Genetic characterization of oropharyngeal trichomonad isolates from wild birds
indicates that genotype is associated to host species, diet and virulence.
1
2
3
Martínez-Herrero, M.C. , Sansano-Maestre, J. ,López Márquez, I. ,
6
1
7
González, J. , Garijo-Toledo, M.M. , Gómez-Muñoz, M.T.
Obón, E.
4
5
, Ponce, C. ,
1
Universidad CEU Cardenal Herrera. Instituto de Ciencias Biomédicas. Facultad de Veterinaria. Departamento
de Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos. Despacho
D-10. Calle Tirant Lo Blanch, 7. CP: 46115. Alfara del Patriarca. Valencia, Spain.
2
Universidad Católica de Valencia. Facultad de Veterinaria y Ciencias Experimentales. Calle Guillem de Castro,
94. CP: 46003. Valencia, Spain.
3
GREFA (Grupo de Rehabilitación de la Fauna Autóctona y su Hábitat). Monte del Pilar s/n. Majadahonda. CP:
28220. Madrid, Spain.
4
Centre de Fauna Salvatge de Torreferrussa, Catalan Wildlife-Service-Forestal Catalana, CP: 08130, Santa
Perpètua de la Mogoda. Barcelona, Spain.
5
Museo de Ciencias Naturales (CSIC). Departamento de Ecología Evolutiva. Calle José Gutiérrez Abascal, 2.
CP: 28006. Madrid, Spain.
6
Centro de Recuperación de Fauna Silvestre de La Alfranca. Finca de La Alfranca s/n. CP: 50195. Pastriz,
Zaragoza. Spain.
7
Universidad Complutense de Madrid. Facultad de Veterinaria. Departamento de Sanidad Animal. Avenida
Puerta de Hierro s/n. CP: 28040. Madrid, Spain.
ABSTRACT
Genetic variation of oropharyngeal trichomonad isolates of wild birds of Spain has been studied. A
total of 1688 samples (1214 of predator birds and 474 of prey species) from Wildlife Recovery
Centers and scientific bird ringing campaigns were analyzed from 2011 to 2013. TYM medium or
InPouch
TM
pockets were used for the growth of the parasite. Overall 20.3% of the cultures were
positive, 11.4% for raptors and 43.3% for prey species. All finches sampled in the study were
negative. Gross lesions were present in 26% of the infected birds, 57.3% of infected predator birds
and 4.9% of infected prey species. The most commonly parasitized species were the goshawk
(Accipiter gentilis) for predator birds and the rock pigeon (Columba livia) for prey species, with 74.5%
and 79.4% of positive samples, respectively. Sequence analysis of the ITS1/5.8S/ITS2 region
revealed the presence of five different genotypes (ITS-OBT-Tg-1, ITS-OBT-Tg-2, ITS-OBT-Tcl-1, ITSOBT-Tcl-2, ITS-OBT-Tvl-5), two of them are new reports (ITS-OBT-Tcl-1, ITS-OBT-Tcl-2). New host
species, not previously analyzed by PCR and sequencing, are also reported: 2 columbids (Columba
palumbus and Streptopelia turtur); 1 corvid (Pica pica) and 13 raptors (Accipiter gentilis, Accipiter
nisus, Asio otus, Bubo bubo, Buteo buteo, Circus aeruginosus, Circus cyaneus, Falco naumanni,
Falco peregrinus, Neophron percnopterus, Otus scops, Pernis apivorus and Strix aluco). A
relationship between genotype and host species was observed (ITS-OBT-Tg-1-raptors, ITS-OBT-Tg2-columbids, ITS-OBT-Tcl-1-turtle doves, ITS-OBT-Tcl-2-goshawks and ITS-OBT-Tvl-5-Egyptian
vulture), but only two genotypes (ITS-OBT-Tg-1; ITS-OBT-Tg-2) were widely distributed among
different bird species. Genotype ITS-OBT-Tg-1 was most frequently found in predator birds and
statistically associated with birds displaying gross lesions although some mixed infections were also
found in symptomatic birds. The diet of the raptor is another factor that determines their susceptibility
to develop the disease, being non-strict ornithophagous species at higher risk than ornithophagous
ones. Genotypes ITS-OBT-Tcl-1, ITS-OBT-Tcl-2 and ITS-OBT-Tvl-5 are reported for the first time in
1
Spain and showed higher genetic homology to other species of the genus Trichomonas (T.
canistomae and T. vaginalis) than to T. gallinae isolates, indicating the possibility of new species
within this genus.
