Superinfection Exclusion of the Ruminant Pathogen Anaplasmamarginale in Its Tick Vector Is Dependent on the Time betweenExposures to the Strains

Susan M. Noh,a,b,c Michael J. Dark,b* Kathryn E. Reif,b Massaro W. Ueti,a Lowell S. Kappmeyer,a Glen A. Scoles,a Guy H. Palmer,b,c

Kelly A. Braytonb,c

Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington, USAa; Program in Vector-Borne Diseases, Departmentof Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USAb; The Paul G. Allen School for Global Animal Health, Washington StateUniversity, Pullman, Washington, USAc

ABSTRACT

The remarkable genetic diversity of vector-borne pathogens allows for the establishment of superinfection in the mammalianhost. To have a long-term impact on population strain structure, the introduced strains must also be transmitted by a vectorpopulation that has been exposed to the existing primary strain. The sequential exposure of the vector to multiple strains fre-quently prevents establishment of the second strain, a phenomenon termed superinfection exclusion. As a consequence, super-infection exclusion may greatly limit genetic diversity in the host population, which is difficult to reconcile with the high degreeof genetic diversity maintained among vector-borne pathogens. Using Anaplasma marginale, a tick-borne bacterial pathogen ofruminants, we hypothesized that superinfection exclusion is temporally dependent and that longer intervals between strain ex-posures allow successful acquisition and transmission of a superinfecting strain. To test this hypothesis, we sequentially exposedDermacentor andersoni ticks to two readily tick-transmissible strains of A. marginale. The tick feedings were either immediatelysequential or 28 days apart. Ticks were allowed to transmission feed and were individually assessed to determine if they wereinfected with one or both strains. The second strain was excluded from the tick when the exposure interval was brief but notwhen it was prolonged. Midguts and salivary glands of individual ticks were superinfected and transmission of both strains oc-curred only when the exposure interval was prolonged. These findings indicate that superinfection exclusion is temporally de-pendent, which helps to account for the differences in pathogen strain structure in tropical compared to temperate regions.

IMPORTANCE

Many vector-borne pathogens have marked genetic diversity, which influences pathogen traits such as transmissibility and viru-lence. The most successful strains are those that are preferentially transmitted by the vector. However, the factors that determinesuccessful transmission of a particular strain are unknown. In the case of intracellular, bacterial, tick-borne pathogens, one po-tential factor is superinfection exclusion, in which colonization of ticks by the first strain of a pathogen it encounters preventsthe transmission of a second strain. Using A. marginale, the most prevalent tick-borne pathogen of cattle worldwide, and its nat-ural tick vector, we determined that superinfection exclusion occurs when the time between exposures to two strains is brief butnot when it is prolonged. These findings suggest that superinfection exclusion may influence strain transmission in temperateregions, where tick activity is limited by season, but not in tropical regions, where ticks are active for long periods.

Vector-borne, antigenically variant bacterial and protozoalpathogens that establish persistent infection, including those

in the genera Anaplasma, Borrelia, Plasmodium, and Trypano-soma, have remarkable amounts of genetic diversity (1–4), whichenables establishment of persistent infection and ongoing trans-mission within a host population. As a consequence of this geneticdiversity within the population, individual mammalian hosts maybe infected with multiple genotypically distinct strains (5–9). Atthe host population level, the success of onward transmission ofdistinct strains determines the overall microbial strain structureover time and space.

Infection of an individual host with multiple strains may occurthrough coinfection, the simultaneous transmission of more thanone strain, or by superinfection, i.e., sequential infection by dis-tinct strains. Within the mammalian host, the adaptive immunesystem has a strong influence on strain structure, especially in thecase of superinfection, where a second strain has to evade theexisting immune response generated against a primary infectingstrain (10, 11). From an epidemiological standpoint, the ability of

a newly introduced strain to overcome preexisting immunity inthe mammalian host and establish infection ensures a large pop-ulation of susceptible hosts. However, to have a long-term impacton strain structure within the population, the introduced second

Received 19 January 2016 Accepted 15 March 2016

Accepted manuscript posted online 18 March 2016

Citation Noh SM, Dark MJ, Reif KE, Ueti MW, Kappmeyer LS, Scoles GA, Palmer GH,Brayton KA. 2016. Superinfection exclusion of the ruminant pathogen Anaplasmamarginale in its tick vector is dependent on the time between exposures to thestrains. Appl Environ Microbiol 82:3217–3224. doi:10.1128/AEM.00190-16.

