Male C57BL/6J mice have higher presence and abundance of Borrelia burgdorferi in their ventral skin compared to female mice.

Borrelia burgdorferi is a tick-borne spirochete that causes Lyme disease in humans. The host immune system controls the abundance of the spirochete in the host tissues. Recent work with immunocompetent Mus musculus mice strain C3H/HeJ found that males had a higher tissue infection prevalence and spirochete load compared to females. The purpose of this study was to determine whether host sex and acquired immunity interact to influence the prevalence and abundance of spirochetes in the tissues of the commonly used mouse strain C57BL/6. Wildtype (WT) mice and their SCID counterparts (C57BL/6) were experimentally infected with B. burgdorferi via tick bite. Ear biopsies were sampled at weeks 4, 8, and 12 post-infection (PI) and five tissues (left ear, ventral skin, heart, tibiotarsal joint of left hind leg, and liver) were collected at necropsy (16 weeks PI). The mean spirochete load in the tissues of the SCID mice was 260.4x higher compared to the WT mice. In WT mice, the infection prevalence in the ventral skin was significantly higher in males (40.0 %) compared to females (0.0 %), and the spirochete load in the rear tibiotarsal joint was significantly higher (4.3x) in males compared to females. In SCID mice, the spirochete load in the ventral skin was 200.0x higher in males compared to females, but there were no significant sex-specific difference in spirochete load in the other tissues (left ear, heart, tibiotarsal joint, or liver). Thus, the absence of acquired immunity greatly amplified the spirochete load in the ventral skin of male mice. It is important to note that the observed sex-specific differences in laboratory mice cannot be extrapolated to humans. Future studies should investigate the mechanisms underlying the male bias in the abundance of B. burgdorferi in the mouse skin.

Tick-to-host transmission differs between Borrelia afzelii strains.

Many vector-borne pathogens establish multiple-strain infections in the vertebrate host and the arthropod vector. Multiple-strain infections in the host influence strain acquisition by naive vectors. Whether multiple-strain infections in the vector influence strain-specific transmission to naive hosts remains unknown. The spirochete, Borrelia afzelii, causes Lyme borreliosis and multiple-strain infections are common in both the tick vector and vertebrate host. Our study used two B. afzelii strains: Fin-Jyv-A3 and NE4049. Donor mice were infected with Fin-Jyv-A3 alone, NE4049 alone, or with both strains. Larval ticks fed on donor mice and molted into nymphal ticks infected with either strain or both strains. These nymphs were fed on test mice to determine whether multiple-strain infections in the nymph influence nymph-to-host transmission (NHT). Multiple-strain infection in the donor mice reduced the acquisition of both strains by ticks by 23%. Thus, a substantial fraction of infected nymphs from the multiple strain treatment were infected with the "wrong" competitor strain rather than the "right" focal strain. As a result, nymphs from the multiple strain treatment were 46% less likely to infect the test mice with the focal strain compared to nymphs from the single strain treatment. However, multiple-strain infection in the nymphal tick had no effect on the NHT of either strain. The nymphal spirochete load of Fin-Jyv-A3 was 1.9 times higher compared to NE4049. NHT of Fin-Jyv-A3 (79%) was 1.5 times higher compared to NE4049 (53%). Our study suggests that B. afzelii strains with higher nymphal spirochete loads have higher NHT. IMPORTANCE For many vector-borne pathogens, multiple-strain infections in the vertebrate host or arthropod vector are common. Multiple-strain infections in the host reduce strain acquisition by feeding vectors. Whether multiple-strain infections in the vector influence strain transmission to the host remains unknown. In our study, we used two strains of the tick-borne spirochete Borrelia afzelii, which causes Lyme borreliosis, to investigate whether multiple-strain infections in the nymphal tick influenced nymph-to-host transmission (NHT) of strains. Multiple-strain infections in mice reduced the acquisition of both B. afzelii strains by nymphal ticks. As a result, nymphs from the multiple strain treatment were less likely to infect naive test mice with the focal strain. Multiple-strain infection in the nymphal ticks did not influence the NHT of either strain. The strain with the higher bacterial abundance in the nymph had higher NHT. Our study suggests that pathogen abundance in the arthropod vector is important for vector-to-host transmission.

