Prevalence of Trichomonas in columbids
Stock doves, wood pigeons, collared doves and turtle doves in our study throughout a range of European sites showed a high prevalence of infection by Trichomonas sp. These results are in agreement with previous findings in the UK .
Although we only had a small sample size of collared doves, the prevalence seems high (67%). Additionally, if we separate the results by countries, Spanish collared doves suggest a much lower prevalence by Trichomonas sp. (33%) than in the UK (86%, see ) and lie closer to the prevalence shown in Iraq (10%, see ), but the samples from Malta showed a prevalence closer to the results shown in the UK (Fig. 1). Both compared studies used swab samples [12, 46] as we did in our survey, thus the differences cannot be linked to the different sample material. Furthermore, Al-Bakry  used a larger sample size of collared doves (n = 40) compared to Lennon et al. , who used seven individuals. Thus, the Iraqi results might be more reliable and a low Trichomonas prevalence in collared doves, as shown in the present study, may be more realistic. However, the small sample size in our study and Lennon et al.  should be interpreted carefully.
In our study, stock doves had a much higher prevalence than previously suggested (22–40% [12, 45]). Furthermore, chicks had higher prevalence of Trichomonas than adults, which is in agreement with Bunbury , who showed the negative impact of trichomonosis in Mauritian Pink pigeon chicks until an age of three months. In the present study, stock dove chicks were between five and 22 days old and therefore, within the most common time for infection with Trichomonas parasites in pigeon chicks .
In turtle doves, a high infection status of 67% was detected, which is 1/3 lower than previously observed in the UK (95%) [R. C. Thomas, unpublished data]. However, if we disregard the prevalence results of defrosted tissue samples from Maltese turtle doves, we would have a prevalence of 93% in turtle doves from Europe (Table 1), thus very similar to results from the UK.
On the other hand, our results revealed much higher prevalence for wood pigeons than previously suggested (47%, see ). However, it needs to be taken into account that the 70% prevalence of Trichomonas infection in German wood pigeons and the very low prevalence in turtle doves from Malta likely underestimates the true prevalence, because samples were not cultured directly, but analysed from tissues after freezing and defrosting. This might cause major differences in the detection rate, because of DNA degradation due to several freezing and thawing cycles, especially for Maltese samples during transportation [47,48,49,50]. Furthermore, Dunn et al.  showed the need to culture Trichomonas samples to reliably detect the infection. However, our results suggest high prevalence, at least for wood pigeons, but note that Maltese tissue samples were treated with a different Taq DNA Polymerase than wood pigeon samples. Thus, it might be the DreamTaq DNA Polymerase was less sensitive to DNA in thawed tissue samples.
Compared to other columbid species like mourning doves (Zenaida macroura), the overall prevalence of Trichomonas protozoan in the species studied here lies at the top of prevalence ranges. Mourning doves showed a very low prevalence of only 5.6% ranging from 4.4 to 10.6% . In the endangered Mauritian pink pigeon the average prevalence of Trichomonas was 50% (ranging from 20 to 82%) , and therefore lower than in turtle doves, which are listed as a vulnerable species . However, such prevalence has already been highlighted as a major threat to pink pigeons’ population recovery . This might be caused by their small distribution range and population size, but also reveals the importance of gaining knowledge about parasite infection, particularly in recovering and declining birds. In the UK, trichomonosis has been indicated as a potential additive factor for the reported population decline of turtle doves since the 1970s . Furthermore, Calderon et al.  already highlighted the decreasing effective population size of turtle doves, which makes them more vulnerable to threats. Thus, the present study suggests a potentially strong impact of Trichomonas on declining turtle doves across western and central Europe and likewise on recovering German stock dove populations.
Turtle doves are likely to be more dependent on anthropogenic food sources at feeding sites than in the 1960s, which was described in detail in the UK . However, this circumstance might not only be limited to Great Britain, since it is linked to increased use of herbicides resulting from agricultural intensification, which has occurred throughout Europe [55, 56]. Furthermore, in the last century a change and intensification in forest management occurred as well , which led to a decreased number of natural breeding holes. That is why nowadays stock doves are largely dependent on artificial nest boxes in Germany [19, 56]. In Hesse, the stock dove population recovered locally, through provision of artificial nest boxes. However, besides increased food stress or insufficient natural breeding sites , turtle doves, stock doves, but also wood pigeons are migratory birds , which is why they may be more exposed to a wider range of parasites and pathogens of different bird species or populations (stop-overs at feeding and water sites) [15, 19, 21, 55]. Moreover, despite the existence of three main migratory flyways , European turtle doves show a lack of genetic structure . That increases the probabilities for higher vulnerability and potential fast spread of infection among turtle dove populations.
Resident species, in contrast, may be less prone to infection, as shown for collared doves in Iraq  and Spain (this study, but see ). On the other hand, collared doves in Malta indicated a 100% Trichomonas prevalence.