Editor: H. L. Drake, University of Bayreuth

Address correspondence to Susan M. Noh, susan.noh@usda.ars.gov.

* Present address: Michael J. Dark, Department of Infectious Diseases andPathology, College of Veterinary Medicine, University of Florida, Gainesville,Florida, USA.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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strain would also need to be transmitted by a vector populationthat has been exposed to the existing primary strain.

The outcomes of vector-borne acquisition and transmissionfor hosts simultaneously exposed to multiple pathogen strainsvary by the specific pathogen and vector pairing. For example,with some exceptions, the overall genetic diversity of West Nilevirus populations is not restricted in the mosquito vector. In onestudy, genetic diversity was reduced in the midgut but recoveredin the salivary glands (12). In a second study, decreased geneticdiversity occurred in the midgut and salivary glands, but onlywhen minor variants were present in the population (13). In thecase of tick vectors, feeding of nymphal Dermacentor andersoniticks on mice infected with up to 90 different genetic variants ofFrancisella novicida resulted in a marked loss of variant diversity,which was attributed to competition for growth-limiting re-sources and to a stochastic bottleneck (14). D. andersoni can ac-quire and readily transmit two strains of Anaplasma marginale byfeeding on an animal infected with those two strains (15). Unlikethe simultaneous exposure to multiple strains under conditions ofcoinfection, the sequential exposure of the vector to multiplestrains frequently prevents establishment of the second strain, aphenomenon termed superinfection exclusion (16–18), and onlythe primary strain is transmitted. As a consequence, superinfec-tion exclusion has the potential to greatly limit genetic diversity inthe host population, which is difficult to reconcile with the ob-served high degree of genetic diversity maintained among manyvector-borne pathogens (6, 19–23).

Prior studies have documented significant differences in A.marginale strain structure between temperate and tropical re-gions, environments that dramatically influence vector ecology.Individual hosts in tropical regions, characterized by long periodsof warm temperatures and high relative humidity, carry multiplegenetically distinct A. marginale strains, and there is broad heterogeneity at the population level (2, 6, 24). In contrast, hosts intemperate regions are most commonly infected with only a singleA. marginale strain, and the overall population strain structure ishomogeneous (25, 26). Unlike temperate regions, in which peakvector activity occurs in a narrow seasonal window, tropical envi-ronments allow prolonged vector activity, during which adultmale ticks, primarily responsible for A. marginale transmission,can intermittently feed on multiple hosts over time, increasing thelikelihood of exposure and potential acquisition of a second strain(27). However, in order to influence strain structure in the popu-lation, the second strain would need to both colonize the tick andbe transmitted, events incompatible with superinfection exclu-sion. We hypothesized that superinfection exclusion is temporallydependent and that longer intervals between strain exposures al-low successful acquisition and transmission of a second, superin-fecting strain. Herein we report testing of the impact of the tem-poral interval on superinfection exclusion and discuss the resultsin the context of the consequences for pathogen strain structure.

MATERIALS AND METHODSHost infection. All protocols involving the use of animals were approvedby the Washington State University and University of Idaho InstitutionalAnimal Care and Use Committees (ASAF numbers 2732 and 2007-47).Age-matched, A. marginale-seronegative Holstein calves were inoculatedintravenously with either the South Idaho or St. Maries strain of A. mar-ginale. Infection was tracked by daily determination of packed cell volume

and microscopic examination of Giemsa-stained blood smears to identifyA. marginale-infected erythrocytes.