Variation among strains of Borrelia burgdorferi in host tissue abundance and lifetime transmission determine the population strain structure in nature.

Pathogen life history theory assumes a positive relationship between pathogen load in host tissues and pathogen transmission. Empirical evidence for this relationship is surprisingly rare due to the difficulty of measuring transmission for many pathogens. The comparative method, where a common host is experimentally infected with a set of pathogen strains, is a powerful approach for investigating the relationships between pathogen load and transmission. The validity of such experimental estimates of strain-specific transmission is greatly enhanced if they can predict the pathogen population strain structure in nature. Borrelia burgdorferi is a multi-strain, tick-borne spirochete that causes Lyme disease in North America. This study used 11 field-collected strains of B. burgdorferi, a rodent host (Mus musculus, C3H/HeJ) and its tick vector (Ixodes scapularis) to determine the relationship between pathogen load in host tissues and lifetime host-to-tick transmission (HTT). Mice were experimentally infected via tick bite with 1 of 11 strains. Lifetime HTT was measured by infesting mice with I. scapularis larval ticks on 3 separate occasions. The prevalence and abundance of the strains in the mouse tissues and the ticks were determined by qPCR. We used published databases to obtain estimates of the frequencies of these strains in wild I. scapularis tick populations. Spirochete loads in ticks and lifetime HTT varied significantly among the 11 strains of B. burgdorferi. Strains with higher spirochete loads in the host tissues were more likely to infect feeding larval ticks, which molted into nymphal ticks that had a higher probability of B. burgdorferi infection (i.e., higher HTT). Our laboratory-based estimates of lifetime HTT were predictive of the frequencies of these strains in wild I. scapularis populations. For B. burgdorferi, the strains that establish high abundance in host tissues and that have high lifetime transmission are the strains that are most common in nature.

Clobetasol increases the abundance of Borrelia burgdorferi in the skin 70 times more in male mice compared to female mice.

Lyme borreliosis is caused by the spirochete Borrelia burgdorferi and is transmitted among vertebrate hosts by Ixodes scapularis ticks in eastern North America. Treatment with topical corticosteroids increases the abundance of B. burgdorferi in the skin of lab mice that have been experimentally infected via needle inoculation. In the present study, female and male C3H/HeJ mice were infected with B. burgdorferi via nymphal tick bite. Infected mice were treated with clobetasol on the skin of the right hindleg on days 35 and 36 post-infection and euthanized at days -2, 1, 3, 5, and 7 post-treatment; a group of control mice was infected but not treated with clobetasol. The spirochete abundance was quantified in 8 mouse tissues including bladder, heart, left hindleg skin, right hindleg skin, dorsal skin, ventral skin, left ear and right ear. Averaged across the 8 mouse tissues, the abundance of B. burgdorferi on days 3 and 5 were 21.4x and 14.4x higher in mice treated with clobetasol compared to the untreated control mice, but there were large differences among tissues. There was a dramatic sex-specific effect of the clobetasol treatment; the peak abundance of B. burgdorferi in the skin (left hindleg, right hindleg, dorsal, ventral) was 72.6x higher in male mice compared to female mice. In contrast, there was little difference between the sexes in the tissue spirochete load in the ears, bladder, and heart. Topical application of clobetasol could increase the sensitivity of direct diagnostic methods (e.g., culture, PCR) to detect B. burgdorferi in host skin biopsies.

Borrelia burgdorferi strain and host sex influence pathogen prevalence and abundance in the tissues of a laboratory rodent host.

