An Emerging Tick-Borne Disease of Humans Is Caused by a Subset of Strains with Conserved Genome Structure

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An Emerging Tick-Borne Disease of Humans Is Caused by a Subset of Strains with Conserved Genome Structure
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Pathogens 2013, 2, 544-555; doi:10.3390/pathogens2030544
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Barbet, Anthony
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The prevalence of tick-borne diseases is increasing worldwide. One such emerging disease is human anaplasmosis. The causative organism, Anaplasma phagocytophilum, is known to infect multiple animal species and cause human fatalities in the U.S., Europe and Asia. Although long known to infect ruminants, it is unclear why there are increasing numbers of human infections. We analyzed the genome sequences of strains infecting humans, animals and ticks from diverse geographic locations. Despite extensive variability amongst these strains, those infecting humans had conserved genome structure including the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface antigen. These data provide potential targets to identify human-infective strains and have significance for understanding the selective pressures that lead to emergence of disease in new species.
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Pathogens 20 1 3 2 544 555 ; doi :10.3390/ pa thogens 2 0 3 0 544 pathogens ISSN 2076 0817 www.mdpi.com/journal/pathogens Artic le An E merging T ick Borne D isease of H umans I s C aused by a S ubset of S trains with C onserved G enome S tructure Anthony F. Barbet 1 ,2 , Basima Al Khedery 1 , Snorre Stuen 3 Erik G. Granquist 4 Roderick F. Felsheim 5 and Ulrike G. Munderloh 5 1 Dep artment of Infectious Diseases and Pathology, University of Florida, Gainesville, FL 32611, USA; E Mail: balkhedery@ufl.edu 2 Emerging Pathogens Institute, Univers ity of Florida, Gainesville, FL 32611 USA 3 Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, Sandnes N 4325, Norway ; E M ail: snorre.stuen@nvh.no 4 Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, Oslo N 0033, Norway; E M ail: erikgeorg.granquist@nvh.no 5 Department of Entomology, Univer sity of Minnesota, St. Paul, MN 55108, USA; E M ail s : felsh001@umn.edu ( R F F ) ; munde001@umn.edu ( U G M ) These authors contributed equally to th is work Author to whom correspondence should be address ed; E M ail: barbet@ufl.edu; Tel.: +1 352 294 4119; Fax: +1 352 392 9704 Received: 17 July 2013; in revised form: 29 August 2013 / Accepted: 2 September 2013 / Published: 1 0 September 2013 Abstract: The prevalence of tick borne diseases is increasing worldwide. One such emerging disease is human anaplasmosis The causative organism, Anaplasma phagocytophilum is known to infect multiple animal species and cause human fatalities in the U.S., Europe and Asia. Although lo ng known to infect ruminants, it is unclear why there are increasing numbers of human infections. We analyzed the genome sequences of strains infecting humans, animals and ticks from diverse geographic locations. Despite extensive variability amongst these strains, those infecting humans had conserved genome structure including the pfam01617 superfamily that encodes the major, neutralization sensitive, surface antigen. These data provide potential targets to identify human infective strains and have signifi cance for understanding the selective pressures that lead to emergence of disease in new spe cies. OPEN ACCESS

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Pathogens 2013 2 545 Keywords: a naplasmosis; tick borne diseases; high throughput sequencing; pfam01617; msp2/p44; comparative genomics 1. Introduction Human exposure to vectors carrying disease agents has been increased by climate and land use changes causing more contact between humans and domestic animals with wildlife reservoirs [ 1 ] One such recently emerging disease is tick borne anaplasmosis that causes infections in multip le animal species [ 2] These include cattle, sheep, goats, horses, dogs, foxes, cats, rodents and most recently, humans. Different strains of the causative organism, Anaplasma phagocytophilum have different host predilections and not all strains can infect all hosts. Small mammals are thought to be a reservoir, infecting immature stages of ticks which act as bridge vectors transferring infection to humans and domestic animals. Cases reported to the U.S. Centers for Disease Con trol and Prevention comprise a flu like febrile illness with some severe sequelae such as multiple organ failure, or severe acute respiratory distress syndrome. In the U.S., case reports increased from 348 cases in 2000 to 1 761 cases in 2010 [3]. The repo rt e d hospitalization rate is 36% [4 ] Granulocytic anaplasmosis (GA) can be treated with antibiotics, but the symptoms, such as headache, fever, and muscle aches are non specific and can be confused with other common diseases such as Lyme, transmitted by t he same species of tick. If left untreated GA can be severe [5 ] resulting in a case fatality rate in the U.S. of up to 3% (CDC data for 2003). Increasingly, there are reports of infections transmitted by blood trans fus ions in the U.S. and Europe [6 7 ] He re we show that, in contrast to the extensive worldwide genomic diversity of A. phagocytophilum strains, human infective strains are a conserved subset. This has implications for understanding the selective pressures that lead to emergence of disease in ne w species and for control of this infection. 2. Results and Discussion 2.1. Comparative Genomics of Nine Strains of A. phagocytophilum Underlying the ecological complexity and variable host tropism of A. phagocytophilum is a large degree of genetic variability. European strains of A. phagocytophilum are highly pathogenic in sheep and cattle, whereas North American strains do not cause disease in domestic ruminants. Some U.S. strains, defined as Ap variant 1 because of a 2 base pair difference in 16S rRNA, are infectious to deer and other ruminants, but do not infect mi ce and are thought not to infect humans [8 9 ] Highly unusual for an obligate intracellular pathogen with a small, 1.5 Mb genome, A. phagocytophilum contain s msp 2/ p44 ) that codes for th e major antigen on its surface [10 ] These hypervariable pseudogene cassettes are recombined into a single expression site which provides N and C terminal conserved sequences, allowing the bacterium to serially express variable an tige ns and eva de host immunity [11 12 ] We applied high throughput genome sequencing to nine strains, derived from humans and different host animal species from the U.S. an d Europe These strains are: two human origin strains from New York

