Trichomonas gallinae (Parabasalia: Trichomonadida) is a widespread flagellated protozoan parasite of birds from various orders, including Columbiformes, Accipitriformes, Strigiformes, Psittaciformes, Falconiformes and Passeriformes (1–3). Among columbiform birds, the rock pigeon (Columba livia) is the main reservoir of this parasite. The most important route of trophozoite transmission between birds is oral by saliva, through shared water and food sources (2, 4, 5). In columbids the predominant way of spreading to nestlings is via crop milk (1). Additionally, predatory birds can become infected by consuming a carrier prey item, since trichomonas trophozoites may survive in carcasses for at least 48 h (2, 4, 5). Trichomonas gallinae can persist for up to 1 h in various water sources, e.g., in gutters and drinkers (6), but higher temperatures (30–35°C) can further prolong its survival (7). Although it is able to form pseudocyst in unfavorable conditions, the moist environment is essential to maintain its infectivity (2).

Trichomonas gallinae is often considered a normal inhabitant (commensal) of the mucosal surface in the upper gastrointestinal tract (2). However, by eliciting inflammation in the underlying tissues or when entering more distally the digestive tract of birds, this protozoan parasite might cause mild to severe lesions depending on strain virulence and host susceptibility. Infection with highly pathogenic strains may lead to death. However, columbiform birds may also be asymptomatic carriers of T. gallinae, ensuring the carefree spread of this protozoon. In addition, if protective immunity develops, affected birds become resistant to a new infection (2, 4, 8, 9).

Regarding pathogenesis, T. gallinae trophozoites establish preferentially in the upper gastrointestinal tract (the oropharynx, esophagus and crop), where they can cause yellowish necrotic lesions. In severe cases trichomonosis can lead to starvation and suffocation. Furthermore, T. gallinae can spread to tissues of the cranium, thorax, and abdomen, as well as of the liver and air sacs causing similar lesions, with deepening tissue involvement, referred to as canker (1). Recently, massive death of passeriform birds (the greenfinch, Chloris chloris, and the goldfinch, Carduelis carduelis) due to infection with a specific lineage of T. gallinae has been reported throughout Europe (3).

Avian trichomonosis has been reported to have a worldwide occurrence (2). In Europe, as on other continents, columbiform birds play the most significant role in the maintenance of T. gallinae (2). The prevalence in western and southern Europe is high among wild columbids (74%) (9), unlike in northern central Europe where only a little more than one third of racing pigeons proved to be PCR positive (10).

The main objective of this study was to investigate the prevalence, genetic diversity and phylogenetic relationships of avian Trichomonas species in Hungary and Romania, where no similar data are available. Sampled hosts included racing and urban feral pigeons as well as other highly urbanized or pet columbiform bird species (Eurasian collared doves: Streptopelia decaocto, common wood pigeons: C. palumbus and ring doves: S. risoria, respectively). The nomenclature used in the context of feral pigeons complies with genetic and ornithological studies on this species (11, 12).

According to the preliminary comparison on the efficacy of molecular detection of T. gallinae, 19 out of 20 swab samples but only 18 out of 20 culture medium samples were PCR positive (i.e., one pigeon was positive only by its culture medium sample vs. two pigeons diagnosed as infected only from their swab samples) (Supplementary Table 1: 7th column, samples TG1-50). Therefore, swab samples were used in the remaining part of the study.

Trichomonas gallinae was detected in all four studied bird species, with an overall prevalence of 73% (72 out of 99) (Table 1). Based on the screening assay, racing feral pigeons had a significantly (P < 0.0001) higher prevalence of T. gallinae infection (95%: 40 of 42) than urban feral pigeons (34%: 11 of 32). Among other columbiform bird species, the rate of PCR-positivity was the highest, 100% among wood pigeons (4 out of 4), followed in decreasing order by ring doves (94%: 15 out of 16) and collared doves (33%: 1 out of 3) (Table 1; Supplementary Table 1). These results also imply that the association of T. gallinae infection with artificially bred, captive columbiform birds (racing feral pigeons and ring doves) was highly significant (P < 0.0001) in comparison with free-living columbiform birds (urban and rural feral pigeons, wood pigeons and collared doves) (55/58 vs. 17/41, respectively).