Tick feeding. Adult male D. andersoni ticks (Reynold’s Creek colony)were used in all experiments because they are primarily responsible for thetransmission of A. marginale. Both A. marginale strains readily infect andare transmitted by these ticks (28–30). Ticks were allowed to feed sequen-tially on hosts infected with either the St. Maries strain or the South Idahostrain and subsequently allowed to feed on a host infected with the recip-rocal strain. Control ticks were exposed to only a single strain. In the firstset of experiments, ticks were allowed to acquisition feed for 3 days duringacute infection (�108 bacteria/ml) on an animal infected with the SouthIdaho strain (animal 1257) or the St. Maries strain (animal 1259). At thesame time, control ticks were fed identically on an uninfected animal(animal 1258). The individual cohorts of ticks fed on animals infectedwith the first strain were then immediately transferred to animals persis-tently infected (�107 bacteria/ml; �0.1% infected erythrocytes) with thereciprocal strain, either the St. Maries strain (animal 1254) or the SouthIdaho strain (animal 1244). The control ticks were allocated to two co-horts and immediately transferred to the same persistently infected ani-mals. All ticks were removed after an additional 3 days of feeding and thenheld for 7 days at 26°C and 94% relative humidity to allow for digestion ofthe blood meal. Ticks were then dissected, and isolated midguts and sali-vary glands were analyzed for infection (n � 30/group). An additional 30ticks per cohort were allowed to transmission feed on individual serone-gative animals. Following the 7-day transmission feed, all ticks were dis-sected and their midguts and salivary glands analyzed.

In the second set of experiments, the design was similar, with theexception that the interval between the first tick feeding and the secondwas 28 days and the length of tick feeds was 7 days. Specifically, ticks wereinitially fed for 7 days on an animal acutely infected (�108 bacteria/ml)with the South Idaho (animal 1207) or St. Maries strain (animal 1240).Removed ticks were held at 26°C and 94% relative humidity for 28 daysand then allowed to feed for an additional 7 days on animals persistentlyinfected with the reciprocal strain, either the St. Maries (animal 1199) orSouth Idaho (animal 1219) strain, prior to analysis.

Strain-specific detection. The strain-specific infection rate (propor-tion of ticks infected with each strain) and evidence of successful trans-mission were determined by PCR followed by Southern blotting. TheSouth Idaho and St. Maries strains of A. marginale are genetically distinctat multiple loci and can be distinguished based on the number and se-quence of tandem repeats in msp1� (25, 26, 31, 32). Importantly, msp1� isinvariant within both the tick and the mammalian host (32). DNA wasextracted from blood, tick salivary gland pairs, and midguts as previouslydescribed (15). A 552-bp segment of the St. Maries strain and a 664-bpsegment of the South Idaho strain were amplified using the forwardmsp1� primer (5=-TTATGGCAGACATTTCCATATACTGTGCAG) andthe reverse msp1� primer (5=-GGAGCGCATCTCTCTTGCC) (26).Probes for msp1� were amplified with the above primers, labeled withdigoxigenin, and used in Southern hybridization assays as previously de-scribed (33).

Statistical analysis. The data were treated as categorical, and a contin-gency analysis was done using JMP, version 10.0.0 (SAS Institute Inc.,Cary, NC). The Pearson chi-square value was calculated and Fisher’s two-tailed exact test used to determine statistical significance.

RESULTSStrain exclusion in the vector following immediate exposure toa second strain. In the first replicate, ticks were allowed to initiallyfeed on an animal infected with the South Idaho strain and thenimmediately exposed to the St. Maries strain (SI/SM ticks). Con-trol ticks were exposed first to an uninfected animal and then tothe St. Maries strain (C/SM ticks). After the acquisition feed, therewas no significant difference (P � 0.27) in the St. Maries infectionrate in the midguts of the SI/SM ticks (73%) compared to theC/SM ticks (60%) lacking exposure to the primary South Idaho

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strain (Fig. 1A). However, there was a marked difference in thesalivary glands. The St. Maries strain infection rate in the C/SMticks was 80%, versus only 7% in the SI/SM cohort (Fig. 1B). This91% reduction in the St. Maries strain as the potentially superin-fecting strain was statistically significant (P � 0.0001). This pat-tern was consistent in the midguts and salivary glands followingtransmission feeding on a naive animal. Specifically, there was nosignificant difference (P � 0.18) in the St. Maries strain infectionrate in the midguts of transmission-fed SI/SM ticks (52%) com-pared to transmission-fed C/SM ticks (69%) (Fig. 1C). There wascomplete exclusion of the St. Maries strain in the salivary glands oftransmission-fed SI/SM ticks (P � 0.0001) (Fig. 1D), while the St.Maries strain infection rate in the transmission-fed C/SM cohortwas 69%.

