Abstract
Multiple-strain pathogens often establish mixed infections inside the host that result in competition between strains. In vector-borne pathogens, the competitive ability of strains must be measured in both the vertebrate host and the arthropod vector to understand the outcome of competition. Such studies could reveal the existence of trade-offs in competitive ability between different host types. We used the tick-borne bacterium Borrelia afzelii to test for competition between strains in the rodent host and the tick vector, and to test for a trade-off in competitive ability between these two host types. Mice were infected via tick bite with either one or two strains, and these mice were subsequently used to create ticks with single or mixed infections. Competition in the rodent host reduced strain-specific host-to-tick transmission and competition in the tick vector reduced the abundance of both strains. The strain that was competitively superior in host-to-tick transmission was competitively inferior with respect to bacterial abundance in the tick. This study suggests that in multiple-strain vector-borne pathogens there are trade-offs in competitive ability between the vertebrate host and the arthropod vector. Such trade-offs could play an important role in the coexistence of pathogen strains.
Keywords: Borrelia afzelii, co-infection, inter-strain competition, Ixodes ricinus, life-history trade-off, transmission
1. Introduction
Many populations of pathogens consist of genetically distinct strains. As a result of this strain diversity, hosts are often infected with multiple strains, a phenomenon known as co-infection, mixed infection or multiple-strain infection [1–3]. Co-infection implies that the strains can interact with each other, which can result in cooperation or competition [3–5]. Empirical evidence for inter-strain competition typically shows that the performance (e.g. abundance, transmission) of a given strain in a co-infection is reduced compared with when it occurs alone [6–10]. Pathogen strains with similar ecological niche requirements will experience intense competition, which can result in competitive exclusion and loss of strain diversity [11–13]. Thus a fundamental task is to understand the mechanisms that can maintain strain diversity in the face of strong within-host competition, and one potential explanation is life-history trade-offs [5,14].
A commonly assumed life-history trade-off for pathogens is between transmission and virulence [15,16], which is essentially a trade-off between current and future reproduction [14]. In mixed infections, competition between strains should select for fast-growing virulent strains that can monopolize the limited host resources and thereby outcompete slow-growing avirulent strains [6,8,17–20]. At the pathogen population level, a diversity of strains can be maintained because virulent strains outcompete avirulent strains in mixed infections, but the reverse is true in single-strain infections [21,22]. Other life-history trade-offs that have been reported for pathogens include trade-offs between multiplication rate (and presumably competitive ability) and persistence in the abiotic environment [23,24], and between competitiveness and ability to colonize new hosts [25]. Given the variety of pathogen life cycles, we should expect a similar diversity of trade-offs between competitive ability and other life-history traits.
Trade-offs in competitive ability might be particularly common in parasites that must infect more than one host type to complete their life cycle [2]. In these complex or vector-borne life cycles, the genes and phenotypes that lead to competitive success in one host type (e.g. vertebrate host) may have little bearing on within-host competition in another host type (e.g. arthropod vector). Despite the plausibility of this idea, there are few studies that have compared the strain-specific competitive ability between different host types [26]. In the case of vector-borne pathogens, numerous studies have investigated interactions between strains in the vertebrate host [6,10,12,27–31], but similar studies in the arthropod vector are rare [32–35], and we are not aware of any studies that have investigated both host types. To investigate these questions, we used Borrelia afzelii, a spirochaete bacterium that requires both a vertebrate host and an arthropod vector to complete its life cycle, as a model system.
Borrelia afzelii belongs to the Borrelia burgdorferi sensu lato (sl) genospecies complex, which includes the aetiological agents of Lyme borreliosis in North America and Eurasia [36,37]. In Europe, Borrelia afzelii is a common genospecies [36,38,39], which is transmitted by the hard tick Ixodes ricinus and uses rodents as reservoir hosts [38,39]. The life cycle of I. ricinus consists of three stages: larva, nymph and adult. The larvae acquire B. afzelii from infected rodents during the larval blood meal, and develop into infected nymphs that transmit the pathogen back to the reservoir host population the following year. The engorged larva and the resultant nymph are therefore the two stages where interactions between strains are most ecologically important. Populations of B. burgdorferi sl consist of multiple strains and mixed infections are common in both the vertebrate host [10,29,40,41] and the tick vector [41–44]. Competition between strains of B. burgdorferi sl in the vertebrate host has been shown in field studies [10,29] and experimental infections [9,45,46]. Field studies on our local I. ricinus population found that approximately 80% of B. afzelii-infected nymphs were infected with multiple strains and that the mean strain richness was 2.4–2.9 strains per nymph [42,44]. In co-infected nymphs, the spirochaete load per strain decreased with increasing strain richness, and this result provides indirect evidence for competition [44]. However, to date there is no direct experimental evidence that competition between strains of B. burgdorferi sl can occur in the tick vector.
