Since tick-borne diseases (TBDs) incidence, both in human and animal populations, is increasing worldwide, there is the need to assess the presence, distribution and prevalence of tick-borne pathogens. Reliable estimates on tick-borne pathogens (TBPs) prevalence represent the public health foundation to create risk maps and take effective prevention and control actions against TBDs. Tick surveillance consists of collecting and testing (usually in pools) thousands of specimens. Construction and analysis of tick pools represent a challenge due to the complexity of tick-borne pathogens and tick-borne diseases ecology. The aim of this study is to provide a practical guideline on appropriate pooling strategies and statistical analysis of infection prevalence through: (i) reporting the different pooling strategies and statistical methodologies commonly used to calculate pathogen prevalence in tick populations and (ii) practical comparison between statistical methods utilising a real dataset of infection prevalence in ticks collected in Northern Italy. Reporting detailed information on tick pool composition and size is as important as the correct TBPs prevalence estimation. Among the prevalence indexes, we suggest using maximum-likelihood estimates of pooled prevalence instead of minimum infection rate or pool positivity rate given the merits of the method and availability of software.

Exploratory behaviour is one of the best-investigated behavioural traits. However, little is known about how differences in familiarity, i.e. in the knowledge and previous experience with a companion can influence the exploration of a novel environment. However, to our knowledge, such a critical feature of the social environment has never been the target of a study relating it to exploratory behaviour in birds. Here we examined if familiarity with a conspecific could affect behavioural responses of individuals confronted with a novel environment. We recorded the latency to land on the ground, latency to feed, time spent feeding and number of sectors visited of 48 female and 48 male house sparrows (Passer domesticus) in an indoor aviary in three contexts: alone (individual context), with an unfamiliar and with a familiar same-sex companion. House sparrows landed sooner on the ground when in the familiar context than when in the individual context. Birds in unfamiliar pairs followed each other less than familiar birds, but this difference diminished with time spent exploring. Moreover, males and females differed in their behavioural responses in the unfamiliar context. Females with a familiar companion landed sooner than when they were paired with an unfamiliar conspecific, whereas only the presence of a companion but not familiarity reduced males latency to land on the ground. Finally, when considering the unfamiliar context males had shorter latencies to forage and thus spent more time eating than females. The presence or absence of a companion and its familiarity with the focal individual influenced differently the behavioural responses of male and female house sparrows in a novel environment. As house sparrows are strongly sociable, the influence of the social environment is likely to be of paramount importance to understand the selective pressures acting on them, particularly in recently colonized areas with ephemeral food sources. Our results shed light on the complex influence that the social environment has on the behavioural responses of a cosmopolitan bird.

Since most avian species have been considered anosmic or microsmatic, olfaction and associated behavioural patterns have hardly been investigated. Most importantly, empirical data on avian olfaction is not equally distributed among species. Initial investigations focused on species with relatively big olfactory bulbs because they were thought to have better olfactory capabilities. Hence, in this study we tested the ability of house sparrows (Passer domesticus) to use chemical cues as parameters to estimate nest features. House sparrows are a commonly used model species, but their olfactory capabilities have not been studied so far. We offered two different odours to males and females, namely the scent of mouse urine (Mus musculus domesticus), representing a possible competitor and a threat to eggs and hatchlings, and the odour of hay, representing a familiar and innocuous odour. The experiment was performed at the sunset to simulate a first inspection to new possible roosting or nesting sites. Interestingly, males but not females preferred to spend significantly more time in front of the hay odour, than in front of the scent of mouse urine. Our results strengthen the hypothesis that oscines can not only perceive odours but also use olfaction to assess the environment and estimate nest site quality.

In the last decades, higher attention has been paid to olfactory perception in birds. As a consequence, a handful of avian species have been discovered to use olfaction in different contexts. Nevertheless, we still have a very limited knowledge about the use of odor cues in avian social life, particularly, in the case of songbirds. Here, we investigate if female house sparrows Passer domesticus show any preference for the odor of kin and nonkin conspecifics and we also test a possible role of familiarity based on male scent in female choice. We performed the experiment with captive birds twice, during the nonbreeding and breeding seasons. Our results show that female house sparrows strongly avoided the odor of unrelated familiar (UF) males, both in the breeding and nonbreeding seasons. Our results suggest recognition for olfactory stimuli related to familiarity and kinship. We suggest that avoidance for UF males is associated with previous experience in this species. Also, we provided further evidence to the use of olfaction in passerine species by using a new experimental setup.

Candidatus Rickettsia vini was originally detected in Ixodes arboricola ticks from Spain, and subsequently reported from several other Western Palearctic countries including Belgium. Recently, the bacterium was isolated in mammalian (Vero) cell culture from macerated male I. arboricola from Czech Republic, but there have been no reports of propagation in tick cells. Here we report isolation in a tick cell line of three strains of Ca. R. vini from I. arboricola collected from nests of great tits (Pants major) in Belgium. Internal organs of one male and two engorged female ticks were dissected aseptically, added to cultures of the Rhipicephalus microplus cell line BME/CTVM23 and incubated at 28 degrees C. Rickettsia-like bacteria were first seen in Giemsa-stained cytocentrifuge smears between 2 and 15 weeks later. Two of the isolates grew rapidly, destroying the tick cells within 2-4 weeks of onward passage in BME/CTVM23 cells, while the third isolate grew much more slowly, only requiring subculture at 4 - 5-month intervals. PCR amplification of bacterial 16S rRNA and Rickettsia gltA, sca4, ompB, ompA and 17-kDa genes revealed that all three isolates were Ca. R. vini, with 100 % identity to each other and to published Ca. R. vini sequences from other geographical locations. Transmission electron microscopy revealed typical single Rickettsia bacteria in the cytoplasm of BME/CTVM23 cells. The Ca. R. vini strain isolated from the male I. arboricola tick, designated Boshoek1, was tested for ability to grow in a panel of Ixodes ricinus, Ixodes scapularis and R. microplus cell lines and in Vero cells. The Boshoek1 strain grew rapidly, causing severe cytopathic effect, in the R. microplus line BME26, the I. ricinus line IRE11 and Vero cells, more slowly in the I. ricinus line IRE/CTVM19, possibly established a low-level infection in the I. ricinus line IRE/CTVM20, and failed to infect cells of any of four I. scapularis lines over a 12-week observation period. This study confirmed the applicability of the simple tick organ-cell line co-cultivation technique for isolation of tick-borne Rickettsia spp. using BME/CTVM23 cells.