KEYWORDS (3-6) Columbids, gross lesions, genotype, protozoan, raptors, Trichomonas.
INTRODUCTION
Avian trichomonosis is a parasitic disease caused by the protozoan Trichomonas gallinae (Rivolta,
1878). This flagelated infects the upper digestive tract of birds, mainly the oropharynx, crop and
oesophagus where it is attached to the mucosa and multiplies by binary fission.
The parasite induces erosions, ulcers and caseous necrotic granulomas in the epithelium of the
digestive tract. Clinical signs of infection can vary from asymptomatic to dysphagia, regurgitation,
sialorrhea/ptyalism and also death by starvation, because animals become unable to feed. These
differences could be explained by the immune status of the host and the virulence of the strains. High
virulent ones (Jone’s Barn and Eiberg) can be lethal in 15 days or less due to the necrotic foci
induced on internal organs such as the liver, while mild and low virulent strains can promote an
adequate immunological response (Narcisi et al., 1991).
The reservoir hosts for T. gallinae are columbid species. The transmission is maintained in the wild by
predation and necrophagy from raptors, by sharing contaminated food or water sources between
columbids as well as columbids and not ornithophagous birds (finches, i.e., Lawson et al., 2011a, b).
The most frequent presentation form is an endemic process with high morbidity but low mortality rates
on adult columbids. For example, 27.5% of adult Seychelles columbids were infected, but no
evidence of clinical signs of the disease were found (Bunbury, 2011). In Spain a low percentage of
macroscopical lesions has also been reported (only one bird of 612) despite the high prevalence
found (44.8%) (Sansano-Maestre et al., 2009). Nevertheless, occasional outbreaks of the disease
causing high mortality have been reported in columbid species, (Greiner and Baxter, 1974; Höfle et
al., 2004; Villanúa et al., 2006; Stimmelmayr et al., 2012). In fact, trichomonosis has been detected as
one of the concerns for the endangered pink pigeon (Columba mayeri) in Mauritius and Bonelli’s
eagle (Aquila fasciata) in Spain and Portugal (Höfle et al., 2000; Real et al., 2000; Swinnerton et al.,
2005; Bunbury et al., 2007). Furthermore, it has been recently described as an emerging disease for
2
passerines, as it has been reported in epidemic focuses involving finch species in the United Kingdom
in 2006 and in Canada in 2008 (Forzán et al., 2010; Robinson et al., 2010; Lawson et al., 2011a).
Besides, migratory routes followed by finches have spread the disease to northern European
countries such as Germany, Sweden, Norway and Finland in 2008 and 2009 (Neimanis et al., 2010;
Lawson et al., 2011b). Fatal cases have also been detected in France in 2010 and in Slovenia and
Austria during 2012 and 2013 (Gourlay et al., 2011; Zadravec et al., 2012; Ganas et al., 2013).
Different studies have analyzed the prevalence of the disease among wild birds through bird ringing
campaigns or at admissions of Wildlife Recovery Centers. Prevalence values vary depending on the
species and geographic location, but high levels of infection (86%) have been found in columbids
such as the Eurasian collared dove (Streptopelia decaocto) and the European turtle dove
(Streptopelia turtur) in England and the rock pigeon (Columba livia) (44.8%) in Spain (SansanoMaestre et al., 2009; Lennon et al., 2013). High prevalence in raptors has been reported with values
of 85% in ornithophagous species like the Cooper’s hawk (Accipiter cooperii) and 65.1% in goshawks
(Accipiter gentilis) (Krone et al., 2005). This parasite constitutes an important cause of infectiousmortality for raptor nestlings, especially in urban areas were columbids are their main prey (Cooper
and Petty, 1988; Rutz et al., 2006, Molina-López et al., 2011; Chi et al., 2013).
The most frequently used region to study intra-specific variation within the Trichomonadidae Family is
the ITS1/5.8S/ITS2, composed by the 5.8S large subunit of the ribosomal RNA and the adjacent
introns ITS1 and ITS2. This region is widely used for phylogenetic purposes and it is recognized as a
strong molecular tool to determine inter- and intra-specific diversity (Felleisen, 1997; Kleina et al.,
2004; Gaspar da Silva et al., 2007; Anderson et al., 2009; Sansano-Maestre et al., 2009;
Grabensteiner et al., 2010; Stimmelmayr et al., 2012; Chi et al., 2013; Ganas et al., 2013).
