Influence of phylogeny, migration and type of diet on the presence of intestinal parasites in the faeces of European passerine birds (Passeriformes)

Migratory and non-migratory passerine birds can carry several pathogens, including parasites, which may cause significant diseases in birds, other animal species and humans. Parasites have been shown to negatively impact many populations of wildlife, and this may become more significant with global temperature changes. This study was performed to investigate the prevalence of intestinal parasites in faecal samples of European passerines. Intestinal parasites identified were statistically associated with passerines phylogenetic classification, migratory habits (migratory, non-migratory) and the type of diet (omnivorous, insectivorous and granivorous). A total of 385 passerines of 42 species were captured and their droppings collected. The prevalence of parasites in faecal samples of passerines was 15.6%. Intestinal parasites were identified in 50/309 (16.2%) migratory passerines and 10/76 (13.2%) non-migratory passerines using the faecal flotation method. Coccidia were most often identified parasites; they were more likely to be present in an omnivorous bird species (p = 0.02). Syngamus spp. was more likely to be detected in omnivorous passerines (p = 0.04). Tits (p = 0.01) and finches (p = 0.006) were less likely to have intestinal parasites present in their faecal samples than passerines classified in other phylogenetic clades. Tits (p = 0.02) and finches (p = 0.008) were also less likely to have coccidia present in their faecal samples. Phylogeny was associated with the presence of parasites in faecal samples of passerines (p = 0.03). The prevalence of parasites, however, was not associated with the migration habit of passerines, but to the type of diet (p = 0.04). Our analysis suggests that the diversity of feeding sources of omnivore passerines exposes them to infection with intestinal parasites to a greater extent than granivore or insectivore passerines.


Infl uence of phylogeny, migration and type of diet on the presence of intestinal parasites in the faeces of European passerine birds (Passeriformes)
Several bird species migrate continental or intercontinental distances to survive seasonal climate changes and/or to be able to breed. In Europe birds migrate from north and central Europe to southern parts of Europe, the Middle East and sub-Saharan Africa, or they roam across the European continent (Hahn et al. 2009). Such migration provides a mean for circulation of parasitic, bacterial and viral pathogens along macro-ecological niches present in Europe, Asia and Africa (Hub á lek 2004, Jellison et al. 2007, Keawcharoen et al. 2008, Fuller et al. 2012. A vast body of research has been published defi ning the epidemiology of diff erent pathogens in migratory passerine bird species (passerines; Passeriformes ) that are, or are potentially harmful for human or other animal species health (Bengis et al. 2004, Hub á lek 2004. Less research, however, has been directed towards studying emerging pathogens, and those that were not considered highly virulent for passerines and are now re-emerging. Recent disease outbreaks in wild bird populations can or are expected to become more signifi cant as a consequence of climate changes (Robinson et al. 2010, Kilpatrick 2011, Fuller et al. 2012. Besides their lethal eff ect, parasite emergence can change host population dynamics and modify coevolution relationships between hosts and their parasites. Th e evolutionary pressure of the host and the parasite contributed to the position of species on the phylogenetic tree (Best et al. 2010).
Studies in European passerines investigated their arthropod ectoparasites (Trilar 2004, Sychra et al. 2008), blood parasites including Plasmodium (Scheuerlein andRicklefs 2004, Levin et al. 2009) and intestinal parasites (Svobodov á 1994, Zinke et al. 2004, Dolnik 2006, L ó pez et al. 2007, Brown et al. 2010, Dolnik et al. 2010. However, the prevalence and infl uence of the overall presence of intestinal parasites has not been investigated. Intestinal parasitic infections are present globally and are a signifi cant cause of illness and disease in animals and humans (Th omas et al. 2005, Haque 2007, Benchaoui 2010. Th ey may become a more signifi cant problem in temperate climates because global temperature patterns may change established parasite -host relationships with an increased parasite development rate and a larger parasite pressure on the host (Tompkins and Begon 1999, Read and Taylor 2001, Hudson et al. 2006). Figure 1. Th e map shows the summer breeding grounds of migrating passerines from north Europe (yellow and red coloured areas north of the green fl ag) that migrate through Slovenia (green fl ag) to their non-breeding grounds (red and yellow coloured areas south of the green fl ag) in winter. Th e yellow to red coloured areas above the green fl ag indicate increase density (congregation) of migrating passerines, whereas the red to yellow coloured areas below the green fl ag indicate decrease density (dispensation) of migrating passerines.