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Pathogens 2013 2 546 and Minnesota, a rodent and dog origin strain from Minnesota, two Ap variant 1 strains from Minnesota, a horse origin strain from California and two sheep origin strains from Norway for which no in vitro cultures are available. In Figure 1 the complete genome sequence of a strain derived from rodents in an area of high prevalence for human disease (Camp Ripley, MN, USA ) is compared with that of the human ApHZ strain (from the state of New York). The figure shows Blast comparisons of the rodent and human strains, particularly the positions of members of the pfam01617 superfamily, to which msp2/p44 belongs. Overall, despite widely separated geographic and species origins, the two genomes are conserved and almost completely syntenic. The majority of the highly repetitive msp2/p44 genes (visualized using partial opacity) are located in the third of the genome closest to the origin of replication (top of figure, changing sign of GC s kew), as rep orted previously [13 ] However, using high stringency Blast comparisons (fifth circle from the outside) differences between the two genomes were primarily localized to this region also and to msp2/p44 genes in particular, showing the propensity of this gen e family to undergo rapid diversification. F igure 1. Genome synteny between two strains of A. phagocytophilum Ap HZ (human origin, NY, USA ) an d ApJM (rodent origin, MN, USA ). The reference strain is ApHZ (GenBank CP000235). The two outermost circles show annotated ApHZ coding sequences, the third circle shows the locations of members of the ApHZ pfam01617 superfamily, the fourth and fifth circles are Blastn comparisons of the ApJM genome with ApHZ using 90% and 99% identity cutoffs respectively, the sixth circle shows GC skew and the innermost circle the numeric genome position. Comparisons were conducted using the CGView server using partial opacity to visualize overlapping hits (darker bars in the green and blue circles) The white bars on the fifth circl e indicate no match.

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Pathogens 2013 2 547 This comparative genomics analysis was extended to all nine strains (Figure 2) Except for Ap variant 1 strains, the U.S. strains had high identities across their entire genomes, with the ApDog strain most similar to human ApHZ by Bl ast analysis The Norwegian sheep and U.S. Ap variant 1 strains had the lowest identities with ApHZ in Blast comparisons, especially in members of the pfam01617 superfamily. Figure 2 Similarities and differences between nine A. phagocytophilum genomes. Roche/454 reads from each genomic DNA were compared with the human ApHZ genome as reference using Blastn and the CGView Comparison Tool. The first to ninth circles compare, respectively, sequencing reads from ApHZ, ApDog, ApJM, ApHGE1, ApMRK, ApC RT35, ApCRT38 1, ApNorV2, ApNorV1 genomic DNAs. The 10th circle shows the locations of members of the ApHZ pfam01617 superfamily. Blast parameters used a query size of 500 bp segments of the reference genome and a Blast expect value of 10 100 Circles are colored according to the percent identities of matches (black to light red, 100 % 90% identical, dark to light blue, 88 % 82% identical, colorless, 0% identical). T he differences in this superfamily were examined more closely in order to determine their ms p2/p44 genomic repertoires and if differences were primarily located in the known pseudoge ne hypervariable region s (Figure 3 ).

