Host dispersal shapes the population structure of a tick-borne bacterial pathogen.

Birds are hosts for several zoonotic pathogens. Because of their high mobility, especially of longdistance migrants, birds can disperse these pathogens, affecting their distribution and phylogeography. We focused on Borrelia burgdorferi sensu lato, which includes the causative agents of Lyme borreliosis, as an example for tick-borne pathogens, to address the role of birds as propagation hosts of zoonotic agents at a large geographical scale. We collected ticks from passerine birds in 11 European countries. B. burgdorferi s.l. prevalence in Ixodes spp. was 37% and increased with latitude. The fieldfare Turdus pilaris and the blackbird T. merula carried ticks with the highest Borrelia prevalence (92 and 58%, respectively), whereas robin Erithacus rubecula ticks were the least infected (3.8%). Borrelia garinii was the most prevalent genospecies (61%), followed by B. valaisiana (24%), B. afzelii (9%), B. turdi (5%) and B. lusitaniae (0.5%). A novel Borrelia genospecies "Candidatus Borrelia aligera" was also detected. Multilocus sequence typing (MLST) analysis of B. garinii isolates together with the global collection of B. garinii genotypes obtained from the Borrelia MLST public database revealed that: (a) there was little overlap among genotypes from different continents, (b) there was no geographical structuring within Europe, and (c) there was no evident association pattern detectable among B. garinii genotypes from ticks feeding on birds, questing ticks or human isolates. These findings strengthen the hypothesis that the population structure and evolutionary biology of tick-borne pathogens are shaped by their host associations and the movement patterns of these hosts.

Getting under the birds' skin: tissue tropism of Borrelia burgdorferi s.l. in naturally and experimentally infected avian hosts.

Wild birds are frequently exposed to the zoonotic tick-borne bacteria Borrelia burgdorferi sensu lato (s.l.), and some bird species act as reservoirs for some Borrelia genospecies. Studying the tropism of Borrelia in the host, how it is sequestered in different organs, and whether it is maintained in circulation and/or in the host's skin is important to understand pathogenicity, infectivity to vector ticks and reservoir competency.We evaluated tissue dissemination of Borrelia in blackbirds (Turdus merula) and great tits (Parus major), naturally and experimentally infected with Borrelia genospecies from enzootic foci. We collected both minimally invasive biological samples (feathers, skin biopsies and blood) and skin, joint, brain and visceral tissues from necropsied birds. Infectiousness of the host was evaluated through xenodiagnoses and infection rates in fed and moulted ticks. Skin biopsies were the most reliable method for assessing avian hosts' Borrelia infectiousness, which was supported by the agreement of infection status results obtained from the analysis of chin and lore skin samples from necropsied birds and of their xenodiagnostic ticks, including a significant correlation between the estimated concentration of Borrelia genome copies in the skin and the Borrelia infection rate in the xenodiagnostic ticks. This confirms a dermatropism of Borrelia garinii, B. valaisiana and B. turdi in its avian hosts. However, time elapsed from exposure to Borrelia and interaction between host species and Borrelia genospecies may affect the reliability of skin biopsies. The blood was not useful to assess infectiousness of birds, even during the period of expected maximum spirochetaemia. From the tissues sampled (foot joint, liver, spleen, heart, kidney, gut and brain), Borrelia was detected only in the gut, which could be related with infection mode, genospecies competition, genospecies-specific seasonality and/or excretion processes.

Characterization Through Multilocus Sequence Analysis of Borrelia turdi Isolates from Portugal.