In the reciprocal experiment, ticks were first acquisition fed ona St. Maries strain-infected animal and then immediately exposedto the South Idaho strain (SM/SI ticks). Control ticks were ex-posed first to an uninfected animal and then to the South Idahostrain (C/SI ticks). In the control ticks (C/SI), the infection rate forthe South Idaho strain was 73% in the midgut and 57% in thesalivary glands (Fig. 2A and B). In marked contrast, none of SM/SItick midguts or salivary glands were infected with the South Idahostrain (Fig. 2A and B). These differences were both statisticallysignificant (P � 0.0001). This did not indicate refractoriness of theticks to infection with either strain, as noted by the permissiveness

of the control ticks for the South Idaho strain (Fig. 2A and B) andthe high infection rates (�97%) in both the midgut and the sali-vary gland for the primary St. Maries strain (Table 1). This patternwas maintained following transmission feeding on a naive animalin that there was complete exclusion of the South Idaho strain inboth the midgut and the salivary glands (P � 0.0001) (Fig. 2C andD). For comparison, the infection rates in the transmission-fedC/SI cohort were 33% and 50% for the midgut and salivary glands,respectively. The infection rate for the primary strain, St. Maries,was �97% for both the midgut and salivary glands, indicating thatthe ticks were permissive to A. marginale colonization (Table 1).

Strain exclusion during transmission. The complete exclu-sion from the salivary glands under conditions of immediate ex-posure to the second strain was supported by transmission feedingof ticks on naive animals. Only the primary strain was detected inthe bovine host following transmission feeding of ticks, regardlessof whether it was the SM/SI or SI/SM cohort of ticks. As expected,the control ticks that had been exposed to only one strain (C/SMor C/SI ticks) transmitted only the St. Maries or South Idahostrain, respectively (Table 1).

Strain exclusion in the vector following delayed exposure to asecond strain. Ticks were allowed to initially feed on an animalinfected with the South Idaho strain and then held for 28 days at

FIG 1 Strain exclusion in the vector following immediate exposure to a sec-ond strain in both acquisition-fed (AF) and transmission-fed (TF) ticks. Theblack bars represent the infection rates in the tick midgut (A and C) andsalivary glands (B and D) for the St. Maries strain in the ticks exposed only tothe St. Maries strain (C/SM) or the ticks exposed first to the South Idaho strainand then to the St. Maries strain (SI/SM). *, P � 0.0001.

FIG 2 Strain exclusion in the vector following immediate exposure to a sec-ond strain in both acquisition-fed (AF) and transmission-fed (TF) ticks. Thegray bars represent the infection rates in the tick midgut (A and C) and salivaryglands (B and D) for the South Idaho strain in the ticks exposed only to theSouth Idaho strain (C/SI) or the ticks exposed first to the St. Maries strain andthen to the South Idaho strain (SM/SI). *, P � 0.0001.

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26°C and 94% relative humidity before being exposed to the St.Maries strain (SI/SM). The infection rates for the St. Maries strainin these ticks were 90% and 70% for the midguts and salivaryglands, respectively (Fig. 3A and B). The infection rates in thecontrol ticks (C/SM), which were not first exposed to the SouthIdaho strain but otherwise were handled identically, were 93%and 83% for the midguts and salivary glands, respectively. Thesedifferences were not statistically significant (P � 0.64 for themidgut; P � 0.22 for the salivary glands).

Analysis of individual ticks, including only those infected, re-vealed that 64% of midguts and 66% of salivary glands were in-fected with both strains, demonstrating superinfection within in-dividual ticks (Fig. 3C and D). Consistent with this high level ofsuperinfection in the salivary glands, a cohort of SI/SM ticks wereallowed to transmission feed for 7 days on a naive animal, andboth strains established infection in the bovine host (Table 1).