A recent study characterized isolates of T. gallinae and described 12 different genotypes of the
ITS1/5.8S/ITS2 region across the United States (Gerhold et al., 2008). In Europe, other studies have
revealed the existence of six variants for this region (Grabensteiner et al., 2010). In Spain two
genotypes were predominant on wild birds, while in England three genotypes have been found so far
(Sansano-Maestre et al.., 2009; Chi et al., 2013).
3
Different studies have reflected little variation among the parasite strains recovered from the same or
similar bird populations, like the columbid hosts pink pigeons (C. mayeri) and Madagascar turtledoves (Streptopelia picturata) in Mauritius (Gaspar da Silva et al., 2007) or the finch outbreaks in the
United Kingdom, Austria and Slovenia (Lawson et al., 2011a and b; Ganas et al., 2013). In California
(USA), it was also found that columbids, raptors, corvids and finches shared a genetically identical
parasite while a different trichomonad species was found in mockingbirds (Mimus polyglottos) with
avian trichomonosis-like lesions (Anderson et al., 2009).
One of the latest epidemic reports in columbids, with 15-20% mortality rate, was produced by two
different pathogenic isolates at the Caribbean, one of them described as Trichomonas-like
parabasalid (Stimmelmayr et al., 2012). Ecco et al. (2012) found five different pathogenic
trichomonads on raptors and passerines in Brazil, belonging to genotypes of T. gallinae, Trichomonas
vaginalis-like and similar to the newly described genus Simplicimonas similis.
Also, the existence of strains with higher similarity to T. vaginalis and T. tenax in Europe, Brazil and
USA has been reported, indicating the possibility of different species of trichomonads in the avian
population (Gerhold et al., 2008; Grabensteiner et al., 2010; Ecco et al., 2012). Recently, Girard et al.
(2014) identified a new species (Trichomonas stableri) of the parasite involved in mortality events of
band-tailed pigeons (Patagioenas fasciata monilis) in USA. This latest discovery reflects the large
diversity of oral trichomonads present on bird species and points out the need for further
investigations on this area.
The genetic variability of the parasite and the correlation with the clinical presentation has been poorly
studied. Previous studies carried out in Spain and United Kingdom employing the sequencing of the
ITS1/5.8S/ITS2 region have shown a relationship between the genetic variant and the presence of
gross lesions (Sansano-Maestre et al., 2009; Lawson et al., 2011a; Chi et al., 2013). In fact, only one
clonal strain was detected in the English outbreaks were it was estimated that 500,000 birds died
directly from trichomonosis (Robinson et al., 2010; Lawson et al., 2011a).
The objectives of our research are to provide information about the occurrence of trichomonads
among wild bird species and to study the genetic variability of oropharyngeal trichomonads obtained
from the animals at scientific ringing campaigns, clinical cases and admissions from some of the most
4
important Wildlife Recovery Centers in Spain. The relationship between genotype, host species, diet
and clinical presentation has been also analyzed.
MATERIAL AND METHODS
Sampling and Geographical location
Before sampling, a letter asking for collaboration was sent to some of the most important Wildlife
Recovery Centers of Spain (those having a large number of admissions) and bird ringing scientists of
nearby areas (Valencian Community, Murcia and Madrid). Those Centers/scientists that answered in
a positive manner were included at this study. Geographical location of the centers is shown at Fig. 1.
A total of 1688 samples were analyzed.
Wildlife Recovery Centers
Wildlife Recovery Centers located at the provinces of Alicante, Barcelona, Murcia, Toledo and
Zaragoza submitted samples from the avian patients that showed tricomonosis-like lesions (Fig. 2).
Samples were taken with a sterile cotton swab, previously moistened with physiologic saline solution
that was gently rubbed over the oropharyngeal cavity and crop. The diagnosis was done by culture
using InPouch
TM
TF Feline pockets (BioMed Diagnostics Inc, White City, Oregon, USA) (Bunbury et
al., 2007; Anderson et al., 2009; Bunbury, 2011; Girard et al., 2014). InPouch
TM
pockets were
inoculated with the oral swab and incubated at 37ºC during 48 hours prior to the submission of the
samples to the laboratories of the Veterinary Faculties of University CEU Cardenal Herrera (Moncada,
Valencia) or University Complutense of Madrid (Madrid), Spain.