The purpose of this study was to test whether strains of B. burgdorferi sl compete inside their rodent host and their tick vector, and whether there was a trade-off in strain-specific competitive ability between the two host types. Mice were infected via tick bite with either one or two strains of B. afzelii. The infected mice were infested with larval ticks and these were allowed to moult into nymphal ticks that carried either single-strain infections or co-infections. The mouse-to-tick transmission success and the spirochaete load in the nymph were used as measures of competitive success in the rodent host and the tick vector, respectively. We predicted that inter-strain competition in the mouse and the nymph would reduce the strain-specific mouse-to-tick transmission success and the strain-specific spirochaete load, respectively. We also predicted that the strain that was competitively superior in the rodent host would be competitively inferior in the tick vector, and vice versa.
2. Material and methods
(a). Mice, ticks, and strains of Borrelia afzelii
Forty female, pathogen-free Mus musculus BALB/c mice aged five weeks were used as the rodent reservoir host. All I. ricinus ticks came from the Borrelia-free laboratory colony that has been maintained at the University of Neuchâtel since 1978. Borrelia afzelii isolates Fin-Jyv-A3 and NE4049 were used in this study, which were obtained from a bank vole (Myodes glareolus) in Finland and an I. ricinus nymph in Switzerland, respectively. We had originally started the study with two Swiss strains, but one of the strains failed and we used strain Fin-Jyv-A3 as a back-up solution. Fin-Jyv-A3 has ospC major group (oMG) A3, multi-locus sequence type (MLST) 676, and strain ID number 1961 in the Borrelia MLST database. Isolate NE4049 has oMG A10, MLST 679, and strain ID number 1887 in the Borrelia MLST database. The purity of these isolates with respect to the oMG allele has been assessed using 454-sequencing. For isolates Fin-Jyv-A3 and NE4049, 137 and 1313 ospC gene sequences were obtained, respectively, and all but one belonged to the correct oMG. We are confident that these isolates are genetically homogeneous and will hereafter refer to them as strains Fin-Jyv-A3 and NE4049.
I. ricinus nymphs infected with either strain Fin-Jyv-A3 or strain NE4049 were created as follows. Female BALB/c mice (n = 5) were infected with one of the two strains via needle inoculation. At four weeks post-infection, Borrelia-free larval ticks from our laboratory colony of I. ricinus were fed on these mice. Engorged larval ticks were placed in individual eppendorf tubes and were allowed to moult into nymphs. At four weeks after the larva-to-nymph moult, a random sample of nymphs was selected for each strain and tested for B. afzelii infection using qPCR. The percentage of nymphs infected with B. afzelii was 70% (7/10) for strain Fin-Jyv-A3 and 71.4% (10/14) for strain NE4049.
(b). Infection of mice via tick bite with one or two strains
The study consisted of experiments 1 and 2, where the focal strains were Fin-Jyv-A3 and NE4049, respectively. In experiment 1, mice were randomly assigned to infection with strain Fin-Jyv-A3 (n = 10 mice) or to co-infection with strains Fin-Jyv-A3 and NE4049 (n = 10 mice). In experiment 2, mice were randomly assigned to infection with strain NE4049 (n = 10 mice) or to co-infection with strains NE4049 and Fin-Jyv-A3 (n = 10 mice). All mice were infected via tick bite. Mice in the co-infection treatments were infested with 5 Fin-Jyv-A3-infected nymphs and 5 NE4049-infected nymphs. Mice in the single-strain infection treatments were infested with 5 Fin-Jyv-A3-infected nymphs or 5 NE4049-infected nymphs. Each mouse in the single-strain infection treatment was also infested with five uninfected nymphs. When nymphs take a blood meal, they secrete saliva that contains immunosuppressive molecules [47]; for this reason, each mouse in the study was infested with the same number of nymphs. Details of the tick infestation procedure have been described elsewhere [48].