Th e purpose of this study was, therefore, to evaluate the prevalence of intestinal parasites in faecal droppings of different species of European migratory and non-migratory passerines, and to associate specifi c parasite prevalence with the birds ' phylogenetic classifi cation and, more specifi cally, with their migratory habit and the type of diet.
Birds were classifi ed into eight clades according to their morphological and molecular characteristics (Table 1) (Cramp 1988, 1992, Cramp and Perrins 1993, 1994a, b, Jancar et al. 1999, Barker et al. 2002, Fregin et al. 2012, McCormack et al. 2013, BirdLife International 2014. Th ey were further classifi ed according to their migrating habits (migratory, non-migratory) and the type of diet (omnivorous, granivorous and insectivorous) ( Table 2) (Cramp 1988, 1992, Cramp and Perrins 1993, 1994a. Birds were captured in the afternoon with mist nets and placed individually in clean cloth bags for no more than 30 min. After the bird was removed from the bag to be ringed, weighed, measured and their species and age determined, the faecal sample was collected from the bag if present. Droppings that corresponded to urine (i.e. lacked faecal material) were rejected (L ó pez et al. 2007). Faecal samples were placed in clean 2 ml vials and stored immediately at 4 ° C to preserve diagnostic parasitic stages. Samples were transported to the laboratory for parasitological analysis on the morning following the sampling.
Th e fl otation method was used (with saline solution density: 1.20) as a diagnostic concentration method to separate parasite particles from faecal debris. Th e solution was left standing for a minimum of 15 min before making the wet mount for the microscopic examination. Th e whole amount of the concentrate from the surface of the solution was placed on a microscope slide and evaluated at 100 ϫ magnifi cation (Maizels and Yazdanbakhsh 2003).

Statistical analysis
Fisher's exact test was used to univariatley associate phylogenetic characteristics, the type of diet and migration to the presence of parasites. Th e association between phylogenetic characteristics and the presence of parasites was also analysed with the logistic regression using the Firth's correction (Heinze and Schemper 2002). Briefl y, Firth's correction was proposed to solve the problem of separation that can occur when studying rare events. Logistic regression was used also to estimate the multivariate association between the type of diet, migratory habits and the presence of parasites. In order to account for the possible confounding eff ect of species and phylogeny, both eff ects were included in separate models as a random eff ect; separate models for both eff ects were estimated as a simultaneous inclusion of both factors was not possible due to scarce presence of parasites. A p-value equal to or less than 0.05 was considered as statistically signifi cant. Th e analysis was performed using the R language for statistical computing ( Ͻ www.r-project.org Ͼ ).
Th e type of diet was univariately associated with the presence of Syngamus spp. (p ϭ 0.05). Syngamus spp. were more likely to be detected in omnivorous than granivorous and insectivorous passerines. However, this also was not strongly statistically supported by our data (OR: 0.1, 95% CI: 0.01 -0.8, p ϭ 0.09).

Discussion
Some studies have revealed the prevalence of coccidial infection in certain species of birds in Europe (Zinke et al. 2004), whereas others have evaluated the role of foraging ecology in coccidian infections (Dolnik et al. 2010). Our study is the fi rst study that investigated the association of intestinal parasites prevalence in European passerines with their phylogeny, migration habits and the type of diet. Our results indicated that the prevalence of intestinal parasites in passerines ' faeces is 15.6%; the prevalence of intestinal parasites is not associated with migration, but is associated with the type of diet and overall phylogenetic relationship.