Based on the long fragment of the 18S rRNA gene, six Trichomonas subtypes were detected in 37 columbiform birds (Table 1). Compared to a reference sequence from North America (GenBank: EU215373), these had four to eight nucleotide differences (Table 2), i.e., 99.5–99.7% (1,471–1,475/1,479 bp) identity. In a smaller geographical context, the 18S rRNA gene sequences obtained in this study from central and southeastern Europe had higher, up to 11 nucleotide differences with conspecific sequences from western and southwestern Europe: only 99.3–99.7% (1,468–1,474/1,479 bp) sequence identity with T. gallinae from passeriform birds sampled in 2019 in France (e.g., MK172846), whereas 99.5–100% (1,443/1,450–1,449/1,449 bp) sequence identity with samples collected from columbiform birds in 2019 in Portugal (e.g., MK932772).

Only two genetic variants were detected among sympatric urban feral pigeons (subtypes B, D in Hungary; A, D in Romania), while four subtypes (B, C, D and the most divergent Hu-TG37) occurred in racing feral pigeons kept in one trading-breeding place (Table 1). Among racing feral pigeons, 18S rRNA gene subtype D was only found in birds that originated from Germany, while in Hungary subtypes A and E were exclusively found in wood pigeons and ring doves, respectively (Table 1). In the latter species, the occurrence of 18S rRNA gene subtypes was related to the origin (breeding place) of captive birds: subtype E was detected in two ring doves of one sampling locality, whereas subtype B in four birds at the other sampling locality (Supplementary Table 1). Clinical signs (lesions in the oropharyngeal cavity or the eyes, conjunctiva) were only associated with subtype D (Supplementary Table 1; Supplementary Figure 1).

Amplification and sequencing of part of the alpha-tubulin gene were successful from at least one sample representing each 18S subtype. Alpha-tubulin gene sequences obtained in this study had a lower (98.5–98.6%: 1,008–1,009/1,023 bp) or a higher level (99.6–99.7%: 1,019–1,020/1,023 bp) of sequence identity to that of an isolate (EU215382) used as a reference. Based on the corresponding amino acid sequences, most mutations in this protein encoding gene were synonymous, but subtypes E and D had a single amino acid difference compared to the reference isolate (valine instead of alanine at position 149, and isoleucine instead of valine at position 34, respectively).

Considering the results of phylogenetic analyses, the topology of the 18S rRNA phylogenetic tree (Figure 1) did not show clear clustering of T. gallinae subtypes obtained in this study according to host species, living place (or keeping modes) of columbiform birds. However, subtype A (identified both in Hungary and Romania) formed a sister group to all other 18S rRNA gene subtypes (B, C, D, E): although with only moderate (65%) support (Figure 1) but confirmed by the alpha-tubulin phylogenetic tree (Figure 2). More importantly, Trichomonas sp. Hu-TG37 (detected in a racing feral pigeon from south Hungary) belonged to the phylogenetic group of T. canistomae and T. tenax with moderate (61%) support, implying, that this is a separate species (in other words: if this isolate would belong to T. gallinae, this species would not be monophyletic). The separation of Trichomonas sp. Hu-TG37 from T. gallinae was also confirmed by the alpha-tubulin phylogenetic tree (Figure 2).

To our knowledge, this is the first study on the genetic diversity of T. gallinae in Hungary, Romania and the whole southeastern European region, complementing previous reports from western, central and southern Europe (see below). Most infected birds in this study did not show clinical signs of trichomonosis except five pigeons (6.9%). A lower prevalence of clinical trichomonosis (0.37%) has been reported in a study involving 612 wild and domestic pigeons (13). Since T. gallinae can cause the death of infected hosts (15), the rarity of symptoms can in part be explained by the death of severely affected birds, which can die before they are examined (13). Furthermore, from an epidemiological point of view, subclinical cases might ensure easier spread of these protozoa. Therefore, screening pigeons for Trichomonas spp. should become an integral part of veterinary practice.

In this study, T. gallinae was detected in all four columbiform bird species examined. The infection rate was 73% which is similar to the 74% overall prevalence reported in wild columbids from western and southern Europe (9). Within the Mediterranean region, in the Iberian Peninsula 44.8% of wild and domestic pigeons were shown to carry T. gallinae (13), but in another study from Spain the prevalence was much higher (79.4%) (16). Furthermore, in the UK other columbiform species including C. palumbus and S. decaocto were also examined and a 60% incidence was reported (17). In Germany, four species (C. livia, C. oenas, C. palumbus, and S. decaocto) were screened and 50% of the birds were infected with T. gallinae (18). The prevalence was 37% among racing pigeons in Poland (10).

In Hungary, based on our screening assay, racing feral pigeons had a significantly higher prevalence of T. gallinae infection than urban feral pigeons (95 vs. 33%). The possible reason for this difference might be that at trading-breeding places birds of different origin are housed close to each other, and the chances for infection are higher if naive racing pigeons can get into contact with carrier birds not only outside, but inside such enclosures, e.g., by sharing food and drinking water or by kissing. Therefore, we propose that similar places play a crucial role as hotspots in the transmission of T. gallinae. To our knowledge, there is no similar study in Europe that compares T. gallinae according to the place where pigeons are kept.