The occurrence of superinfection within the vector was repli-cated using the reciprocal order of the strain pair. In the SM/SIticks, the infection rates for the South Idaho strain in the midgutsand salivary glands were 43% and 53%, respectively (Fig. 4A andB). These rates were not significantly different (P � 0.05) fromthose for the control C/SI ticks, in which the infection rates were67% and 77% for the midgut (P � 0.07) and salivary glands (P �0.06), respectively. Similar to the SI/SM cohort and including onlyinfected ticks, superinfection was common, with 39% of midguts

and 50% of salivary glands being infected with both strains (Fig.4C and D).

DISCUSSION

We accept the hypothesis that A. marginale strain superinfectionexclusion in the tick vector is temporally dependent. This is sup-ported by the following three lines of evidence: (i) statisticallysignificant exclusion of the second strain occurred when the ex-posure interval was brief but not when the interval was prolonged,(ii) both midguts and salivary glands of individual ticks were su-perinfected when the exposure interval was prolonged, and (iii)transmission of two strains occurred only when the exposure in-terval was prolonged. The occurrence of superinfection exclusionand the failure to transmit the second strain following sequentialexposure are consistent with a previous study using geneticallydistinct Virginia and Oklahoma strains in adult male Dermacentorvariabilis ticks (17). This confirmation of superinfection exclusionwithin a short time window is in agreement with population-based studies of A. marginale strain structure (2, 6). Vallejo Es-querra et al. reported that infected cattle, a natural host for A.marginale, in tropical regions of Mexico carried multiple strains(mean of 3.3 � 1.2 strains per animal), with up to six strainsdetected in an individual (2). In contrast, most cattle in temperateregions were infected with a single strain (mean of 1.3 � 0.5

TABLE 1 Infection rates in tick midguts and salivary glands

Cohort

% infected ticksa

Transmissiond

Midgut Salivary glands

SMonly

SMtotalb

SIonly

SItotalc

Bothstrains

Totalinfectionrate

SMonly

SMtotalb

SIonly

SItotalc

Bothstrains

Totalinfectionrate

Acquisition feeding,no hold

C/SM 60 60 0 0 0 60 80 80 0 0 0 80 NASI/SM 0 73 20 93 73 93 0 7 90 97 7 97 NAC/SI 0 0 73 73 0 73 0 0 57 57 0 57 NASM/SI 97 97 0 0 0 97 100 100 0 0 0 100 NA

Transmission feeding,no hold

C/SM 69 69 0 0 0 69 69 69 0 0 0 69 SMSI/SM 3 52 48 97 48 100 0 0 100 100 0 100 SIC/SI 0 0 33 33 0 33 0 0 50 50 0 50 SISM/SI 100 100 0 0 0 100 97 97 0 0 0 97 SM

Acquisition feeding,28-day hold

C/SM 93 93 0 0 0 93 83 83 0 0 0 83 NASI/SM 30 90 3 63 60 93 7 70 27 90 63 97 NASM/SI 50 87 7 43 37 93 40 87 7 53 47 93 NAC/SI 0 0 67 67 0 67 0 0 77 77 0 77 NA

Transmission feeding,28-day hold

SM/SI 59 88 0 29 29 88 47 88 0 41 41 88 SM, SIa SM, St. Maries strain; SI, South Idaho strain.b Includes superinfected ticks and ticks infected only with the St. Maries strain.c Includes superinfected ticks and ticks infected only with the South Idaho strain.d Strain(s) transmitted to a naive host. NA, not applicable.

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strains per animal), with a maximum of two strains in an individ-ual (25, 26).

