IAI Accepts, published online ahead of print on 6 October 2014 Infect. Immun. doi:10.1128/IAI.02537-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Anaplasma marginale superinfection attributable to pathogen strains with distinct

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genomic backgrounds

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Eduardo Vallejo Esquerra1, David R. Herndon2, Francisco Alpirez Mendoza3,§ Juan Mosqueda4, and Guy H. Palmer1*

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Washington 99164 USA;

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Service, United States Department of Agriculture, Pullman, Washington 99164 USA;

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Paul G. Allen School for Global Animal Health, Washington State University, Pullman, 2

Animal Diseases Research Unit, Agricultural Research

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Programa de Salud Animal, INIFAP-CIRGOC, La Posta, Vera Cruz, México;

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Universidad Autónoma de Querétaro, Campus Juriquilla, México.

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*Corresponding Author

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Paul G. Allen School for Global Animal Health, Washington State University, Pullman,

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Washington 99164-7090

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Tel: (509) 335-6033

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Fax: (509) 335-6328

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E-mail: [email protected]

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§

Dr. Alpirez is deceased.

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Abstract

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Strain superinfection occurs when a second pathogen strain infects a host already

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infected with a primary strain. Understanding of the selective pressures that drive strain

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divergence, which underlies superinfection, and allow penetration of a new strain into a

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host population are critical knowledge gaps relevant to shifts in infectious disease

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epidemiology. In endemic regions with high prevalence of infection, broad population

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immunity develops against Anaplasma marginale, a highly antigenically variant

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rickettsial pathogen, and creates strong selective pressure for emergence of and

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superinfection with strains that differ in their Msp2 variant repertoire. The strains may

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emerge either by msp2 locus duplication and allelic divergence on an existing genomic

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background or by introduction of a strain with a different msp2 allelic repertoire on a

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distinct genomic background. To answer this question, we developed a multi-locus

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typing assay, based on high throughput sequencing of non-msp2 target loci, to

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distinguish among strains with different genomic backgrounds. The technical error level

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was statistically defined based on the percentage of perfect sequence matches of

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clones of each target locus and validated using experimental single strains and strain

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pairs. Testing of A. marginale positive samples from endemic tropical regions identified

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individual infections that contained unique alleles for all five targeted loci. The data

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revealed a highly significant difference in the number of strains per animal in the tropical

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regions as compared to infections in temperate regions and strongly supported the

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hypothesis that transmission of genomically distinct A. marginale strains predominates

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in high prevalence endemic areas.

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Introduction

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Microbial strain structure is dynamic over space and time. For pathogens, shifts in

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structure characterized by emergence of genotypically unique strains and loss of others

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results in changing patterns of disease, whether within an individual or a population.

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However the scale of space and time differs markedly among pathogens depending on

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multiple factors including pathogen-specific mechanisms of genetic change and

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effective pathogen population size. For example, RNA viruses such as lentiviruses are

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capable of rapidly generating mutations throughout the genome due to the large number

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of progeny emanating from a single virus within an infected cell and the minimal

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proofreading during replication (1-3).

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have both limited number of progeny per replicative cycle and stricter control on

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genomic fidelity between generations (4,5). These differences in the time required to

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generate genetic variants aside, the successful emergence of a new genotypically

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unique strain is dependent on the strength of the selective pressure acting on the

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pathogen population (6).

In contrast, bacteria and eukaryotic parasites

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Immunity is a strong selective pressure for newly emergent genetic variants.

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Within an individual host the immune response selects for new variants; for viral

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pathogens this includes true genomic variants whereas higher order microbial

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pathogens most typically use differential expression within a more stable genome (3-

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5,7,8).

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pressure and has been postulated to be a driver for diversification in strain structure

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across the spectrum of microbial pathogens (9,10). In this model, the development of

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immunity within a temporally and spatially defined host population against an existing

However at the host population level, immunity is also a strong selective

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strain provides strong selection for an antigenically distinct strain that infects the host

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population regardless of the pre-existing immunity (11).

