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.

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.

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.

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.

The abundance of the Lyme disease pathogen Borrelia afzelii declines over time in the tick vector Ixodes ricinus.

BACKGROUND: The population dynamics of vector-borne pathogens inside the arthropod vector can have important consequences for vector-to-host transmission. Tick-borne spirochete bacteria of the Borrelia burgdorferi (sensu lato) species complex cause Lyme borreliosis in humans and spend long periods of time (>12 months) in their Ixodes tick vectors. To date, few studies have investigated the dynamics of Borrelia spirochete populations in unfed Ixodes nymphal ticks. METHODS: Larval ticks from our laboratory colony of I. ricinus were experimentally infected with B. afzelii, and killed at 1 month and 4 months after the larva-to-nymph moult. The spirochete load was also compared between engorged larval ticks and unfed nymphs (from the same cohort) and between unfed nymphs and unfed adult ticks (from the same cohort). The spirochete load of B. afzelii in each tick was estimated using qPCR. RESULTS: The mean spirochete load in the 1-month-old nymphs (~14,000 spirochetes) was seven times higher than the 4-month-old nymphs (~2000 spirochetes). Thus, the nymphal spirochete load declined by 80% over a period of 3 months. An engorged larval tick acquired ~100 spirochetes, and this population was 20 times larger in a young, unfed nymph. The spirochete load also appeared to decline in adult ticks. Comparison between wild and laboratory populations found that lab ticks were more susceptible to acquiring B. afzelii. CONCLUSION: The spirochete load of B. afzelii declines dramatically over time in domesticated I. ricinus nymphs under laboratory conditions. Future studies should investigate whether temporal declines in spirochete load occur in wild Ixodes ticks under natural conditions and whether these declines influence the tick-to-host transmission of Borrelia.

Inefficient co-feeding transmission of Borrelia afzelii in two common European songbirds.

The spirochete bacterium Borrelia afzelii is the most common cause of Lyme borreliosis in Europe. This tick-borne pathogen can establish systemic infections in rodents but not in birds. However, several field studies have recovered larval Ixodes ricinus ticks infected with B. afzelii from songbirds suggesting successful transmission of B. afzelii. We reviewed the literature to determine which songbird species were the most frequent carriers of B. afzelii-infected I. ricinus larvae and nymphs. We tested experimentally whether B. afzelii is capable of co-feeding transmission on two common European bird species, the blackbird (Turdus merula) and the great tit (Parus major). For each bird species, four naïve individuals were infested with B. afzelii-infected I. ricinus nymphal ticks and pathogen-free larval ticks. None of the co-feeding larvae tested positive for B. afzelii in blackbirds, but a low percentage of infected larvae (3.33%) was observed in great tits. Transstadial transmission of B. afzelii DNA from the engorged nymphs to the adult ticks was observed in both bird species. However, BSK culture found that these spirochetes were not viable. Our study suggests that co-feeding transmission of B. afzelii is not efficient in these two songbird species.

Multistrain Infections with Lyme Borreliosis Pathogens in the Tick Vector.

Mixed or multiple-strain infections are common in vector-borne diseases and have important implications for the epidemiology of these pathogens. Previous studies have mainly focused on interactions between pathogen strains in the vertebrate host, but little is known about what happens in the arthropod vector. Borrelia afzelii and Borrelia garinii are two species of spirochete bacteria that cause Lyme borreliosis in Europe and that share a tick vector, Ixodes ricinus Each of these two tick-borne pathogens consists of multiple strains that are often differentiated using the highly polymorphic ospC gene. For each Borrelia species, we studied the frequencies and abundances of the ospC strains in a wild population of I. ricinus ticks that had been sampled from the same field site over a period of 3 years. We used quantitative PCR (qPCR) and 454 sequencing to estimate the spirochete load and the strain diversity within each tick. For B. afzelii, there was a negative relationship between the two most common ospC strains, suggesting the presence of competitive interactions in the vertebrate host and possibly the tick vector. The flat relationship between total spirochete abundance and strain richness in the nymphal tick indicates that the mean abundance per strain decreases as the number of strains in the tick increases. Strains with the highest spirochete load in the nymphal tick were the most common strains in the tick population. The spirochete abundance in the nymphal tick appears to be an important life history trait that explains why some strains are more common than others in nature. IMPORTANCE: Lyme borreliosis is the most common vector-borne disease in the Northern Hemisphere and is caused by spirochete bacteria that belong to the Borrelia burgdorferi sensu lato species complex. These tick-borne pathogens are transmitted among vertebrate hosts by hard ticks of the genus Ixodes Each Borrelia species can be further subdivided into genetically distinct strains. Multiple-strain infections are common in both the vertebrate host and the tick vector and can result in competitive interactions. To date, few studies on multiple-strain vector-borne pathogens have investigated patterns of cooccurrence and abundance in the arthropod vector. We demonstrate that the abundance of a given strain in the tick vector is negatively affected by the presence of coinfecting strains. In addition, our study suggests that the spirochete abundance in the tick is an important life history trait that can explain why some strains are more common in nature than others.