The epidemiology of tick-borne disease is complex and istightly linked to the life cycle of the tick. Regardless, in both trop-ical and temperate regions, adult male ticks serve as the primaryvectors because they feed on multiple hosts while seeking females(27). In temperate regions, Dermacentor spp., which are three-host ticks, are the primary vectors of A. marginale. While larvaeand nymphs are competent vectors, they preferentially feed onrabbits and rodents and thus are unlikely to be exposed to A.marginale. In tropical regions, the one-host ticks Rhipicephalusspp. are common vectors of A. marginale, and all life stages feed oncattle and remain on the host during molting. The only life stagelikely to move between animals, and thus to transmit the patho-gen, is the male as it searches for mates. In temperate regions, adultmale ticks are active during a relatively brief period of severalweeks in May and early June, when temperatures are warm andhumidity is relatively high. Consequently, transmission occurs fora limited time each year, and superinfection exclusion in the vec-tor likely contributes to the maintenance of low strain diversity inthe host population. However, within tropical regions, ticks maybe continuously active for several months, allowing for super-infection in the tick and subsequent simultaneous transmis-sions of multiple strains, resulting in coinfection of the ani-mals. Once animals within a herd become coinfected, ticks canthen acquire multiple strains through either superinfection orcoinfection, and the number of animals acquiring multiple

strains rapidly increases. Ultimately, both coinfection and su-perinfection play a role in the establishment of multiple strainsper animal (Fig. 5).

Although the temporal pattern was observed for each of thereciprocal strain pair replicates, there was evidence of strain dif-ferences. Specifically, when exposure to the two strains was imme-diately sequential and the St. Maries strain was the primary strain,the South Idaho strain was completely excluded from both themidguts and salivary glands. In contrast, the St. Maries strain wasable to establish superinfection in the tick midgut when the SouthIdaho strain was the primary strain. Including only infected ticks,79% of the midguts and 7% of the salivary glands were superin-fected following the SI/SM immediately sequential acquisitionfeeding. Although this did not translate into successful transmis-sion of the St. Maries strain, this greater level of successful super-infection may result in transmission for intermediate exposuretimes.

Evidence of strain hierarchy is also supported by the strongerability of the St. Maries strain to establish superinfection than thatof the South Idaho strain following the 28-day exposure interval.There was only a 3% drop in the St. Maries infection rate in themidgut when St. Maries served as the secondary strain comparedto when it was the only strain (Fig. 3A). In contrast, there was a24% drop in the South Idaho infection rate when it served as thesecondary strain compared to when it served as the only strain(Fig. 4A). Similarly, in the salivary glands, there was a 13% de-crease in St. Maries strain infection when it served as the second-

FIG 3 Lack of strain exclusion and establishment of superinfection in thevector following delayed exposure to the St. Maries strain. (A and B) Blackbars represent the infection rates in the tick midgut (A) and salivary glands(B) for the St. Maries strain in the ticks exposed only to the St. Maries strain(C/SM) or the ticks exposed first to the South Idaho strain and then to theSt. Maries strain (SI/SM). (C and D) Analyses of individual midguts andsalivary glands indicated that the secondary strain, in this case, St. Maries,was able to establish superinfection, as represented by stripes in the piechart. The gray regions represent the proportions of tissues infected withthe South Idaho strain only, and the black regions represent tissues infectedwith the St. Maries strain only.

FIG 4 Lack of strain exclusion and establishment of superinfection in thevector following delayed exposure to the South Idaho strain. (A and B) Graybars represent the infection rates in the tick midgut (A) and salivary glands (B)for the South Idaho strain in the ticks exposed only to the South Idaho strain(C/SI) or the ticks exposed first to the St. Maries strain and then to the SouthIdaho strain (SM/SI). (C and D) Analyses of individual midguts and salivaryglands indicated that the secondary strain, in this case, South Idaho, was able toestablish superinfection, as indicated by stripes in the pie charts. The grayregions represent the proportions of tissues infected with the South Idahostrain only, and the black regions represent tissues infected with the St. Mariesstrain only.

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ary strain compared to when it was the only strain (Fig. 3B) and a24% drop in the South Idaho strain (Fig. 4B) when it served as thesecondary strain. These observations may underlie, at least in part,the predominance of specific strains within the host populationeven when there is evidence of multiple strains circulating amonghosts. While there is no evidence of a competitive transmissionadvantage for specific strains during either acute or persistent bac-teremia in the host, marked strain differences resulting in prefer-ential strain transmission can occur within the vector. Both themidgut and salivary glands have been reported to serve as barriersthat select for highly transmissible strains (34). These data suggestthat the salivary glands may be a more stringent barrier than themidgut, as the St. Maries strain was able to establish a high level ofsuperinfection in the midgut, but not in the salivary glands, in thepresence of the South Idaho strain. The differences observed bothbetween strains and between tick organs in preferential superin-fection of these sites by the St. Maries strain support the role of thevector in population strain structure.