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Pathogens 2013 2 548 F igure 3 Gene variation in a region rich in p44/msp2 pseudogenes close to the origin of replication in nine genomes of A. phagocytophilum Roche/454 pyrosequencing reads from each of the nine genomes are shown aligned with the annotated reference ApHZ genome (bottom) between positions 1 450 and 1 462 kbp. The sequencing reads are derived from: ApHZ, ApDog, ApHGE1, ApJM, ApMRK ApCRT35, ApCRT38 1, ApNorV1, ApNorV2 (bottom to top panels respectively). The positions of six msp2/p44 pseudogenes are marked by arrows at the top and shown in their respective reading frames at the bottom. The hypervariable regi ons LAKT .. LAKT, present in five of the six pseudogenes, are indicated in red. Gaps in coverage indicate no aligning reads over the central hypervariable regions in some strains. Similar to the rodent strain ApJM, the msp2/p44 repertoires of a U.S. human strain, ApHGE1 and the ApDog strain from the same area of Minnesota were closely related and slightly different from the New York human strain ApHZ (8/95 or 9/95 different msp2/p44 pseudogenes determined as <90% identical Tabl e 1 ). In contrast, strains infecting horses or ruminants (Ap variant 1 strains) in the U.S. no wn to be pathogenic to sheep [14 ] shared fewer msp2/p44 pseudogenes with human strains with the two Norwegian s heep strains having almost totally different repertoi res from human strains Differences between individual members of the msp2/p44 repertoire were located in the known central hypervariable region between flan king consensus amino acids LAKT .. LAKT (Figure 3 )

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Pathogens 2013 2 549 Table 1. Genome structural relationships between Anaplasma strains. Strain ApHZ ApHGE1 ApJM ApDog ApMRK ApCRT35 ApCRT38 ApNorV1 ApNorV2 Different msp2/p44 pseudogenes from ApHZ 0/95 8/95 8/95 9/95 49/95 71/95 75/95 92/95 89/95 ANIm 100 98.81 98.84 98.79 97.76 96.28 96.21 94.92 95.87 Tetra 1.000 0.999 0.999 0.999 0.998 0.996 0.996 0.995 0.996 Strain AmFL AmStM AcIs ApHZ ANIm 100 98.86 88.89 77.82 Tetra 1.000 0.999 0.989 0.821 The top panel compares strains with ApHZ, the lower panel compares strains and species with AmFL. Abbreviations: ANIm, Average % genome nucleotide identity using Mummer; Tetra, correlation coefficient of tetranucleotide signature frequencies; AmFL, Anaplas ma marginale Florida strain; AmStM, A. marginale St. Maries Idaho strain; AcIs, Anaplasma centrale Israel strain; Ap, A. phagocytophilum strains. Two additional quantitative measures of genome structural relationships compare the average nucleotide identit ies or the tetranucleotide frequencies between two genomes. The latter method is independent of alignment algorithms. The proposed threshold for prokaryotic species separation is 94% for average nucleotide identity and 0.990 for the correlation coefficient of tetranu cl eotide signature frequencies [15 ] It is evident from Table 1 that the existing taxonomy of Anaplasma species meets these definitions. However, it is apparent that by these measures also, the U.S. human, dog and rodent strains are closely rela ted (average nucleotide identity 98.79 % 98.84% with ApHZ). The Norwegian ruminant strains are again most distinct from ApHZ (average nucleotide identity 94.92 % 95.87%), agreeing with the similar divergence observed in their pfam01617 superfamily genes. 2.2. Comparison of the msp2/p44 Family among Strains of A. phagocytophilum Although these data showed that two U.S. A. phagocytophilum strains infecting humans, as well as a dog and rodent strain were similar to one another and different from strains infec ting ruminants, we wanted to analyze this more globally using a larger dataset. To do this, we took advantage of the fact that conserved regions of msp2/p44 have been used as sensitive PCR diagnostic targets because of the large copy number of the pfam0161 7 superfamily. More than 500 partial pseudogene sequences are present in GenBank, from humans, ticks and animal strains derived from multiple regions throughout the U.S., Europe and Asia. From these sequences it is possible to extract the hypervariable reg ion of msp2/p44 flanked by sequence encoding a consensus LAKT on either end, facilitating alignment [16 ]. Importantly, for this analysis it is necessary to recognize that multiple different hypervariable regions exist in a single genome because of the ~100 distinct pseudogene cassettes present. Therefore, it is not sufficient to simply analyze phylogenetic trees and apparent evolutionary relationships between all msp2/p44 sequences. If one wishes to compare global repertoires with the genome sequenced human ApHZ strain, it is necessary to align each msp2/p44 sequence with all ~100 ApHZ strain genomic pseudogene cassettes to find the best fit (maximum sequence identity). If strains are related to ApHZ, one expects to find one or more ApHZ pseudogenes with hig h identity to other strain msp2/p44 s. Our analyses, therefore, employed a matrix where every available msp2/p44 polypeptide (661 total