Although the prevalence of parasites in migratory passerines was higher than in non-migratory passerines, the diff erence was not found to be signifi cant. Climate is an important determinant of transmission of parasitic infections, with adequate moisture and warm temperature essential for ova persistence and larval development in the soil or on surfaces (Brooker et al. 2006). It is expected that migratory birds have a greater chance to acquire parasite infections because by migrating they constantly dwell in a relatively warm environment throughout the year, which does not inhibit the parasite life cycle. Th ey can be infected by parasites throughout the year, whereas non-migratory birds remaining in temperate latitudes are less likely to be infected during the cold winter period when parasites are not active. However, the eff ect of winter can be reduced in intensively cultivated habitats (farming) with concentrated bird or other animal population and higher microenvironmental temperatures (Tompkins and Begon 1999, Anderson 2000, Read and Taylor 2001, Hudson et al. 2006, Benchaoui 2010.
Most studies regarding the transmission of pathogens or potential pathogens are aimed at informing the human community about the risks for the disease transmission from birds to humans and/or farm animals. Seasonal bird migration has been associated with the geographic distribution of viruses (Eastern equine encephalitis virus, West Nile to a substantial decline or even an extinction of the species (Holmstad et al. 2006, Vergara et al. 2011, Martinez-Padilla et al. 2012. Successful transmission of parasites is dependent on exposure of susceptible hosts to free-living infective stages, which are the specifi c parasite life cycle forms capable of infecting the host (infectious parasite particles). Parasites can be transmitted through animal (arthropod) vectors or invade feeding grounds (Anderson 2000). Some parasitic infective larvae are known to selectively ascend food plants to increase their chance to invade the bird host (Saunders et al. 2001). As evident from this study, insectivorous and omnivorous passerines are likely to be exposed to a variety of infectious parasite particles during their feeding, whereas granivorous passerines had only coccidia present in their faeces. Granivorous passerines are less likely to consume invertebrate vectors or seeds contaminated with infectious nematode/cestode parasite particles.
Omnivorous passerines are more likely to be infected with coccidia ( Eimeria spp. and Isospora spp.). Th ey are opportunistic feeders and often feed in the ecological niches, which are populated by humans or intensively cultivated by farming and are, therefore, more likely to be contaminated with greater number of infectious parasite particles (Mennerat et al. 2010). In such an environment birds can transmit parasites to mammals; transmission of Ascaris spp. from chickens to pigs was reported by Permin et al. (2000).
Th e presence of Syngamus spp. was associated with the type of diet. Our data also indicated that Syngamus spp. was more likely to be detected in omnivore passerines. Syngamus spp. is the gapeworm of poultry and many species of wild birds (Anderson 2000, Permin et al. 2000. Wild birds may serve as the source of Syngamus infections in outbreaks on poultry and game-bird farms, and vice versa (Anderson 2000). It is possible that omnivores come in closer contact with farmed bird species when seeking food, therefore, entering the concentrated source of Syngamus spp.
Parasites can negatively aff ect many species of wildlife and are the evolutional partner in the development of the host -parasite interaction (Tompkins andBegon 1999, Read andTaylor 2001). It is important to realise that erratic changes in the environment can cause acute adaptations in microbes/parasites or in the host, which can be expressed with a variable degree of lethality for the host population (Casadevall and Pirofski 2000, Maizels and Yazdanbakhsh 2003, Hudson et al. 2006). If evolution is given time , the host -parasite relationship can co-evolve and again become harmonious enabling sustainability of both populations over time (Maizels andYazdanbakhsh 2003, Holmstad et al. 2006, Vergara et al. 2011, Martinez-Padilla et al. 2012. Evaluation of faeces for evidence of parasites in small birds is challenging (Dolnik 2006). A fl otation method was used to concentrate faecal parasitic forms in this study. Passerine birds produced small amounts of faeces ( Ͻ 1 g) and fed on abundant fruit/seed on their autumn migration, which discoloured their faeces and rendered sedimentation or a smear investigation less appropriate. Flotation method used in this study resulted in a clean sample preparation for microscopic examination with a minimal amount of distracting discoloration. Some parasite eggs, however, do not fl oat in saline solution (Th ienpont et al. 1979), and may have been missed using this method only.