Experimental infections with different pathogen strains give insight into pathogen life history traits. The purpose of the present study was to compare variation in tissue infection prevalence and spirochete abundance among strains of Borrelia burgdorferi in a rodent host (Mus musculus, C3H/HeJ). Male and female mice were experimentally infected via tick bite with one of 12 strains. Ear tissue biopsies were taken at days 29, 59 and 89 postinfection, and seven tissues were collected at necropsy. The presence and abundance of spirochetes in the mouse tissues were measured by quantitative polymerase chain reaction. To determine the frequencies of our strains in nature, their multilocus sequence types were matched to published data sets. For the infected mice, 56.6% of the tissues were infected with B. burgdorferi. The mean spirochete load in the mouse necropsy tissues varied 4.8-fold between the strains. The mean spirochete load in the ear tissue biopsies decreased rapidly over time for some strains. The percentage of infected tissues in male mice (65.4%) was significantly higher compared to female mice (50.5%). The mean spirochete load in the seven tissues was 1.5× higher in male mice compared to female mice; this male bias was 15.3× higher in the ventral skin. Across the 11 strains, the mean spirochete loads in the infected mouse tissues were positively correlated with the strain-specific frequencies in their tick vector populations. The study suggests that laboratory-based estimates of pathogen abundance in host tissues can predict the strain composition of this important tick-borne pathogen in nature.

Parvovirus enteritis and other risk factors associated with persistent gastrointestinal signs in dogs later in life: a retrospective cohort study.

BACKGROUND: Parvoviral enteritis (PE) is a viral gastrointestinal (GI) infection of dogs. Recovery from PE has been associated with persistent GI signs later in life. The objectives of this study were: (i) To determine whether dogs that have recovered from PE (post-parvo dogs) had an increased risk of persistent GI signs compared to uninfected control dogs. (ii) To investigate the lifestyle and clinicopathologic factors that are associated with persistent GI signs in post-parvo dogs. METHODS: A total of 86 post-parvo dogs and 52 age-matched control dogs were enrolled in this retrospective cohort study. Many years after hospitalization for PE, the owners were interviewed about the health and habits of their dogs using a questionnaire. We used generalized linear mixed effects models to test whether parvovirus enteritis and other risk factors are associated with owner-recognized general health problems in all dogs and with owner-recognized persistent GI signs in post-parvo dogs. RESULTS: The prevalence of persistent GI signs was significantly higher in post-parvo dogs compared to control dogs (57% vs 25%, P < 0.001). Markers of disease severity at the time of hospital admission such as neutropenia, low body temperature (BT), and treatment with an antiemetic medication (metoclopramide) were significant risk factors for persistent GI signs in post-parvo dogs. For example, PE-affected dogs that were hypothermic at hospital admission (BT of 37.2 °C) were 16.6 × more likely to have GI signs later in life compared to hyperthermic dogs (BT of 40.4 °C). The presence of persistent GI signs in post-parvo dogs was a risk factor for health problems in other organ systems. CONCLUSIONS: Parvovirus enteritis is a significant risk factor for persistent GI signs in dogs highlighting the importance of prevention. The risk factors identified in the present study may guide future investigations on the mechanisms that link parvovirus enteritis to chronic health problems in dogs.

Beech tree masting explains the inter-annual variation in the fall and spring peaks of Ixodes ricinus ticks with different time lags.

BACKGROUND: The tick Ixodes ricinus is an important vector of tick-borne diseases including Lyme borreliosis. In continental Europe, the nymphal stage of I. ricinus often has a bimodal phenology with a large spring peak and a smaller fall peak. There is consensus about the origin of the spring nymphal peak, but there are two alternative hypotheses for the fall nymphal peak. In the direct development hypothesis, larvae quest as nymphs in the fall of the same year that they obtained their larval blood meal. In the developmental diapause hypothesis, larvae overwinter in the engorged state and quest as nymphs one year after they obtained their larval blood meal. These two hypotheses make different predictions about the time lags that separate the larval blood meal and the density of questing nymphs (DON) in the spring and fall. METHODS: Inter-annual variation in seed production (masting) by deciduous trees is a time-lagged index for the density of vertebrate hosts (e.g., rodents) which provide blood meals for larval ticks. We used a long-term data set on the masting of the European beech tree and a 15-year study on the DON at 4 different elevation sites in western Switzerland to differentiate between the two alternative hypotheses for the origin of the fall nymphal peak. RESULTS: Questing I. ricinus nymphs had a bimodal phenology at the three lower elevation sites, but a unimodal phenology at the top elevation site. At the lower elevation sites, the DON in the fall was strongly correlated with the DON in the spring of the following year. The inter-annual variation in the densities of I. ricinus nymphs in the fall and spring was best explained by a 1-year versus a 2-year time lag with the beech tree masting index. Fall nymphs had higher fat content than spring nymphs indicating that they were younger. All these observations are consistent with the direct development hypothesis for the fall peak of I. ricinus nymphs at our study site. Our study provides new insight into the complex bimodal phenology of this important disease vector. CONCLUSIONS: Public health officials in Europe should be aware that following a strong mast year, the DON will increase 1 year later in the fall and 2 years later in the spring. Studies of I. ricinus populations with a bimodal phenology should consider that the spring and fall peak in the same calendar year represent different generations of ticks.