Four weeks after the nymphal challenge, ear tissue biopsies and blood samples were taken from each mouse to confirm their B. afzelii infection status [48]. A Borrelia-specific qPCR assay was used on the ear tissue samples to determine the presence of B. afzelii spirochaetes. A Serion Elisa classic Borrelia burgdorferi IgG/IgM immunoassay was used on the blood samples to determine the presence of Borrelia-specific IgG antibodies. The two infection phenotypes were 100% congruent.
(c). Host-to-tick transmission
Five weeks after the nymphal challenge, each mouse was infested with approximately 100 Borrelia-free xenodiagnostic larvae from our laboratory colony of I. ricinus. Blood-engorged larvae were kept in individual Eppendorf tubes, and were allowed to moult into xenodiagnostic nymphs under standard laboratory conditions (20–25°C, 12 h light : 12 h dark). To maintain high humidity, each tube contained a piece of moistened paper towel. Four weeks after the larva-to-nymph moult, 10 live nymphs were randomly selected from each mouse and frozen at −20°C. To test whether the spirochaetes were viable, we placed up to three xenodiagnostic nymphs in BSK medium for all B. afzelii-infected mice that had sufficient numbers of ticks. The cultures were checked using a dark field microscope on a weekly basis for the presence of live spirochaetes.
(d). DNA extraction
Four-week-old nymphs were crushed in the TissueLyser II using a previously described protocol [48]. The crushed nymphs were digested with proteinase K at 56°C overnight. The DNA of the nymphs was extracted using Qiagen DNeasy 96 Blood and Tissue kit well plates and following the Qiagen protocol. Each plate contained two negative DNA extraction controls (Anopheles gambiae mosquitoes). DNA from the mouse ear samples was extracted using Qiagen DNeasy Blood & Tissue mini spin columns and following the Qiagen protocol. Ear tissue DNA and nymph DNA were eluted into 65 µl of water.
(e). General and strain-specific qPCR assays
Each tick was tested with three independent qPCR assays. A Borrelia-diagnostic qPCR assay that targets a 132 bp fragment of the highly conserved flagellin gene was used to determine infection and quantify the total spirochaete load in each nymph. We developed two strain-specific qPCRs that allowed us to detect and quantify the oMG allele A3 of strain Fin-Jyv-A3 or the oMG allele A10 of strain NE4049. Each strain-specific qPCR used the same primers to amplify a 143 bp fragment of the ospC gene, but used a different strain-specific probe to detect the two different oMG alleles (see electronic supplementary material for details). To validate the ospC qPCR assays, we created communities that differed in the percentage of the A3 and A10 alleles. This independent validation experiment showed that the ospC qPCR assays were highly reliable at estimating the frequencies of each of the two oMG alleles (see electronic supplementary material for details).
The qPCRs were done using a LightCycler 96 Multiwell Plate white (Roche). All the plates contained negative controls for the DNA extraction (mosquito DNA), negative controls for the qPCR (water), and four standards containing 102, 103, 104, and 105 gene copies. All the controls (and standards) were run in duplicate (or triplicate) in each plate. The quantification of copy gene numbers in the samples was done using the LightCycler 96 Software (Roche). For each qPCR assay, a sample of 81 ticks was tested twice to determine the repeatability of the assay. The repeatability of the log10-transformed gene copy number for each of the three independent qPCR assays was very high (see electronic supplementary material). The estimates of the ospC gene copy numbers were also highly correlated with the estimates of the flagellin gene copy numbers (see electronic supplementary material). These results indicate that our methods of estimating the strain-specific spirochaete load in the nymphal ticks are highly reliable.
3. Statistical analyses
All the statistical analyses were done using R v. 3.4.2.
(a). General statistical approach
The two measures of strain-specific fitness include host-to-tick transmission and the spirochaete load inside the nymphal tick. The experimental design of the study contains two fixed factors that are orthogonal to each other: focal strain (two levels: Fin-Jyv-A3, NE4049) and infection treatment (two levels: single, co-infection). To determine the effect of competition, the performance of the focal strain (host-to-tick transmission and spirochaete load in the nymphal tick) was compared between the single strain infection and the co-infection. A significant interaction between the focal strain and the infection treatment indicates that each focal strain is affected differently by the presence of the co-infecting strain.
Generalized linear mixed effects (GLME) models with binomial errors or linear mixed effects (LME) models with normal errors were used to analyse the response variables. The focal strain (two levels: Fin-Jyv-A3, NE4049), the infection treatment (two levels: single, co-infection) and their interaction were fixed factors, and mouse identity was included as a random factor. To determine statistical significance, models that differed with respect to the fixed factor of interest were compared using a log-likelihood ratio test (LLR).