As the disease occurs worldwide and is rapidly spreading, i.e. among wild finches , it seems likely that especially turtle doves and stock doves are also infected in other European countries. Regarding turtle doves, information on the disease in eastern Europe would be especially interesting, since, to our knowledge, no data on prevalence are available yet from the eastern European distribution range. Furthermore, the transmission may be reduced where no supplementary food is provided for endangered or vulnerable species and gamebirds .
Phylogenetic relationships among Trichomonas lineages
Several attempts have been made to classify the genetic diversity of Trichomonas parasites in birds, with ensuing different nomenclatures (Table 2). Out of a total of seven genetic lineages found in the present study, three lineages were found in all columbid species we examined: lineages II , P and C/V/N [42, 44] (Fig. 2, Additional file 4: Figure S1). Of those, lineages II  and P belong to different Trichomonas species (T. tenax and Trichomonas sp., which grouped within T. canistomae ). However, species identification, based on morphological methods, may need to be revised and complemented as more genetic data are available. Since several Trichomonas species have been reported in birds based solely on morphology [12, 33, 43, 51,], the nomenclature for Trichomonas might also need revision as more molecular-based phylogenetic analyses are available. However, judging by the overall occurrence of these three lineages, our findings suggest a widespread distribution of those lineages across columbids [12, 33]. For instance, lineages II  and C/V/N [42, 44] were also predominant in species from the UK, Austria and the USA [12, 55]. Lineage II  even infected the same host species as shown in the UK  (Table 2).
Lineage C/V/N [42, 44] has also been described as “genotype A”, an apparently non-pathogenic Trichomonas lineage  with a global and frequent occurrence. It was previously found in turtle doves and wood pigeons , and we here additionally demonstrate infection in stock doves.
Regarding existing literature and previously described lineages, lineages O, P and Q might be newly detected lineages, because they were not described in previous studies [12, 22, 33, 42, 44, 57]. The samples from lineage P sequenced for the Fe-hydrogenase gene also clustered in a distinct and apparently new group of Fe-hydrogenase sub-lineage P1 (S4). Furthermore, lineages O and Q appear distinct to lineages A/B and C/V/N [42, 44], thus they may not be as common or widespread as lineages II, P and C/V/N [12, 42, 44], because they were only found in one German stock dove sample (O) and one Italian turtle dove sample (Q). Additionally, lineage III , found in stock doves and turtle doves, was identical to assigned reference sequences isolated from feral pigeons and was only found in Austria and the UK [42, 58]. Thus, we confirm its presence in turtle doves from Malta, Italy and Spain and in stock doves from Germany.
Only, lineage A/B  grouped with a potentially fatal lineage (genotype B) [12, 16, 19, 22], which was also responsible for the finch trichomonosis epizootic in the UK  (Table 2). This lineage, which is often lethal, has also been detected in turtle doves, wood pigeons and other non-passerines [12, 16, 22, 27] and we here confirm its presence in stock doves from Germany and resident collared doves from Malta. A fatal case of trichomonosis in a stock dove from Germany has been described previously  based on necrotic lesions and Trichomonas presence in microscopic analyses . The lethal course of the disease hints to an infection by lineage A/B , although it was not genetically confirmed.
The lethal character of trichomonosis was described also in the Mauritian pink pigeon, a resident species on Mauritius similar to the collared doves on Comino (Malta) . Thus, at least on Comino, the collared dove population might decrease in the future due to this potentially pathogenic lineage. Some years ago, the collared dove population already crashed due to an outbreak of a disease, which affected the region around the beak (B. Metzger, personal communication). No further description of the illness is known, but it is possible that it was trichomonosis.
Two genotypes of T. gallinae have previously been proposed to exhibit a differential in pathogenicity. Lineage C/V/N [42, 44] is synonymous with genotype A, described by Sansano-Maestre  as a wide-spread Trichomonas lineage, with mild or no pathogenicity. Lineage A/B  is synonymous with genotype B  described as possessing a more severe pathogenicity. Regarding all other lineages, pathogenicity has not been assessed. For this purpose, transmission experiments would be very helpful to see, if birds show signs of active trichomonosis and if they recover from infection and acquire possible immunity [25, 26].
Phylogenetic analysis of Trichomonas lineages from stock dove siblings
To our knowledge, this is the first report of phylogenetic examination of Trichomonas from stock dove nestlings belonging to the same nest. Both findings (siblings), were infected by the same lineage as well as by different lineages; this can be explained by disease transmission via crop milk feeding, as both parents share chick feeding . Thus, if both parents are infected by the same Trichomonas lineage, chicks receive the same Trichomonas pathogen. If both parents are infected by Trichomonas but carry different lineages, their chicks might be infected by different pathogens as well. Additionally, studies have shown [14, 59, 60], that individual birds may carry more than one Trichomonas lineage, so it is possible we only sequenced one strain when more than one was present. Here, we found a T. gallinae and T. canistomae-like lineage in two siblings of a stock dove nest. A coinfection of an apparently non-pathogenic T. gallinae (genotype A)  lineage plus a T. tenax-like strain was found previously in pigeons .