A systematic review of all admissions was carried out at the Wildlife Recovery Centers of GREFA
(Grupo de Rehabilitación de la Fauna Autóctona y su Habitat) in Madrid, “La Granja de El Saler” in
Valencia and the breeding center for lesser kestrel (Falco naumanni) of DEMA (Defensa y Estudio del
Medio Ambiente) in Badajoz. TYM medium (Trypticase-Maltose-Yeast Extract) was used for the
screening of oral trichomonads (Diamond, 1957).
Scientific ringing campaigns
5
Adults and juvenile birds of both, passerine granivorous species and raptors, were tested by culture
using TYM medium. Samples were submitted to the above mentioned laboratories during the
following 24 hours after collection. Scientific ringing campaigns were scheduled by local ornithological
organizations (ANSE-Asociación de Naturalistas del Sureste, SEO/Birdlife-Sociedad Española de
Ornitología, Pit-Roig, Albufera and Monticola ringing groups from the CMA-SEO/Birdlife and SVOSociedad Valenciana de Ornitología) at the provinces of Alicante, Madrid, Murcia and Valencia.
Special authorizations were obtained for capture and ringing of raptor nestlings (goshawk and
Eurasian eagle-owl, Bubo bubo) that were included in different research projects. Due to the recent
outbreaks and spread of the infection among Passeriformes in northern Europe (Neimanis et al.,
2010; Lawson et al., 2011b), some of these species were also studied: greenfinch (Carduelis chloris),
chaffinch (Fringilla coelebs) and Eurasian blue tit (Cyanistes caeruleus).
Parasite culture
Oropharyngeal swab samples were inoculated in the correspondent medium (TYM/In pouch),
incubated at 37ºC and examined daily during 15 days using an inverted microscope to check the
presence of motile trophozoites. Subsequent passages in TYM medium were done to perform
axenization of the parasite. The composition of the medium was as follows: 20 g of trypticase (SigmaAldrich, St. Louis, Missouri, USA), 10 g of D(+)-maltose (Sigma-Aldrich, St. Louis, Missouri, USA), 10
g of yeast extract (Sigma-Aldrich, St. Louis, Missouri, USA), 1 g of L-cysteine (Sigma-Aldrich, St.
Louis, Missouri, USA), 0.1 g of ascorbic acid (Sigma-Aldrich, St. Louis, Missouri, USA) and 10% of
inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, Missouri, USA) per liter. After mixing all the
ingredients, the pH was adjusted to 6 and sterilized by filtration through a filter of 0.22 µm
(Millipore, Billerica, Massachusetts, USA). Antibiotics and antimycotic supplements were used to
avoid contamination: 24 mL of nystatin/l (10,000 IU/mL; Sigma-Aldrich, St. Louis, Missouri, USA),
ticarcillin, vancomycin and ceftiofur, 36 mg/L each
(Sigma-Aldrich, St. Louis, Missouri, USA) were
added. Storage at -80ºC using 5% of DMSO (dimethyl sulphoxide, Sigma-Aldrich, St. Louis, Missouri,
USA) in TYM medium was carried out to preserve the isolates for further analysis.
DNA extraction
6
Genomic DNA was extracted from parasite cultures using a commercial DNA extraction kit (DNeasy
Blood and Tissue Extraction Kit; QIAGEN, Valencia, California, USA). Parasites were recovered from
1 mL of culture (ranging from 40,000 to 1,200,000 trophozoites) and centrifuged at 350 x g for 3 min.
The supernatant was discarded and the pellet was washed twice in phosphate buffered saline pH 7.4.
The pellet was processed following the manufacturer’s instructions. DNA was stored at -20ºC until
use.
PCR of the fragment ITS1/5.8S/ITS2
PCR was done using primers TFR1 (5’-TGCTTCAGCTCAGCGGGTCTTCC-3’) and TFR2 (5’CGGTAGGTGAACCTGCCGTTGG-3’, Felleisen, 1997). The reaction was done in a final volume of 50
µL, containing 5 µL of 10x buffer, 1.5 mM MgCl2, 2 mM dNTP, 2 µM of each primer, 2.5 IU Taq
polymerase (MP, Thomas Scientific, Swedesboro, New Jersey, USA) and 5 µL of genomic DNA. The
PCR protocol started with an initial step to activate the enzyme at 95ºC for 9 minutes, followed by 40
cycles of denaturation at 94ºC for 30 seconds, annealing at 66ºC for 30 seconds, extension at 72ºC
for 30 seconds and a final extension step at 72ºC for 15 minutes. Electrophoresis was done in a 1.5%
agarose gel stained with ethidium bromide (0.5 µL/mL) at 80V for 35 minutes. Ten µL of each sample
was loaded and gels were observed under ultraviolet light.