(b). Host-to-tick transmission of Borrelia afzelii-infected nymphs
Host-to-tick transmission refers to the percentage of nymphs that acquired B. afzelii during their larval blood meal. The flagellin qPCR was used to determine the infection status of the ticks. Of the 346 ticks, 22 ticks (distributed over 17 different mice) were excluded from the analysis because they had contradictory results between the flagellin qPCR and the ospC qPCR. The strain-specific ospC qPCR was used to determine the presence of the focal strain in the ticks. Tick infection status with the focal strain was modelled as GLME model with binomial errors.
(c). Spirochaete loads of the nymphs
The analyses were done on the subset of infected nymphs. The gene copy number estimated from the flagellin qPCR assay was adjusted to give an estimate of the total spirochaete load for the entire nymph. For nymphs that were co-infected with strains Fin-Jyv-A3 and NE4049, the estimates of the strain-specific spirochaete loads from the ospC qPCR assays were constrained to sum to the total spirochaete load estimated by the flagellin qPCR assay (see the electronic supplementary material for details). The strain-specific spirochaete loads were log10-transformed to improve their fit to the normal distribution. The log10-transformed spirochaete load of the focal strain was modelled as an LME model with normal errors.
4. Results
(a). Infection success
Three of the 40 mice in the study were excluded: two mice (S5 and S12) died during the study, and one mouse (S37) did not become infected with B. afzelii following the nymphal challenge. In the co-infection treatment, four mice (S17, S20, S25 and S29) were only infected with strain Fin-Jyv-A3. Two of these four mice had been challenged with at least one NE4049-infected nymph. These four mice and their nymphs were excluded from the analyses because the aim of our study was to test whether strains in co-infected mice and co-infected nymphs experienced competition. Including these four mice in the statistical analyses made the results more statistically significant (see the electronic supplementary material). The final analysis therefore included 301 nymphs from 33 B. afzelii-infected mice (table 1). For a subsample of 29 infected mice, we obtained live cultures of B. afzelii from one or more nymphs; this result indicates that the mice transmitted live spirochaetes to the nymphs.
Table 1.
The proportion of B. afzelii-infected nymphs is shown for each of the 33 mice in the study. For each mouse, the geometric mean spirochaete load for the subset of infected nymphs and the 95% confidence interval (95% CI) are also shown. Here A3 and A10 refer to strains Fin-Jyv-A3 and NE4049, respectively.
| exp | strains | mouse ID | infected nymphs/total nymphs (%) | spiro load mean |
spiro load 95% CI |
|---|---|---|---|---|---|
| 1 | A3 | S01 | 8/8 (100.0%) | 18 408 | 6613–51 235 |
| 1 | A3 | S02 | 9/9 (100.0%) | 4885 | 1861–12 824 |
| 1 | A3 | S03 | 9/9 (100.0%) | 9309 | 3546–24 436 |
| 1 | A3 | S04 | 6/8 (75.0%) | 9513 | 2917–31 023 |
| 1 | A3 | S06 | 6/7 (85.7%) | 1672 | 513–5454 |
| 1 | A3 | S07 | 8/8 (100.0%) | 6722 | 2415–18 710 |
| 1 | A3 | S08 | 8/9 (88.9%) | 9523 | 3421–26 504 |
| 1 | A3 | S09 | 6/7 (85.7%) | 8643 | 2650–28 185 |
| 1 | A3 | S10 | 7/8 (87.5%) | 4771 | 1597–14 251 |
| 1 | A3 + A10 | S11 | 8/10 (80.0%) | 2188 | 786–6089 |
| 1 | A3 + A10 | S13 | 10/10 (100.0%) | 8730 | 3494–21 809 |
| 1 | A3 + A10 | S14 | 7/9 (77.8%) | 1963 | 657–5863 |
| 1 | A3 + A10 | S15 | 9/10 (90.0%) | 4786 | 1823–12 565 |
| 1 | A3 + A10 | S16 | 10/10 (100.0%) | 4898 | 1961–12 236 |
| 1 | A3 + A10 | S18 | 10/10 (100.0%) | 5117 | 2048–12 783 |