The mechanisms that allow or block strain superinfection inthe time-dependent and, to a lesser degree, tick organ-specificfashion described in the present work are unknown and representa significant knowledge gap. Within the arthropod vector, a sec-ond strain may be excluded due to a limited number of host cells,particularly in the salivary glands. Alternatively, antibody in the

blood meal may block uptake of the second strain in the midgut.Other possibilities include changes in midgut or salivary glandphysiology, entry receptor cycling, an induced innate immuneresponse, competition for limited nutrients, or a combination ofthese processes. Transcriptomic analysis of tick responses to A.marginale infection revealed upregulated candidates in multiplecellular pathways (35). Of these, three genes with high identity tokey metabolic pathway enzymes allowed enhanced A. marginaleinfection rates when silenced using small interfering RNA(siRNA), consistent with a normal role in limiting infection.These genes include TC22382, which has significant identity toarthropod NADH-ubiquinone reductases and is thought to re-spond to increased energy demands. TC17129 has a conservedregion of glutamine synthetase, which plays a role in nitrogenmetabolism. TC16059 has identity to arthropod aldehyde dehy-drogenase and may play a role in NAD(P)�-dependent enzymesthat play a wide role in metabolic pathways. Importantly, thetime-dependent nature of superinfection exclusion suggests thatthe limiting resource regenerates over time, as a nutrient or mem-brane receptor would. Examining how vector molecules and path-ways are regulated over time provides a new approach to identifydeterminants of superinfection exclusion as well as possible tar-gets to block transmission.

FIG 5 Impacts of superinfection exclusion (A) and superinfection permissiveness (B) in the tick on strain structure in the animal host population. The modelinitiates with two strains in the host population (individual hosts are indicated by boxes) and shows the predominant strain (blue boxes), the minor or recentlyintroduced strain (yellow boxes), and uninfected hosts (gray boxes). Individual ticks are represented by ovals and were infected with a single strain or, underpermissive conditions, superinfected (divided circles). Over time, permissiveness for strain superinfection in the tick leads to a prevalence of hosts carrying bothstrains and to both superinfected ticks and coinfected ticks (gradient circles), resulting in the majority of hosts carrying more than a single strain due tosuperinfection (divided boxes) or coinfection (gradient boxes). The outcome in panel A is representative of infected hosts in temperate regions, where 94 to 100%of hosts have been reported to carry only a single strain and the mean number of strains per host is 1.3 � 0.5, with a maximum of two strains in a single host (2,25). The outcome in panel B is representative of infected hosts in tropical regions, where �75% of hosts have been reported to carry multiple strains, with meannumbers of strains ranging from 2.1 � 1.4 to 3.5 � 2.1 and with up to six strains in a single host (24).

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ACKNOWLEDGMENTS

We acknowledge the excellent technical help of Xiaoya Cheng and DeirdreMercado Ducken. We appreciate the expertise of Ralph Horn, James Al-lison, and Kathy Mason in animal care and handling and tick rearing.

FUNDING INFORMATIONThis work, including the efforts of Susan M. Noh, Kathryn Elizabeth Reif,Massaro W. Ueti, Lowell Kappmeyer, and Glen A. Scoles, was funded byUnited States Department of Agriculture (5348-32000-027-00D and5348-32000-033-00D). This work, including the efforts of Kelly A. Bray-ton, was funded by United States Department of Agriculture-NRICGP(2005-35604-15440). This work, including the efforts of Michael J. Dark,was funded by HHS | National Institutes of Health (NIH) (K08AI064162). This work, including the efforts of Guy H. Palmer, was fundedby HHS | National Institutes of Health (NIH) (R37 AI44005).

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