Borrelia afzelii Infection in the Rodent Host Has Dramatic Effects on the Bacterial Microbiome of Ixodes ricinus Ticks.

The microbiome of blood-sucking arthropods can shape their competence to acquire and maintain infections with vector-borne pathogens. We used a controlled study to investigate the interactions between Borrelia afzelii, which causes Lyme borreliosis in Europe, and the bacterial microbiome of Ixodes ricinus, its primary tick vector. We applied a surface sterilization treatment to I. ricinus eggs to produce dysbiosed tick larvae that had a low bacterial abundance and a changed bacterial microbiome compared to those of the control larvae. Dysbiosed and control larvae fed on B. afzelii-infected mice and uninfected control mice, and the engorged larvae were left to molt into nymphs. The nymphs were tested for B. afzelii infection, and their bacterial microbiome underwent 16S rRNA amplicon sequencing. Surprisingly, larval dysbiosis had no effect on the vector competence of I. ricinus for B. afzelii, as the nymphal infection prevalence and the nymphal spirochete load were the same between the dysbiosed group and the control group. The strong effect of egg surface sterilization on the tick bacterial microbiome largely disappeared once the larvae molted into nymphs. The most important determinant of the bacterial microbiome of I. ricinus nymphs was the B. afzelii infection status of the mouse on which the nymphs had fed as larvae. Nymphs that had taken their larval blood meal from an infected mouse had a less abundant but more diverse bacterial microbiome than the control nymphs. Our study demonstrates that vector-borne infections in the vertebrate host shape the microbiome of the arthropod vector. IMPORTANCE Many blood-sucking arthropods transmit pathogens that cause infectious disease. For example, Ixodes ricinus ticks transmit the bacterium Borrelia afzelii, which causes Lyme disease in humans. Ticks also have a microbiome, which can influence their ability to acquire and transmit tick-borne pathogens such as B. afzelii. We sterilized I. ricinus eggs with bleach, and the tick larvae that hatched from these eggs had a dramatically reduced and changed bacterial microbiome compared to that of control larvae. These larvae fed on B. afzelii-infected mice, and the resultant nymphs were tested for B. afzelii and for their bacterial microbiome. We found that our manipulation of the bacterial microbiome had no effect on the ability of the tick larvae to acquire and maintain populations of B. afzelii. In contrast, we found that B. afzelii infection had dramatic effects on the bacterial microbiome of I. ricinus nymphs. Our study demonstrates that infections in the vertebrate host can shape the tick microbiome.

Infection with Borrelia afzelii and manipulation of the egg surface microbiota have no effect on the fitness of immature Ixodes ricinus ticks.