| 1 | A3 + A10 | S19 | 8/10 (80.0%) | 6839 | 2457–19 036 |
| 2 | A10 | S31 | 6/8 (75.0%) | 5888 | 1806–19 202 |
| 2 | A10 | S32 | 8/10 (80.0%) | 9094 | 3267–25 312 |
| 2 | A10 | S33 | 10/10 (100.0%) | 9162 | 3667–22 890 |
| 2 | A10 | S34 | 8/10 (80.0%) | 2563 | 921–7134 |
| 2 | A10 | S35 | 6/10 (60.0%) | 2247 | 689–7329 |
| 2 | A10 | S36 | 9/9 (100.0%) | 4986 | 1900–13 090 |
| 2 | A10 | S38 | 6/10 (60.0%) | 614 | 188–2003 |
| 2 | A10 | S39 | 7/9 (77.8%) | 1820 | 609–5436 |
| 2 | A10 | S40 | 7/10 (70.0%) | 2138 | 716–6387 |
| 2 | A10 + A3 | S21 | 7/9 (77.8%) | 4169 | 1396–12 453 |
| 2 | A10 + A3 | S22 | 10/10 (100.0%) | 5808 | 2325–14 509 |
| 2 | A10 + A3 | S23 | 8/9 (88.9%) | 6978 | 2507–19 423 |
| 2 | A10 + A3 | S24 | 10/10 (100.0%) | 6223 | 2491–15 547 |
| 2 | A10 + A3 | S26 | 10/10 (100.0%) | 7907 | 3165–19 753 |
| 2 | A10 + A3 | S27 | 7/7 (100.0%) | 7268 | 2433–21 712 |
| 2 | A10 + A3 | S28 | 8/9 (88.9%) | 6550 | 2353–18 232 |
| 2 | A10 + A3 | S30 | 8/9 (88.9%) | 2447 | 879–6813 |
(b). Comparison of fitness between strains Fin-Jyv-A3 and NE4049
For the mice infected with one strain, the host-to-tick transmission of Fin-Jyv-A3 (91.8% = 67/73) was significantly higher than NE4049 (77.9% = 67/86; GLME LLR: p = 0.021). The nymphal spirochaete load of strain Fin-Jyv-A3 (n = 67, mean = 7109, 95% CI = 4947–10 216) was twice that of strain NE4049 (n = 67, mean = 3497, 95% CI = 2434–5026; LME LLR: p = 0.038). Thus, in single-strain infections, strain Fin-Jyv-A3 outperformed strain NE4049 in both phenotypes.
For the co-infected mice, 91.5% of the nymphs (130/142) were infected with B. afzelii. Of the 130 infected nymphs, 17.7% (23/130) carried strain Fin-Jyv-A3 alone, 34.6% (45/130) carried strain NE4049 alone and 47.7% (62/130) carried both strains. For these 142 nymphs, host-to-tick transmission was 59.9% (85/142) for strain Fin-Jyv-A3 and 75.4% (107/142) for strain NE4049. Thus in co-infected mice, strain NE4049 was the superior competitor because it had higher host-to-tick transmission than strain Fin-Jyv-A3.
(c). Effect of competition on host-to-tick transmission
The effect of co-infection on host-to-tick transmission was analysed separately for each focal strain (figure 1) because the interaction between the focal strain and infection treatment was significant (GLME LLR: p = 0.0134). In experiment 1, host-to-tick transmission of strain Fin-Jyv-A3 was significantly lower for the co-infected mice (49.3% = 34/69), compared to the single strain mice (91.8% = 67/73; figure 1; GLME LLR: p < 0.001). In experiment 2, host-to-tick transmission of strain NE4049 was lower but not significantly so for the co-infected mice (69.9% = 51/73) compared to the single strain mice (77.9% = 67/86; figure 1; GLME LLR: p = 0.487). There was a significant negative effect of competition on the host-to-tick transmission of strain NE4049 when all 10 mice in the co-infection treatment were included in the analysis (see electronic supplementary material).
Figure 1.
Co-infection reduces host-to-tick transmission for two strains of B. afzelii. In experiments 1 and 2, the focal B. afzelii strains are Fin-Jyv-A3 and NE4049, respectively. Experiment 1 shows that host-to-tick transmission of strain Fin-Jyv-A3 is reduced in the presence of strain NE4049. Experiment 2 shows that host-to-tick transmission of strain NE4049 is not affected in the presence of strain Fin-Jyv-A3. Each data point represents the mean for a single mouse (n = 33 mice). Shown are the medians (grey line), the 25th and 75th percentiles (edges of the box), the minimum and maximum values (whiskers), and the outliers (solid circles). (Online version in colour.)