virus, Infl uenza A virus, Newcastle disease virus), bacteria ( Anaplasma phagocytophilum , Borrelia burgdorferi , Campylobacter jejuni , Pasteurella multocida , Clostridium botulinum , Mycobacterium avium ), as well as protozoa and parasites (Hub á lek 2004). Fewer studies, however, had the objective to recognize and follow pathogenic elements, including parasites that can be harmful only to birds. Knowing and following the prevalence and intensity of parasitic infection in any animal species may be important for future studies evaluating how global climate change may have infl uenced rates of parasites ' prevalence (Tompkins andBegon 1999, Smallridge andBull 2000). In the recent study by Lawson et al. (2011), trichomonosis was identifi ed as a newly emerging signifi cant disease in greenfi nch Carduelis chloris population in England and continental Europe. Trichomonas gallinae , a protozoan parasite responsible for trichomonosis, was often isolated from pigeons, doves and birds of prey, and was not considered dangerous for garden birds (Dovc et al. 2004). However, it was recently recognized as a significant factor in an epidemic mortality and population decline of greenfi nches Carduelis chloris and chaffi nches Fringilla coelebs in UK/Europe (Lawson et al. 2011). Th erefore, it is important that existing pathogens and potentially pathogenic organisms for specifi c bird population are recognized, including intestinal parasites (Th omas et al. 2005, Haque 2007, Benchaoui 2010. Our results show that Fringillidae (clade 7), which include greenfi nches, together with tits (clade 4: Certhiidae, Paridae and Aegithalidae), are less likely to have parasites in their faeces than passerines classifi ed in other clades. It is not possible to clearly defi ne the reasons for clades 4 and 7 to be less likely positive for intestinal parasites in their faeces. Passerines from clades 4 and 7 are nonmigratory birds and, based on their biology, less likely to come in close contact with concentrated source of infectious parasitic particles in intensively cultivated farming environments.
Taxonomic diversifi cation of passerines has been accompanied by extensive diversity, and has proved diffi cult to classify (Cramp 1988, 1992, Cramp and Perrins 1993, 1994a, b, Jancar et al. 1999, BirdLife International 2014. Molecular methods have put some more order into the taxonomy (Fregin et al. 2012, McCormack et al. 2013). However, passerines origin, phylogeny, biogeography, anatomical and morphological characteristics, migration habits and the type of diet in combination or individually can infl uence the presence and dissemination of intestinal parasites in passerines ' faeces. Th ese characteristics were all utilized to classify passerines in this study (Table 1).
Several parasites were detected in our bird faecal samples: Eimeria spp., Isospora spp., Trichostrongylus tenuis , Syngamus spp., cestoda ova and ascarid ova. Captive (Svobodov á 1994, Quiroga et al. 2000 and wild (Svobodov á 1994, McQuistion 2000 passerines can be infected with Eimeria spp. and Isospora spp., which can cause a serious clinical disease and high mortality (Giacomo et al. 1997). Less is known about the infection with Trichostrongylus tenuis , Syngamus spp., cestoda and ascarids in passerines. Th ey can, however, signifi cantly aff ect birds ' health and behaviour and render them less competitive and more vulnerable in their environment, which is evident through reduced fi tness to feed, court and/or breed. It can lead to either an evolutionary adaptation(s) of the host to a specifi c parasite, or Th e presence of parasites in passerine faeces was not strongly infl uenced by migration, which is intriguing because non-migratory passerines should not be exposed to infective parasite stages during the winter, whereas migratory passerines are constantly pressure by parasite infection in the warm environment.
One quarter of all European breeding passerines and near-passerine birds migrate from the European continent to sub-Saharan Africa in autumn each year, which is estimated to be over 2 billion birds (Moreau 1972, Hahn et al. 2009). Such migration can have an impact on local communities and can aff ect the health of birds or other animals, and humans. Perhaps most importantly in the long term, warmer global temperatures may favour parasite development and infectivity, which may signifi cantly impact the health of several animal populations. Th erefore, studies like ours are important. We need to investigate and follow the prevalence of parasites and other etiological factors that can have a negative eff ect on passerine bird populations. In short, only by knowing the present epidemiological situation off ers the possibility to examine the infl uence of global warming on the prevalence of parasites in passerines.