Arthropod vectors carry vector-borne pathogens that cause infectious disease in vertebrate hosts, and arthropod-associated microbiota, which consists of non-pathogenic microorganisms. Vector-borne pathogens and the microbiota can both influence the fitness of their arthropod vectors, and hence the epidemiology of vector-borne diseases. The bacterium Borrelia afzelii, which causes Lyme borreliosis in Europe, is transmitted among vertebrate reservoir hosts by Ixodes ricinus ticks, which also harbour a diverse microbiota of non-pathogenic bacteria. The purpose of this controlled study was to test whether B. afzelii and the tick-associated microbiota influence the fitness of I. ricinus. Eggs obtained from field-collected adult female ticks were surface sterilized (with bleach and ethanol), which reduced the abundance of the bacterial microbiota in the hatched I. ricinus larvae by 28-fold compared to larvae that hatched from control eggs washed with water. The dysbiosed and control larvae were subsequently fed on B. afzelii-infected or uninfected control mice, and the engorged larvae were left to moult into nymphs under laboratory conditions. I. ricinus larvae that fed on B. afzelii-infected mice had a significantly faster larva-to-nymph moulting time compared to larvae that fed on uninfected control mice, but the effect was small (2.4% reduction) and unlikely to be biologically significant. We found no evidence that B. afzelii infection or reduction of the larval microbiota influenced the four other life history traits of the immature I. ricinus ticks, which included engorged larval weight, unfed nymphal weight, larva-to-nymph moulting success, and immature tick survival. A retrospective power analysis found that our sampling effort had sufficient power (> 80%) to detect small effects (differences of 5% to 10%) of our treatments. Under the environmental conditions of this study, we conclude that B. afzelii and the egg surface microbiota had no meaningful effects on tick fitness and hence on the R0 of Lyme borreliosis.

Competition between strains of Borrelia afzelii in the host tissues and consequences for transmission to ticks.

Pathogen species often consist of genetically distinct strains, which can establish mixed infections or coinfections in the host. In coinfections, interactions between pathogen strains can have important consequences for their transmission success. We used the tick-borne bacterium Borrelia afzelii, which is the most common cause of Lyme disease in Europe, as a model multi-strain pathogen to investigate the relationship between coinfection, competition between strains, and strain-specific transmission success. Mus musculus mice were infected with one or two strains of B. afzelii, strain transmission success was measured by feeding ticks on mice, and the distribution of each strain in six different mouse organs and the ticks was measured using qPCR. Coinfection and competition reduced the tissue infection prevalence of both strains and changed their bacterial abundance in some tissues. Coinfection and competition also reduced the transmission success of the B. afzelii strains from the infected hosts to feeding ticks. The ability of the B. afzelii strains to establish infection in the host tissues was strongly correlated with their transmission success to the tick vector. Our study demonstrates that coinfection and competition between pathogen strains inside the host tissues can have major consequences for their transmission success.

Masting by beech trees predicts the risk of Lyme disease.

BACKGROUND: The incidence of Lyme borreliosis and other tick-borne diseases is increasing in Europe and North America. There is currently much interest in identifying the ecological factors that determine the density of infected ticks as this variable determines the risk of Lyme borreliosis to vertebrate hosts, including humans. Lyme borreliosis is caused by the bacterium Borrelia burgdorferi sensu lato (s.l.) and in western Europe, the hard tick Ixodes ricinus is the most important vector. METHODS: Over a 15-year period (2004-2018), we monitored the monthly abundance of I. ricinus ticks (nymphs and adults) and their B. burgdorferi s.l. infection status at four different elevations on a mountain in western Switzerland. We collected climate variables in the field and from nearby weather stations. We obtained data on beech tree seed production (masting) from the literature, as the abundance of Ixodes nymphs can increase dramatically 2 years after a masting event. We used generalized linear mixed effects models and AIC-based model selection to identify the ecological factors that influence inter-annual variation in the nymphal infection prevalence (NIP) and the density of infected nymphs (DIN). RESULTS: We found that the NIP decreased by 78% over the study period. Inter-annual variation in the NIP was explained by the mean precipitation in the present year, and the duration that the DNA extraction was stored in the freezer prior to pathogen detection. The DIN decreased over the study period at all four elevation sites, and the decrease was significant at the top elevation. Inter-annual variation in the DIN was best explained by elevation site, year, beech tree masting index 2 years prior and the mean relative humidity in the present year. This is the first study in Europe to demonstrate that seed production by deciduous trees influences the density of nymphs infected with B. burgdorferi s.l. and hence the risk of Lyme borreliosis. CONCLUSIONS: Public health officials in Europe should be aware that masting by deciduous trees is an important predictor of the risk of Lyme borreliosis.