(d). Comparison of the total nymphal spirochaete load between single strain and co-infection treatments
In experiments 1 and 2, there was no significant difference in the total spirochaete load (as estimated by the flagellin qPCR) between the nymphs that had fed as larvae on the co-infected mice and the nymphs that had fed as larvae on the mice infected with a single strain (see the electronic supplementary material for details).
(e). Effect of competition on the nymphal spirochaete load
The nymphal spirochaete load of the focal strain is the number of spirochaetes of that strain in the nymph. With respect to the log10-transformed spirochaete load, focal strain (LME LLR: p = 0.009) and infection treatment (LME LLR: p = 0.003) were significant, but their interaction was not (LME LLR: p = 0.715; figure 2). For focal strain Fin-Jyv-A3, the nymphal spirochaete load in the co-infection group (n = 34, mean = 3257, 95% CI = 1918–5531) was reduced by more than half compared to the single strain infection group (figure 2; n = 67, mean = 7109, 95% CI = 4879–10 368). For focal strain NE4049, the nymphal spirochaete load in the co-infection group (n = 51, mean = 1929, 95% CI = 1252–2972) was reduced by almost half compared to the single strain infection group (figure 2; n = 67, mean = 3497, 95% CI = 2396–5094). The parameter estimates of the LME model also show a 50% reduction in the nymphal spirochaete load of the focal strain when the mouse was co-infected with another strain.
Figure 2.
Co-infection reduces the spirochaete load in I. ricinus nymphs for two strains of B. afzelii. In experiments 1 and 2, the focal B. afzelii strains are Fin-Jyv-A3 and NE4049, respectively. Experiment 1 shows that the nymphal spirochaete load of strain Fin-Jyv-A3 is reduced by 50% in the presence of strain NE4049. Experiment 2 shows that the nymphal spirochaete load of strain NE4049 is reduced by 50% in the presence of strain Fin-Jyv-A3. Each data point represents the mean for a single mouse (n = 33 mice). Shown are the medians (grey line), the 25th and 75th percentiles (edges of the box), the minimum and maximum values (whiskers), and the outliers (solid circles). (Online version in colour.)
For the 62 nymphs that were co-infected with both strains, the mean spirochaete load of Fin-Jyv-A3 (mean = 3286, 95% CI = 2217–4872) was significantly higher than that of NE4049 (mean = 1543, 95% CI = 1041–2287; LME LLR: p = 0.0002). Thus in co-infected ticks, strain Fin-Jyv-A3 was the superior competitor because it had a higher spirochaete load than strain NE4049.
5. Discussion
Our study found that the competitive ability of each strain differed depending on the host type in the life cycle of this important tick-borne pathogen. In co-infected mice, strain NE4049 was the superior competitor because it had higher host-to-tick transmission than strain Fin-Jyv-A3. In contrast, in co-infected ticks, strain Fin-Jyv-A3 was the superior competitor because its spirochaete load was higher than strain NE4049. To our knowledge, this study is the first demonstration that in multi-strain pathogens with a vector-borne life cycle, the winner of inter-strain competition in the vertebrate host can be the loser of inter-strain competition in the arthropod vector.
One of the central questions in ecology is to understand the factors that allow a community of species (or strains) to persist over time [5,49–52]. We and others have previously shown that a dozen strains of B. afzelii can coexist at the spatial scale of a soccer field [41,42,44,53]. Two independent long-term studies on B. afzelii in ticks and reservoir hosts have shown that the community of strains is stable over a time period of at least a decade [53,54]. Thus, the central question is how a dozen strains of B. afzelii can persist in the same local Lyme disease system [42,44,53]. A general explanation for coexistence is the presence of trade-offs where the competitive hierarchy between genotypes is reversed between different states [14]. Below, we give three examples of how the competitive hierarchy could be reversed between two or more strains. First, trade-off between performance when alone versus co-infection; for example, in single infections, strain Fin-Jyv-A3 had higher host-to-tick transmission than strain NE4049, but in mixed infections the relationship was reversed (figure 1). Second, trade-off between different host types in the life cycle; for example, strain NE4049 outcompeted strain Fin-Jyv-A3 in the rodent host but the relationship was reversed in the tick vector. Third, intransitive competition relationships between strains; for example, strain A beats B, B beats C and C beats A. In summary, there are different trade-offs that could stabilize a community of a dozen B. afzelii strains coexisting in the same local Lyme disease system.