All four examined wood pigeons were infected. Although the sample size was limited, this apparently high (100%) infection rate is similar to what was reported among wood pigeons in Germany (70%) (9) and the Iberian Peninsula (83.3%) (19). This high prevalence is likely associated with urbanization of wood pigeons in Hungary, resulting in close contact of birds (e.g., via sharing common drinking sources) in green areas where the number of wood pigeons has recently increased significantly (20).

Concerning other studied bird species, the prevalence was also high (94%) among ring doves in Hungary. There is a lack of literature data on trichomonosis of both collared dove and ring dove in Europe, despite the fact that ring doves were found to be susceptible to T. gallinae during experimental infection (21). In the Caribbean an outbreak was reported (22), drawing the attention of veterinarians to the necessity to monitor T. gallinae in this bird species.

Based on the long fragment of the 18S rRNA gene, six Trichomonas subtypes were detected in columbiform birds in Hungary and Romania. Compared to a reference sequence, they had up to eight nucleotide differences, meaning that the maximum genetic difference was low (0.5%) compared to what was reported from North America (3.4%) (8) and even from Austria, a country neighboring Hungary (2.9%) (23). In Hungary, two genetic variants were detected among urban feral pigeons, vs. four 18S rRNA gene subtypes occurred in racing feral pigeons kept in the same trading-breeding place, highlighting the epidemiological importance of similar facilities in general.

It is noteworthy that each bird from which T. gallinae was sequenced, carried a single 18S rRNA gene subtype, as also demonstrated repeatedly with different modes of detection (Supplementary Table 1, 8th column: samples TG1-50). Since it was reported that T. gallinae triggers premunition (24) and immunity lasts until the loss of infection (1), the a priori presence of any variant probably protected the relevant birds during a heterologous challenge, which is likely to occur in the environment of a pigeon trading-breeding place. It is also highly relevant to note that among racing pigeons, 18S rRNA gene subtype D was only found in pigeons that originated from Germany. We suspect that subtype D was already present in these birds when imported to Hungary and probably premunition protected them from becoming infected in the pigeon trading place by other variants.

The clinical signs relevant to trichomonosis were only associated with 18S rRNA gene subtype D in Hungary. There are several reports indicating that certain haplotypes are highly correlated with more severe lesions in various bird species (13, 16, 25). However, no close correlation has been found in terms of pathogenicity and geographical distribution when columbids were examined across Europe (9).

Considering the results in a phylogenetic context, the topology of the 18S rRNA phylogenetic tree did not show clear clustering of T. gallinae subtypes from this study according to host species, living place or keeping mode of columbiform birds. Clustering of Trichomonas sp. Hu-TG37 to the phylogenetic group of T. canistomae and T. tenax suggests that it might represent a new species. This separate position of Trichomonas sp. Hu-TG37 was confirmed also by the alpha-tubulin phylogenetic tree. It is known that some Trichomonas spp. show high genetic diversity depending on bird species, and few of these infections are caused by variants/species closely related to T. vaginalis, T. tenax (8, 23) or T. canistomae (16). Trichomonas sp. Hu-TG37 identified for the first time in the present study belongs to the phylogenetic group of Trichomonas spp. infecting (among the others) domestic carnivores. This calls for further epidemiological studies on the possible contact between dogs and pigeons (e.g., via water deposited in gardens in drinking bowls) and its role in the transmission of these protozoan parasites.

In conclusion, this is the first report in Hungary and Romania on the prevalence and 18S rRNA gene subtypes of T. gallinae in various columbiform birds using molecular methods. The results suggest that most of these variants are not host-specific and do not cause clinical signs. The highest degree of genetic diversity and high prevalence of infection was observed among racing pigeons and captive ring doves, thereby highlighting the epidemiological importance of pigeon/dove trading-breeding places.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the animal study because all columbiform birds in this study were handled and sampled during regular veterinary care, therefore no ethical permission was needed. Written informed consent was obtained from the owners for the participation of their animals in this study.

BT-S: study design, DNA extraction, data analysis, and manuscript writing. GS, AS, ÁP, and KB: sample collection. NT: PCR tests and sequencing. JK: phylogenetic analyses. ÁK and ÁJ: contribution to sample collection. JV: supervision and contribution to molecular analyses. SH: conceptualization, study design, primer design, GenBank processing, and manuscript writing. All authors contributed to the article and approved the submitted version.