Sentinel surveillance of Lyme disease risk in Canada, 2019: Results from the first year of the Canadian Lyme Sentinel Network (CaLSeN).

BACKGROUND: Lyme disease is an emerging vector-borne zoonotic disease of increasing public health importance in Canada. As part of its mandate, the Canadian Lyme Disease Research Network (CLyDRN) launched a pan-Canadian sentinel surveillance initiative, the Canadian Lyme Sentinel Network (CaLSeN), in 2019. OBJECTIVES: To create a standardized, national sentinel surveillance network providing a real-time portrait of the evolving environmental risk of Lyme disease in each province. METHODS: A multicriteria decision analysis (MCDA) approach was used in the selection of sentinel regions. Within each sentinel region, a systematic drag sampling protocol was performed in selected sampling sites. Ticks collected during these active surveillance visits were identified to species, and Ixodes spp. ticks were tested for infection with Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, Babesia microti and Powassan virus. RESULTS: In 2019, a total of 567 Ixodes spp. ticks (I. scapularis [n=550]; I. pacificus [n=10]; and I. angustus [n=7]) were collected in seven provinces: British Columbia, Manitoba, Ontario, Québec, New Brunswick, Nova Scotia and Prince Edward Island. The highest mean tick densities (nymphs/100 m2) were found in sentinel regions of Lunenburg (0.45), Montréal (0.43) and Granby (0.38). Overall, the Borrelia burgdorferi prevalence in ticks was 25.2% (0%-45.0%). One I. angustus nymph from British Columbia was positive for Babesia microti, a first for the province. The deer tick lineage of Powassan virus was detected in one adult I. scapularis in Nova Scotia. CONCLUSION: CaLSeN provides the first coordinated national active surveillance initiative for tick-borne disease in Canada. Through multidisciplinary collaborations between experts in each province, the pilot year was successful in establishing a baseline for Lyme disease risk across the country, allowing future trends to be detected and studied.

Climate and tree seed production predict the abundance of the European Lyme disease vector over a 15-year period.

BACKGROUND: To predict the risk of tick-borne disease, it is critical to understand the ecological factors that determine the abundance of ticks. In Europe, the sheep tick (Ixodes ricinus) transmits a number of important diseases including Lyme borreliosis. The aim of this long-term study was to determine the abiotic and biotic factors driving the annual abundance of I. ricinus at a location in Switzerland where Lyme borreliosis is endemic. METHODS: Over a 15-year period (2004 to 2018), we monitored the abundance of I. ricinus ticks on a monthly basis at three different elevations on Chaumont Mountain in Neuchâtel, Switzerland. We collected climate variables in the field and from nearby weather stations. We obtained data on beech tree seed production from the literature, as the abundance of Ixodes nymphs can increase dramatically two years after a masting event. We used AIC-based model selection to determine which ecological variables drive annual variation in tick density. RESULTS: We found that elevation site, year, seed production by beech trees two years prior, and mean annual relative humidity together explained 73.2% of the variation in our annual estimates of nymph density. According to the parameter estimates of our models, (i) the annual density of nymphs almost doubled over the 15-year study period, (ii) changing the beech tree seed production index from very poor mast (1) to full mast (5) increased the abundance of nymphs by 86.2% two years later, and (iii) increasing the field-collected mean annual relative humidity from 50.0 to 75.0% decreased the abundance of nymphs by 46.4% in the same year. Climate variables collected in the field were better predictors of tick abundance than those from nearby weather stations indicating the importance of the microhabitat. CONCLUSIONS: From a public health perspective, the increase in nymph abundance is likely to have increased the risk of tick-borne disease in this region of Switzerland. Public health officials in Europe should be aware that seed production by deciduous trees is a critical driver of the abundance of I. ricinus, and hence the risk of tick-borne disease.

Competition Among Gardnerella Subgroups From the Human Vaginal Microbiome.