To our knowledge, this study is also one of the first experimental demonstrations that strains of a vector-borne pathogen experience competitive interactions inside the arthropod vector. Our study adds to a growing literature on interactions between strains of vector-borne pathogens within their arthropod vectors [33,35]. A study on mixed infections of rodent malaria parasites in mosquitoes found that malaria strains had a greater chance to establish infection and reach a high density if the mosquito was already infected by another strain, which is an example of cooperation or facilitation [33]. A study on the tick-borne bacterium Francisella novicida found that a wild-type strain excluded other strains from establishing infection in the tick vector [35]. Our results are in agreement with our previous study, which found indirect evidence for competition between strains of B. afzelii in wild I. ricinus nymphal ticks [44]. That study found that the number of spirochaetes per strain decreased as the strain richness increased inside the nymphs [44]. Negative associations between the spirochaete abundances of some strains inside the nymphal tick were also shown [44]. The present study found a large effect size of inter-strain competition in the tick vector; co-infection in the tick reduced the spirochaete abundance of each strain by 50%. A critical question is whether competition between strains in the nymph influences the strain-specific nymph-to-host transmission success.
Competition between strains in the tick vector could have important consequences if the observed reductions in spirochaete load influence strain-specific nymph-to-host transmission. During the nymphal blood meal, spirochaetes migrate from the midgut to the salivary glands [55,56], and the nymph inoculates about 100 spirochaetes into the vertebrate host [57]. This small inoculum size suggests that competition between strains in the nymphal tick could influence the strain-specific nymph-to-host transmission success. We have recently shown that B. afzelii strains that establish high spirochaete loads in wild I. ricinus nymphs are more common in nature [44]. This observation suggests that strains with high nymphal spirochaete loads are more competitive and have higher nymph-to-host transmission success [44]. Previous studies on other vector-borne pathogens have shown that the arthropod vector can limit the genetic diversity of strains that is transmitted to the vertebrate host [35,58]. For example, Rego et al. [58] used a set of genetically tagged clones of B. burgdorferi sensu stricto (ss) to show that co-infected ticks transmit a subset of clones to the rodent host. This result shows that the arthropod vector can act as a genetic bottleneck when they transmit mixed infections to the vertebrate host. The vector bottleneck would be even more important, if some strains are better than others at achieving vector-to-host transmission from co-infected vectors.
Competition between microbial species or strains can occur by three different mechanisms: interference, exploitation and apparent competition [4,59–62]. In interference competition, microbes produce toxic substances that weaken the performance (transmission, growth, reproduction) of other species or strains [63–65]. This mechanism is unlikely because B. burgdorferi sl does not produce toxic substances [66]. Exploitation competition happens when pathogen strains compete over limited host resources such as nutrients or space [8,67]. For example, many species of free-living and pathogenic bacteria compete over iron [68,69]. A recent theoretical study of the microbiome found that rapid population expansion of bacteria followed by extrinsic resource limitation by the host exacerbates competitive interactions and enhances community stability [52]. This condition is likely to be met in ixodid ticks where a single resource pulse (the blood meal) is followed by a rapid expansion of the microbial community [70–72]. Others and we have shown that following the larval blood meal, the population of B. burgdorferi sl expands rapidly from an initial inoculum of about 100 bacteria to a spirochaete population size inside the nymph that ranges from 2000 to 32 000 cells [73–76]. We have also shown that the spirochaete loads in the nymphs decrease dramatically over time, further suggesting that the resources inside the nymphal midgut are limiting [74]. Thus, exploitation competition over limited resources probably underlies the competitive interactions between the two strains of B. afzelii observed in this study. Finally, in apparent competition, the host immune response triggered by one strain affects the fitness of another strain [31,77]. Ticks contain immune defences such as antimicrobial peptides and phagocytic cells [78–81]. The observation that the total spirochaete load inside the nymph is low, suggests that the tick immune system restricts the spirochaete population inside the nymphal midgut to certain limits.
Our study found direct evidence for competitive interactions between strains of B. afzelii in the rodent reservoir host. This result is in agreement with other studies on the North American Lyme disease system of B. burgdorferi ss, I. scapularis ticks and Peromyscus leucopus mice [9,45] that have shown that co-infection reduces host-to-tick transmission success. In addition, we found evidence for asymmetric competition: co-infection reduced host-to-tick transmission of strain Fin-Jyv-A3 by approximately 50%, but had no effect on the host-to-tick transmission of strain NE4049. This result is not novel, as one of the studies on B. burgdorferi ss also found evidence of asymmetric competition [9]. If we include the four mice in the co-infected group that became infected with strain Fin-Jyv-A3 alone, competition significantly reduced host-to-tick transmission by approximately 30% in both strains (see electronic supplementary material for details). In summary, there is strong evidence that co-infection of strains of B. burgdorferi sl in the rodent host reduces host-to-tick transmission success.