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We have been investigating how infection prevalence and associated population

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immunity drives strain structure using Anaplasma marginale, an α-proteobacterium that

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infects wild and domestic ruminants (12). A. marginale establishes persistent infection

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within an animal by antigenic variation in the immunodominant surface protein, Major

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Surface Protein (MSP)-2, which is generated via gene conversion using a genomic

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repertoire of distinct msp2 alleles (13-16).

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revealed that although A. marginale has a “closed core” genome with essentially the

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same gene content among all strains, the repertoire of msp2 alleles is distinct (17,18).

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Importantly, strains with a distinct msp2 allelic repertoire have been shown

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experimentally to be capable of strain superinfection, in which a second strain

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establishes infection in an animal already infected with a primary strain (15,19,20).

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Superinfection requires that the second strain contain at least one unique msp2 allele

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and that this be expressed at superinfection (19,21).

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experimental observations, we have more recently identified a significantly higher

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number and diversity in expressed msp2 alleles in A. marginale infections from high

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prevalence, high population immunity tropical regions as compared to low prevalence,

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low population immunity temperate regions (22).

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Genomic comparison of multiple strains

Consistent with these

The increase in msp2 allelic diversity in the high prevalence tropical regions may

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arise from two different scenarios (Fig. 1).

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establishes broad population immunity and thus selects for a distinct strain B that has a

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unique msp2 allelic repertoire. Alternatively, strain A may expand its allelic repertoire

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In the first, the theoretical strain A

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on the same genomic background to generate a closely related strain A’. This latter

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possibility is supported by msp2 allelic gene duplication within a single strain and the

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presence of differing numbers of msp2 loci and alleles among strains (16-19). Either

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strain A’ or B would generate the observed increase in the number and diversity of

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expressed msp2 alleles within the high prevalence tropical regions (22). In the present

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study, we address this question by developing and validating a multi-locus strain typing

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approach and testing whether strain superinfection in tropical regions is linked to strains

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with the same or different genomic backgrounds.

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Materials and Methods

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Identification of loci with strain-specific alleles: Target loci that had previously been

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shown to be broadly conserved among strains (17,18,23) were initially screened for

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strain-specific alleles using the complete genome sequences of the St. Maries

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(GenBank accession no. CP000030) and Florida strains (CP0011079).

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were then screened against the whole-genome shotgun data contigs from the

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Oklahoma (AFMX00000000.1), Washington-Okanogan (AFMW00000000.1) and South

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Idaho strains (AFMY00000000.1) with the corresponding target loci using blastn, blastx

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and tblastn analysis. Target sequences across the five strains were compiled using the

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VECTOR NTI software suite (Invitrogen) and single nucleotide polymorphisms (SNPs)

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and insertion/deletions (indels) identified. Forward and reverse primers were designed

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for the identical conserved regions flanking the most informative SNPs and indels:

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omp5, 5’-CTGGAAAGCTGCACAGGATG-3’ and 5’-CGACGCTTCCGCAAACATAC-3’;

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omp9, 5’-GGCAATTCCAATCATGTGCG-3’ and 5’-CAAGCTGTGAAGTCACTACACG-

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3’;

omp12,

5’-CTAGCGCTATGTTGCATGCATC-3’

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Candidates

and

5’-

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ACGCAAATTCAGATCACAGGG-3’; omp13, 5’-CAAGCAGATCCACAGCATCAATTC-3’

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and

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and 5’-CCACTTATTTCCACAATCTCCATGC-3’ (Supplemental Figure 1).

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regions are all 18 months) pastured in high prevalence, tropical regions in the Mexican states

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of Veracruz and Chiapas (26,27). Samples were collected in Medellin, Veracruz (19° 03'

Independent replicates of an uncloned strain (St.

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N, 96° 09' W; annual precipitation of 1,418 mm), La Concordia, Chiapas (16º 07' N, 92º

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41' W; annual precipitation of 1450 mm, and Tonala, Chiapas (16º 06' N, 93º 45' W;

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annual precipitation of 1953 mm). Samples from temperate areas were obtained from

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adult cattle (>18 months) pastured in temperate areas in the U.S.: Tonasket,

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Washington (48° 42' N, 119° 26' W; annual precipitation of 383 mm) and Burns, Oregon

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(43° 35' N, 119° 03' W; annual precipitation of 278 mm). Cattle had not been vaccinated

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to prevent A. marginale infection nor treated to clear infection.