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Pathogens 2013 2 550 including those encoded by ApHZ pseudogene cassettes) was aligned with every other, generating 218,791 alignments having a mean amino acid sequence identity of 48.2%. These percentage sequence identities can be rapidly analyzed using a 661 (row) 661 (column) spreadsheet. Surprisingly, despite the differences in msp2/p44 repertoires observed previously, all 91 available msp 2/p44 human origin sequences from widely dispersed U.S. locations (states of New York, Massachusetts, Wisconsin and Minnesota) encoded polypeptides with a mean maximum percentage identity with ApHZ of 97.2% (Table 2). Further, in analyzing the available ms p2/p44 sequences it was notable that, world wide, other human origin strains also achieved similar levels of sequence identity with ApHZ (e.g., 98.1% mean maximum percentage amino acid identity of a dataset from Japan comprising 27 human origin msp2/p44 va riants, no significant difference with ApHZ). In contrast, A. phagocytophilum strains from Europe and Asia from non human sources had mean maximum identities of <76% (significantly different from ApHZ, Table 2). Table 2 Comparison of msp2/p44 variants fro m different geographic locations with the human ApHZ strain. Country Source Number of msp2/p44 Variants Analyzed Mean Maximum % a.a. Identity with ApHZ ( + / Std.Dev.) Significantly different from U.S. Human U.S.A. Human 91 97.2 (7.0) U.S.A. Dog 27 94.8 (9.8) No U.S.A. Horse 29 94.2 (7.3) No U.S.A. Bear 4 92.4 (4.1) N/A U.S.A. Woodrat 88 78.4 (13.8) Yes U.S.A. Ruminant, tick (Ap variant 1) 34 86.5 (14.7) Yes Norway Sheep 54 66.6 (6.4) Yes Sweden Dog 19 64.3 (7.9) Yes U.K. Goat 20 67.2 (8.2) Yes U.K. Sheep 19 65.1 (6.7) Yes Czech Republic Human, Roe deer, Perdix, Ixodes ricinus 6 86.6 (14.5) N/A [Human only] [2] [97.8][(2.0)] N/A Japan Sika deer 17 39.3 (3.0) Yes Japan Ixodes persulcatus 87 69.0 (6.9) Yes Japan Ixodes ovatus 22 71.8 (15.5) Yes Japan Haemaphysalis formosensis 9 75.6 (14) N/A Japan Human 27 98.1 (7.2) No China Human 2 100 (0) N/A Kruskal Wallis One Way Analysis of Variance on Ranks ( p Comparisons versus a Control group ( p 0.05). N/A: Not applied to sample sizes <10. 3. Experimental 3.1. Origin of A. phagocytophilum Strains The origins of the A. phagocytophilum strains used in this study are as follows: ApHZ, human, Westchester County, New York; HGE1, human, Minnesota; ApJM, meadow jumping mouse ( Zapus

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Pathogens 2013 2 551 hudsonius ), Camp Ripley, Minnesota; ApDog, Minnesota; ApCRT35, Camp Ripley tick ( Ixodes scapularis ), Minnesota (defined previously as Ap variant 1); ApCRT38 1, Camp Ripley tick ( I. scapul aris ), Minnesota (Ap variant 1); ApMRK, horse, California; ApNorV1, sheep, Norway; ApNorV2, sheep, Norway. A. phagocytophilum genomic DNA was prepared from in vitro cultured organisms or directly from infected sheep (Norwegian str ai ns), as described previo usly [17 ] 3.2. Ethics Statement The experimental study in sheep was approved by the Norwegian Animal Research Authority. 3.3 Genome Sequencing and Bioinformatics Genomic DNA was sequenced on the Roche/454 Genome Sequencer using non paired and 3 kb paired en d libraries, also as described [17 ] Mean genome coverage with respect to ApHZ varied between 31.3X and 72.1X. The ApJM sequence was finished using manual inspection for conflicts and mismatched paired ends and PCR to fill gaps. All sequences were compa red with ApHZ for regions of identity u sing Blastn analysis in CGView [18 19 ] Differences in the msp2/p44 repertoires between strains were defined using a method previously validated using the pfam01617 superfamily and two completely Sanger sequenced geno mes of Anaplasma marginale [20 ] Briefly, this method uses Mosaik to align individual reads and generate BAM format files to detect gaps in alignment (no coverage) with respect to the annotated reference sequence. In A. marginale this method detected all different pseudogenes having <90% nucleotide identity with the reference genome. In homologous comparisons between ApHZ Roche/454 reads and the ApHZ genome all msp2/p44 pseudogenes were detected as present (Table 1). Comparisons of average nucleotide ident ities and the correlation coefficients of tetranucleotide signature frequencies between genomes were conducted using Jsp ecies software, as described [15 ]. 3.4 Analysis of msp2/p44 R epertoires To analyze global msp2/p44 repertoires, deposited msp2/p44 sequences were downloaded from GenBank following Blast searches; several large datasets are also av ailabl e from published studies [16 21 25 ] The sequences were each trimmed to that encoding the hypervariable region, where possible using the consensus fl anking LAKT residues, and the polypeptides were aligned ( all against all) with MATGAT [26 ] This generates the alignments that can be inspected for accuracy, and allows export of all percent identity values to a spreadsheet. This spreadsheet was analyzed i n Excel for the relatedness of the different datasets. The percent identity of each variant msp2/p44 sequence with every ApHZ msp2/p44 polypeptide encoded by a pseudogene was reported and, from that, the mean best match with ApHZ (maximum percent identity) determined for each dataset. As the percentage identities were not normally distributed and the population variances were unequal, significant differences between groups were determined by n onparametric methods (Table 2).