Competitive exclusion occurs when one pathogen strain prevents another strain from establishing infection in the host [11–13]. In the present study, four of the 20 mice in the co-infection treatment became infected with strain Fin-Jyv-A3 but not strain NE4049. This result suggests competitive exclusion: strain Fin-Jyv-A3 prevented strain NE4049 from establishing infection in the host. There is some evidence that competitive exclusion is important in the field [29]. A field study on mixed strain infections of B. afzelii in bank voles found that strains carrying genetically similar ospC alleles were less likely to co-occur in the same host, a pattern that is consistent with competitive exclusion [29]. We have shown that the majority of I. ricinus nymphs carry mixed strain infections [42,44], which means that simultaneous exposure to multiple strains is common in populations of wild reservoir hosts. Thus our observation that competitive exclusion occurred in 20.0% of mice simultaneously exposed to two strains, suggests that this phenomenon could be important in the field.
In nature, Ixodes nymphs are frequently infected with multiple strains of a given species of B. burgdorferi sl [40–44]. Our work on a wild population of I. ricinus in Neuchâtel, Switzerland found that approximately 80% of nymphs infected with B. afzelii carried more than one strain [42,44]. In the present study, the rodents were infected with a maximum of two strains whereas in the field, rodents are often infected with more than two strains [10,29,54]. These differences in strain richness in the rodent host explain why the percentage of co-infected nymphs in our experiment (43.7%) was lower than what we have observed in nature. Our results are in agreement with a recent experimental study using the North American Lyme disease system, which found that 24.5% of nymphs that had fed as larvae on co-infected mice acquired both strains [9]. A field study in North America also showed that larval I. scapularis ticks that feed on wild reservoir hosts often acquire multiple strain infections [40]. A remaining question is why some nymphs acquire a subset of strains present in the vertebrate host whereas other nymphs acquire the complete set of strains?
In the present study, competition between strains of B. afzelii was shown in both the rodent host and the tick vector. Competition in the rodent host reduced host-to-tick transmission of strain Fin-Jyv-A3 but not strain NE4049. Competition in the tick vector reduced the bacterial abundance of both strains by 50%, but strain Fin-Jyv-A3 had a higher abundance than strain NE4049. Thus, strain NE4049 was the superior competitor in the rodent host, whereas strain Fin-Jyv-A3 was the superior competitor in the tick vector. Future studies should investigate whether inter-strain competition in the tick has important consequences for the strain-specific tick-to-host transmission success.
Supplementary Material
Supplementary Material
Supplementary Material
Acknowledgements
The authors sincerely thank Alessandro Belli and Kheirie Kabalan for their help during the experiment. This study is part of the PhD thesis of D.G. The authors thank Cindy Bregnard, Gaël Hauser, Giacomo Zilio and two anonymous reviewers for their comments, which greatly improved this manuscript.
Ethics
The commission that is part of the ‘Service de la Consommation et des Affaires Vétérinaires (SCAV)’ of Canton Vaud, Switzerland evaluated and approved the ethics of this study. The Veterinary Service of the Canton of Neuchâtel, Switzerland issued the animal experimentation permit used in this study (NE04/2016).
Data accessibility
All data and code used in this study are available in the electronic supplementary material.
Authors' contributions
D.G. and M.J.V. conceived and designed the study. D.G. and A.S. conducted the experiment and performed the molecular work. A.S., A.G.-C. and J.D. helped develop the ospC-specific qPCR. J.D. did 454-sequencing on isolates Fin-Jyv-A3 and NE4049 to confirm their purity. C.C. isolated Fin-Jyv-A3 and created the nymphs infected with this isolate. O.R. helped with the experimental infections. D.G. conducted the statistical analyses. D.G. and M.J.V. wrote the manuscript. All authors read and approved the final version of the manuscript.
Competing interests
The authors declare no competing interests.
Funding
This work was supported by a Swiss National Science Foundation grant to M.J.V. (FN 31003A_141153), the Kone foundation, and a grant from the doctoral programme of the University of Jyväskylä to C.C.
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Supplementary Materials
Data Availability Statement
All data and code used in this study are available in the electronic supplementary material.