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confirmed positive using either direct detection of A. marginale bacteremia or by

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serological screening using msp5 C-ELISA (VMRD). DNA was extracted, processed,

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and sequenced as described above.

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Results

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Identification of loci with strain-specific alleles:

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genomic background of a strain independent of the variable msp2 allelic repertoire were

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identified by screening the complete genome sequences of the St. Maries (GenBank

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CP000030) and Florida strains (CP0011079).

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encoded alleles specific to each strain (18,23). The single nucleotide substitutions

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between the two strains ranged from 1-46 with nucleotide differences due to indels

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ranged from 3-21 (Table 1). Each of these omps had previously been shown to be

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invariant within a strain during long-term persistent infection and among different

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animals infected with the same strain (18). The candidate loci were then compared

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among three additional strains, South Idaho, Washington-Okanogan, and Oklahoma,

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using sequences that had been generated by shotgun sequencing and previously

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deposited in GenBank (AFMY00000000.1, AFMX00000000.1, AFMW00000000.1).

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All samples were

Loci that would define the stable

Five omp loci were identified that

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While allelic identity was observed among individual loci between specific strain pairs,

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the inclusion of five target loci ensured that there were differences at multiple loci

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between any given strain pair (Table 1). Importantly, three of these strains (St. Maries,

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South Idaho, Washington-Okanogan) are from within the same region in the northwest

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U.S. and are transmitted both naturally and experimentally by the same vector,

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Dermacentor andersoni (25, 28-30), suggesting that the ability of the five targets to

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distinguish among strains is conserved even among closely related strains. Primers

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were designed in the conserved regions among these five strains to encompass the

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largest number of strain-specific SNPs and indels (Table 2; Supplemental Figure 1).

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Identification of single strain infections:

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detected by high throughput sequencing of the target alleles with the predicted allele

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represented by perfect matches. The paired read consensus sequences obtained for

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each allele ranged from 2,229 to 8,080: omp5 (2,769 sequences in the Florida strain to

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5,931 in the St. Maries strain); omp9 (2,229, Florida to 3,083, Washington-Okanogan);

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omp12 (2,908, St. Maries to 4,101, Florida); omp13 (3,342, Florida to 4,072, St. Maries);

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omp14 (4,371, Washington-Okanogan to 8,080, Florida).

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without a single nucleotide mismatch) were >90% for all alleles in all strains with a

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range from 90.4% for omp12 to 94.9% for omp9, both in the Washington-Okanogan

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strain.

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sequences generated: for the Florida strain there were 94.2% perfect match sequences

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out of 2100 total for omp9 and 92.7% perfect match sequences out of 8080 total for

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omp14.

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reported for a comparison set of four microbial genomes using MiSeq, reflecting the

The identity of each strain was accurately

Perfect matches (those

The percentage of perfect matches was independent of the number of

This percentage of perfect match sequences was higher than previously

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inclusion of only consensus sequences with no disagreement in overlap (31).

To

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determine the “technical error level” associated with sequencing of the five loci, clones

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of each target allele were obtained for the St. Maries strain and sequenced using the

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same procedure in three biological replicates.

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sequences for the cloned alleles were: omp5, 93.8±0.9; omp9, 93.9±1.3; omp12,

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92.4±0.4; omp13, 93.8±0.5; and omp14, 93.8±1.1.

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perfect match sequences for the five alleles as compared to previously published

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genomes (31) is reflected in the higher percentage using cloned omps, consistent with

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the designed sequence lengths being appropriate for strain differentiation using a multi-

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locus approach. The percentage of perfect match sequences for each allele was not

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significantly different between cloned omp and the same omp in the uncloned St. Maries

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strain: omp5, p=0.2; omp9, p=0.2; omp12, p=0.4; omp13, p=0.1; omp14, p=0.2 (two

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sample T-test assuming unequal variances). The criterion for identifying an allele was

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set at a proportion of perfect match sequences that exceeded the technical error level

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by a minimum of three standard deviations.