Gardnerella spp. are hallmarks of bacterial vaginosis, a clinically significant dysbiosis of the vaginal microbiome. Gardnerella has four subgroups (A, B, C, and D) based on cpn60 sequences. Multiple subgroups are often detected in individual women, and interactions between these subgroups are expected to influence their population dynamics and associated clinical signs and symptoms of bacterial vaginosis. In the present study, contact-independent and contact-dependent interactions between the four Gardnerella subgroups were investigated in vitro. The cell free supernatants of mono- and co-cultures had no effect on growth rates of the Gardnerella subgroups suggesting that there are no contact-independent interactions (and no contest competition). For contact-dependent interactions, mixed communities of 2, 3, or 4 subgroups were created and the initial (0 h) and final population sizes (48 h) were quantified using subgroup-specific PCR. Compared to the null hypothesis of neutral interactions, most (69.3%) of the mixed communities exhibited competition. Competition reduced the growth rates of subgroups A, B, and C. In contrast, the growth rate of subgroup D increased in the presence of the other subgroups. All subgroups were able to form biofilm alone and in mixed communities. Our study suggests that there is scramble competition among Gardnerella subgroups, which likely contributes to the observed distributions of Gardnerella spp. in vaginal microbiomes and the formation of the multispecies biofilms characteristic of bacterial vaginosis.

Maternal Antibodies Provide Bank Voles with Strain-Specific Protection against Infection by the Lyme Disease Pathogen.

Multistrain microbial pathogens often induce strain-specific antibody responses in their vertebrate hosts. Mothers can transmit antibodies to their offspring, which can provide short-term, strain-specific protection against infection. Few experimental studies have investigated this phenomenon for multiple strains of zoonotic pathogens occurring in wildlife reservoir hosts. The tick-borne bacterium Borrelia afzelii causes Lyme disease in Europe and consists of multiple strains that cycle between the tick vector (Ixodes ricinus) and vertebrate hosts, such as the bank vole (Myodes glareolus). We used a controlled experiment to show that female bank voles infected with B. afzelii via tick bite transmit protective antibodies to their offspring. To test the specificity of protection, the offspring were challenged using a natural tick bite challenge with either the maternal strain to which the mothers had been exposed or a different strain. The maternal antibodies protected the offspring against a homologous infectious challenge but not against a heterologous infectious challenge. The offspring from the uninfected control mothers were equally susceptible to both strains. Borrelia outer surface protein C (OspC) is an antigen that is known to induce strain-specific immunity. Maternal antibodies in the offspring reacted more strongly with homologous than with heterologous recombinant OspC, but other antigens may also mediate strain-specific immunity. Our study shows that maternal antibodies provide strain-specific protection against B. afzelii in an ecologically important rodent reservoir host. The transmission of maternal antibodies may have important consequences for the epidemiology of multistrain pathogens in nature.IMPORTANCE Many microbial pathogen populations consist of multiple strains that induce strain-specific antibody responses in their vertebrate hosts. Females can transmit these antibodies to their offspring, thereby providing them with short-term strain-specific protection against microbial pathogens. We investigated this phenomenon using multiple strains of the tick-borne microbial pathogen Borrelia afzelii and its natural rodent reservoir host, the bank vole, as a model system. We found that female bank voles infected with B. afzelii transmitted to their offspring maternal antibodies that provided highly efficient but strain-specific protection against a natural tick bite challenge. The transgenerational transfer of antibodies could be a mechanism that maintains the high strain diversity of this tick-borne pathogen in nature.

Susceptibility to infection with Borrelia afzelii and TLR2 polymorphism in a wild reservoir host.