Borrelia turdi is a spirochete from the Borrelia burgdorferi complex, first reported in Japan, that has been increasingly detected in Europe. This genospecies is mostly associated with avian hosts and their ornithophilic ticks such as Ixodes frontalis. In this study, we isolated B. turdi from five I. frontalis feeding on Turdus merula, Turdus philomelos, Parus major and Troglodytes troglodytes, and one Ixodes ricinus feeding on a T. merula in Portugal. These isolates were genetically characterised according to their 5S-23S rRNA intergenic spacer, 16S rRNA and through typing of seven housekeeping genes (multilocus sequence typing). Multilocus sequence analyses revealed that the strains isolated in our study, although belonging to B. turdi genospecies, are not identical to the B. turdi reference strain Ya501. Instead, our strains are separated into a clear defined group, suggesting that the European samples diverged genetically from the strain originally detected in Japan. Population analysis of 5S-23S rRNA sequences can further resolve subpopulations within B. turdi, but more samples from a large geographical scale and host range would be needed to assess potential phylogeographical patterns within this genospecies.

Reliability of steatocrit as an indicator of intestinal health in young birds - Relationships with morphology and growth rate of canary Serinus canaria nestlings.

Intestinal health and capacity to efficiently absorb nutrients from diet, including fat, has been recently suggested as a promising physiological health indicator in wild birds, due to its association with gut parasitism and feather coloration. However, little information is available about the sources of variation of non-absorbed fat in birds' faeces, measured by the acid steatocrit technique, and particularly in nestling birds. We assessed steatocrit in captive nestling canaries Serinus canaria and evaluated if it was affected by breed and its relationship with growth (linear growth rate, LGR) and body mass. We also assessed the presence of coccidia infections. Steatocrit differed significantly between day 9 and 14 of the nestling period, being lower when nestlings were 14 days old. Age must be taken into account when assessing steatocrit in young birds; this variation may be due to a development of the digestive system with age or the amount of fat provided later in the nestling period being more adequate to the nestlings' needs. Steatocrit was higher in nestling canaries from yellow lipochromic than red lipochromic breeds. No coccidia infections were detected. Steatocrit did not affect LGR, but steatocrit measured at day 9 was positively related with body mass at day 14. The positive relationship between steatocrit and body mass in canary nestlings suggests that nestlings in breeding facilities may be fed a larger amount of fat than that they can absorb. This physiological assessment of intestinal health may be a better general health indicator in wild adult birds exposed to a higher level and diversity of parasites.

Experimental infection by microparasites affects the oxidative balance in their avian reservoir host the blackbird Turdus merula.

By draining resources, microparasites can negatively affect the host fitness, which in turn can result in reduced transmission when virulence leads to reductions in host population size. Therefore, for a microparasite to persist in nature, the level of harm it can do to its host is expected to be limited. We tested this hypothesis for tick-borne Borrelia burgdorferi sensu lato (s.l.) infections in the blackbird Turdus merula, one of the most important avian reservoir hosts in Europe. Experimental and observational data were combined to examine the physiological effects caused by B. burgdorferi s.l. infection in blackbirds. Pathogen-free blackbirds were exposed to B. burgdorferi s.l.-infected Ixodes ricinus and I. frontalis nymphs, and compared with a control group (exposed to naïve laboratory-derived I. ricinus nymphs). Their physiological status was evaluated before and after infection with B. burgdorferi s.l., through a set of immunological (erythrocyte sedimentation rate, haptoglobin, white blood cell count and heterophil/lymphocyte ratio), oxidative stress (glutathione peroxidase activity, protein carbonyls and nitric oxide) and general body condition variables (body condition, glucose and haematocrit). Infected males showed higher levels of oxidative damage to proteins (increased levels of protein carbonyls), decreased glutathione peroxidase activity and increased body mass. Infected females had higher levels of glutathione peroxidase activity after infection by B. burgdorferi s.l. than the control group. No significant effects of B. burgdorferi s.l. infection were detected on erythrocyte sedimentation rate, haptoglobin, heterophil/lymphocyte ratio, nitric oxide, glucose and haematocrit. The first experimental study on the effects of B. burgdorferi s.l. on its avian reservoir hosts shows that these bacteria may inflict non-negligible physiological costs. We speculate that during energetically demanding periods, these physiological costs may reduce host fitness and affect pathogen transmission.