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Identification of multiple strain infections:

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would distinguish among strains, strain pairs were mixed to mimic strain superinfection

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and then sequenced. In each of three strain pairs, St. Maries and Florida, St. Maries

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and Washington-Okanogan, and Florida and Washington-Okanogan, each strain could

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be definitively identified (Table 3). When the omp allele differed between the two paired

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strains, the two alleles each were detected with a percentage of perfect matches that

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exceeded the technical error level in all cases. When the omp allele was common

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between the two paired strains (omp 5, St. Maries and Washington-Okanogan; omp 12

The percentage of perfect match

Thus the higher percentage of

To determine if the multi-locus approach

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and omp13, Florida and Washington-Okanogan), the common allele was detected with

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a percentage of perfect match sequences that exceeded 90% (Table 3).

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Strain composition in naturally occurring superinfections: Infections from two tropical

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regions in Mexico were examined, Veracruz and Chiapas. The infection prevalence

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within these high humidity, high temperature regions has been reported as the highest

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in Mexico, consistent with strong selective pressure for strain superinfection (26,27).

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Multi-locus typing revealed multiple omp alleles in at least one locus for 21/23 tropical

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region infections, 16/16 for all infections obtained in Veracruz and 5/7 from Chiapas

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(Supplemental Table 1). The number of unique alleles per infection, both for each

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individual locus and collectively across all loci, was significantly greater for tropical

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region infections as compared to temperate region infections (Table 4). This reflected

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both an increased percentage of loci with more than a single allele and an increase in

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the number of alleles at each locus (Table 4). Correspondingly, the maximal mean

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number of strains per animal in the tropical region was 3.3±1.2 with up to six strains in a

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single infected animal. This was significantly higher (p=0.0004, Students’ unpaired t-

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test) in the tropical regions as compared to infections in two temperate regions, with a

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maximal mean of 1.3±0.5 with a maximum of two strains in an individual infected

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animal.

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Discussion

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We accept that individual animals within endemic regions are infected with multiple

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strains of different genetic backgrounds. The following four lines of evidence support

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this conclusion. First, the multi-locus typing assay detected only one allele per target

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omp locus when applied to experimental single strain infections. Second, the assay

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detected the two predicted alleles in experimental paired strain infections and, when

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there was a common allele between two strains, only the common allele was detected.

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Unique alleles were detected with percentages of perfect sequence matches that

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exceeded the mean plus 3 standard deviations of the technical error level as

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determined for cloned omps and as represented by the omps in experimental single

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strain infections. Third, individual infections from the high prevalence tropical regions

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were identified that contained unique alleles for all five target omp loci. As each omp

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locus in itself represents an independent detection event, the probability that the unique

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alleles for all five target loci reflects sequencing error is extraordinarily low (p2 alleles at locus (maximum number) Tropical 2.4±1.0 (4) 19/23 10/23 omp 5a Temperate 1.3±0.5 (2) 3/8 0/8 Tropical 2.8±1.2 (5) 19/23 13/23 omp 9a Temperate 1.0±0.0 (1) 0/4 0/4 Tropical 2.7±1.1 (5) 18/23 13/23 omp 12a Temperate 1.1±0.4 (2) 1/8 0/8 Tropical 2.2±1.0 (4) 18/23 8/23 a omp 13 Temperate 1.1±0.4 (2) 1/8 0/8 Tropical 2.7±1.4 (6) 17/23 12/23 omp 14a Temperate 1.1±0.4 (2) 1/8 0/8 a The greater number of alleles per tropical region infection as compared to the temperate region infection was statistically significant for all loci collectively, p

Anaplasma marginale superinfection attributable to pathogen strains with distinct genomic backgrounds.

Strain superinfection occurs when a second pathogen strain infects a host already infected with a primary strain. The selective pressures that drive s...
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