospc allele L in the North might be explained by the absence of appropriate local hosts species for which B. burgdorferi ospc allele L strains might form a chronic infection. Another spatial cluster of ticks carrying B. burgdorferi ospc allele L (3.2%) was recently observed in Canada in adult I. scapularis ticks that were collected from companion animals and humans (19). While B. burgdorferi ospc allele L is one of two most prevalent alleles in the southeastern U.S.A., it is the less prevalent one in north-eastern U.S.A. and shares the 13-14 position out of 17 ospc alleles detected in I. scapularis in Canada, most probably being transferred there from the United States (19). Obviously, B. burgdorferi ospc allele L strains are consistently reintroduced to the new regions by infected ticks carried by migratory birds. And in this process the South is the source and the North is the sink for B. burgdorferi ospc allele L. The source-sink connection might be true as well for the other ospc alleles maintained either by cryptic or endemic cycles among non-human species. Multiple studies show association of ospc alleles with strain invasiveness. The following grouping was proposed: a) ospc alleles A, B, I and K are responsible for systemic disease in humans; b) alleles G, H, J, N and T cause a local infection at the tick bite site, but not invasive disease; c) alleles D, E, F and L very rarely, if ever, cause human disease (9, 26, 29, 30). However, results presented by Lagal et al (10) and Alghaferi et al (1) showed that ospc alleles of invasive isolates from humans and small mammals are clustered with noninvasive reference alleles. This fact contradicts the ospc invasiveness hypothesis. 6

134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 Results of the present study suggest that a) globally rare B. burgdorferi ospc L allele is closely associated with non-human biting tick and is very common in the southeastern U.S.A.; b) B. burgdorferi ospc allele L strains show high host-specificity to rodents; their distribution in the U.S.A is, most probably, related to distribution of reservoir hosts, P. gossypinus and S. hispidus, or alternative rodent hosts; c) it was found in previous studies that B. burgdorferi ospc allele L strains are not infectious to four principal reservoir host species in the hyperendemic northeastern region, Peromyscus leucopus (white-footed mouse), Tamias striatus (eastern chipmunk), Blarina brevicauda (short-tailed shrew), and Sciurus carolinensis (gray squirrels) (3). However, analyzed in this study B. burgdorferi ospc allele L strains showed the ability to disseminate in two of the most common natural reservoir hosts in the south, the cotton mouse (P. gossypinus) and cotton rat (Sigmodon hispidus). It is possible, that the interspecific variation in the vertebrate immune system may provide resistance to infection by certain ospc alleles (3). Further analyses of interactions of B. burgdorferi ospc allele L strains and vertebrate hosts, most probably, are worthwhile. Acknowledgment The credits for term source-sink introduced in this study goes to anonymous Reviewer of our manuscript. This work was supported by the GSU Foundation (U.S.A.), grants CZB1-2963-CB-09 to M.G. and CZB1-2966-CB-09 to N.R. and J.H.O. from CRDF Global (U.S.A.), partially by grant R37AI-24899 from National Institute of Health, and with institutional support RVO: 60077344 from Biology Centre, Institute of Parasitology to N.R., M.G., and L.G. (Czech Republic). 7

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Table 1. Borrelia burgdorferi ospc allele L strains isolated from Ixodes affinis and rodent hosts in southeastern U.S.A.: date of collection, location of origin, culture source and GenBank accession numbers of selected genomic loci. Bb ss strain Collection date Collection locality Culture source GenBank # 16S rrna GenBank # 5S-23S IGS GenBank # 16S-23S ITR GenBank # OspC SCCH24 Oct-95 Charleston County, SCCH 30 Oct-95 Charleston County, SCI 1 Apr-93 St. Catherines Island, Georgia SI 14 Mar-95 Sapelo Island, Georgia SI 16 Mar-95 Sapelo Island, Georgia SI 17 Mar-95 Sapelo Island, Georgia S. hispidus (ear clip) EU025032 EU001290 JF739947 JF723228 P. gossypinus EU025035 EU001293 JF739950 JF723232 (ear clip) P. gossypinus EU025040 EU001298 JF739952 JF723234 (ear clip) EU025045 EU001303 JF739954 JF723236 (unfed female) (unfed male) EU025047 EU001305 JF739956 JF723238 EU025048 EU001306 JF739957 JF723239 (unfed male)

SI 19 Mar-95 Sapelo Island, Georgia SCW 9 Aug-94 Charleston County, SCW 12 Sep-94 Charleston County, SCW 43 May-95 Charleston County, SCW 44 May-95 Charleston County, (unfed male) S. hispidus (bladder) P. gossypinus (ear clip) (unfed female) (unfed female) EU025046 EU001304 JF739959 JF723241 EU025053 EU001311 JF739965 JF723247 EU025054 EU001312 JF739966 JF723248 EU025055 EU001313 JF739968 JF723250 EU025056 EU001314 JF739969 JF723251 Downloaded from http://aem.asm.org/ SCW 48 Jun-95 Charleston County, SCW 54 Aug-95 Charleston County, SCW 60 Oct-95 Charleston County, (unfed female) (unfed female) (unfed male) EU025058 EU001316 JF739971 JF723253 EU025060 EU001318 JF739973 JF723255 EU025064 EU001322 JF739977 JF723259 on December 11, 2018 by guest

SCW 61 Oct-95 Charleston County, SCGT 16 Dec-95 Georgetown County, (unfed male) P. gossypinus (ear clip) EU025059 EU001317 JF739978 JF723260 EU025038 EU001296 JF739983 JF723265 Downloaded from http://aem.asm.org/ on December 11, 2018 by guest