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Pathogens 2013 2 552 4. Conclusions These data show that there is genome diversity worldwide within the A. phagocytophilum species that extends close to some proposed guidelines for species discrimination. However, strains infecting humans are a subset with more conserved genome structure th an the species overall, and this includes their repertoires of msp2/p44 genes. This subset of A. phagocytophilum strains does not differ significantly from the U.S. ApHZ strain and is closely related to strains infecting U.S. domestic dogs. The mean seropr evalence (using a conserved msp2/p44 peptide as antigen) in the U.S. among 479,640 dogs was 4.8% with prevalence >50% in some counties in the Northeast and Midwest, corresponding to the location o f the majority of human cases [2 7 ] These data have signific ance in several areas. First, msp2/p44 encodes a protein that induces neutralizing antibodies against homologous strains of A. phagocytophilum [2 8 2 9 ] The repertoire differences between strains reflect an evolving antigenic system with adaptive pressures imposed by an array of different persistently infected hosts. In contrast, humans are incidental dead end hosts that do not impose significant pressur e for change. The similarities between human origin strains in the U.S., Europe and Asia suggest that humans may not be susceptible to many of the circulating wildlife strains. They may become susceptible when selection pressures in small mammal reservoir hosts cause evolution of novel strains that allow invasion and survival in humans. Rodents are important reservoirs for a multitude of human disease agents, vector borne or not, and are known to be infected with strains of A. phagocytophilum hav i ng differe nt species tropisms [30 ] Rodent control has historically been emphasized as a means to control human disease outbreaks. The underlying evolutionary drivers remain to be identified but could reflect the long standing association ond, these data have practical significance for control of this emerging infection. Despite the ubiquity of A. phagocytophilum strains and positive serology world wide, it is necessary to focus on the prevalence and transmission of a smaller, human infecti ve subset. Differentiation of such strains from the high prevalence background may be possible using genomic PCR targets or selected msp2/p44 polypeptides for serology. In order to accomplish this goal it is necessary to acquire genome sequences from multi ple human origin strains on different continents. The technology now exists to do this. Acknowledgments We thank Anna M. Lun dgren for technical support, Savita Shanker for high throughput DNA sequencing, David Allred and Dan Brown for reading the manuscrip t and to Dan Brown for suggesting the use of Jspecies software. This investigation received support from grants RO1 GM081714 and GM081714 03S1. Draft and complete s equences obtained in this project are being made available thro ugh the NCBI Bioproject portal [31] as bioprojects PRJNA158483, 163167, 163169, 171710, 183838, 216999, 217002, 217033, and 217037. Conflict s of Interest The authors declare no conflict of interest

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Pathogens 2013 2 555 30. Foley, J.E.; Nieto, N.C.; Massung, R.; Barbet, A.; Madigan, J.; Brown, R.N. Distinct ecologicall y relevant strains of Anaplasma phagocytophilum Emerg. Infect. Dis. 2009 15 84 2 8 43. 31. The NCBI Bioproject Portal. Available online: www.ncbi.nlm.nih.gov/bioproject (accessed on 5 September 2013 ). 20 1 3 by the authors; licensee MDPI Basel, S witzerland. This article is an o pen a ccess article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).