The study of polymorphic immune genes in host populations is critical for understanding genetic variation in susceptibility to pathogens. Controlled infection experiments are necessary to separate variation in the probability of exposure from genetic variation in susceptibility to infection, but such experiments are rare for wild vertebrate reservoir hosts and their zoonotic pathogens. The bank vole (Myodes glareolus) is an important reservoir host of Borrelia afzelii, a tick-borne spirochete that causes Lyme disease. Bank vole populations are polymorphic for Toll-like receptor 2 (TLR2), an innate immune receptor that recognizes bacterial lipoproteins. To test whether the TLR2 polymorphism influences variation in the susceptibility to infection with B. afzelii, we challenged pathogen-free, lab-born individuals of known TLR2 genotype with B. afzelii-infected ticks. We measured the spirochete load in tissues of the bank voles. The susceptibility to infection with B. afzelii following an infected tick bite was very high (95%) and did not differ between TLR2 genotypes. The TLR2 polymorphism also had no effect on the spirochete abundance in the tissues of the bank voles. Under the laboratory conditions of our study, we did not find that the TLR2 polymorphism in bank voles influenced variation in the susceptibility to B. afzelii infection.

Arousal from hibernation and reactivation of Eptesicus fuscus gammaherpesvirus (EfHV) in big brown bats.

Many viruses that cause serious and often fatal disease in humans have spilled over from bats. Recent evidence suggests that stress may enhance virus shedding by bats increasing the possibility of transmission to other species. To understand the reasons for spillover is therefore important to determine the molecular pathways that link stress to virus reactivation and shedding in bats. We recently isolated and characterized a gammaherpesvirus (Eptesicus fuscus herpesvirus, EfHV) autochthonous to North American big brown bats. Since herpesviruses are known to reactivate from latent infections in response to a wide variety of stressors, EfHV presents us with an opportunity to study how physiological, behavioural or environmental changes may influence the big brown bats' relationship with EfHV. To understand the biology of the virus and how the extended periods of torpor experienced by these bats during hibernation along with the stress of arousal might influence the virus-host relationship, we attempted to detect the virus in the blood of wild-caught non-hibernating bats as well as captive bats arising from hibernation. We compared the prevalence of EfHV in the blood (using PCR) and EfHV-specific antibodies (using ELISA) between captive hibernating bats and wild-caught non-hibernating bats. We detected EfHV only in the blood of captive hibernating bats (27.8% = 10/36) and not in wild-caught non-hibernating bats (0.0% = 0/43). In contrast, the EfHV-specific antibody titres were higher in the non-hibernating bats compared to the hibernating bats. Our study suggests that: (a) viral DNA in blood indicates reactivation from latency, (b) long periods of hibernation lead to suppression of immunity, (c) stress of arousal from hibernation reactivates the virus in bats with lower levels of anti-viral immunity (indicated by humoral immune response), and (d) levels of anti-viral immunity increase in non-hibernating bats following reactivation.

Competition Between Strains of Borrelia afzelii in Immature Ixodes ricinus Ticks Is Not Affected by Season.

Vector-borne pathogens often consist of genetically distinct strains that can establish co-infections in the vertebrate host and the arthropod vector. Co-infections (or mixed infections) can result in competitive interactions between strains with important consequences for strain abundance and transmission. Here we used the spirochete bacterium, Borrelia afzelii, as a model system to investigate the interactions between strains inside its tick vector, Ixodes ricinus. Larvae were fed on mice infected with either one or two strains of B. afzelii. Engorged larvae were allowed to molt into nymphs that were subsequently exposed to three seasonal treatments (artificial summer, artificial winter, and natural winter), which differed in temperature and light conditions. We used strain-specific qPCRs to quantify the presence and abundance of each strain in the immature ticks. Co-infection in the mice reduced host-to-tick transmission to larval ticks and this effect was maintained in the resultant nymphs at 1 and 4 months after the larva-to-nymph molt. Competition between strains in co-infected ticks reduced the abundance of both strains. This inter-strain competition occurred in the three life stages that we investigated: engorged larvae, recently molted nymphs, and overwintered nymphs. The abundance of B. afzelii in the nymphs declined by 40.5% over a period of 3 months, but this phenomenon was not influenced by the seasonal treatment. Future studies should investigate whether inter-strain competition in the tick influences the subsequent strain-specific transmission success from the tick to the vertebrate host.

Competition between strains of Borrelia afzelii inside the rodent host and the tick vector.

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.