Sequence analysis and phylogenetic trees
PCR amplification products were purified with a commercial kit (MinElute PCR Purification Kit;
QIAGEN, Valencia, California, USA) and submitted for sequencing to the laboratories Sistemas
Genómicos, S. A. (Paterna, Valencia, Spain). The reaction was completed in an automatic sequencer
3730XL DNA Analyzer (Applied Biosystems, Foster City, California, USA) with the ABI PRISM®
BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA).
Chromatograms were manually checked in both directions and assembled using Lasergene SeqMan
software version 7.0.0 (DNASTAR, Madison, Wisconsin, USA). Nucleotide BLAST (Basic Local
Alignment Search Tool; URL: http://blast.ncbi.nlm.nih.gov/Blast.cgi/) version 2.2.29 was used to
compare
the
identity
of
the
sequences
at
GenBank
database
(URL:
http://www.ncbi.nlm.nih.gov/genbank/). Parameters were set for Megablast algorithm (optimization for
highly similar sequences) and low complexity region filter.
7
A total of 84 isolates were analyzed by PCR and sequencing of the ITS1/5.8S/ITS2 region (Tables 1
and 2). New sequences or sequences found in host not previously described were submitted to
GenBank database (with accession numbers from KF993680 to KF993707 and KJ776739 to
KJ776743. Supplementary table 1). We included two samples from goshawks (isolates R30/07 and
R31/07) obtained in 2007, since the phylogenetic analysis revealed that they constituted a newly
distinct genotype. Table 3 shows the genotypes previously described by other authors and the new
genotypes found in this study. We had used a codification system based on BLAST sequence
homology results to clarify the genetic group considering the genotypes found in the literature. The
code (ITS-OBT-Tx-n) indicates region of genome (ITS: ITS1/5.8S/ITS2), source (OBT: Oropharyngeal
Bird Trichomonad), organism homology group (Simplicimonas spp.; Tcl: T. canistomae-like; Tg: T.
gallinae; Ts: T. stableri; Trichomonadida spp.; Tt: T. tenax; Tvl: T. vaginalis-like) and subgroup
number (n: 1, 2, etc, a new number was employed when genetic homology was less than 99%).
Sequences less than 200 bp have not been included in the table.
To construct the phylogenetic trees, sequences were selected by its different genotype and the
presence of gross lesions. A total of seven isolates were used, with accession numbers: KF99369192, KF993700-01 and KF993705-07 (Supplementary table 1). Sequences of ITS1/5.8S/ITS2 region
published
by
other
authors
available
at
GenBank
database
(URL:
http://www.ncbi.nlm.nih.gov/genbank/) and with a minimum length of 290 nucleotides were included
(Felleisen, 1997, Kutisova et al., 2005, Gaspar da Silva et al., 2007, Gerhold et al., 2008, Anderson et
al., 2009, Sansano-Maestre et al., 2009, Grabesteiner et al., 2010 and Robinson et al., 2010, Chi et
al., 2013, Girard et al., 2014) (Supplementary table 2). Also, a Pentatrichomonas hominis sequence
was added as an outgroup reference (Cepicka et al., 2005). The analysis involved 26 nucleotide
sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All the positions containing gaps
and missing data were eliminated. There were a total of 256 positions in the final dataset. Multiple
alignment of the sequences was done employing Clustal W algorithm of MEGA software version 5.05
(Tamura et al., 2011). The average Jukes-Cantor distance was 0.094. Evolutionary distances were
computed with Maximum Composite Likelihood method for Neighbour-Joining tree and Tamura-3
parameter model for Maximum Likelihood tree (Fig. 3 and 4). Boostrap of 2000 replicates was
employed to estimate trees reliability.
8
Statistical analysis
Analysis of risk factors was done using categorical data of host bird species (predator bird/prey),
presence of gross lesions compatible with trichomonosis (other causes of lesions were not
investigated), genotype (ITS-OBT-Tg-1/ITS-OBT-Tg-2) and diet in predator birds (ornitophagous/nonstrict ornitophagous). The presence of lesions was not determined in two animals; therefore, two
samples were excluded from the statistic analysis. WinEpiscope software version 2.0 (URL:
http://www.clive.ed.ac.uk/winepiscope/) was used for the estimation of Odds Ratio (OR) tests and
95% Confidence Interval (CI). The presence of interaction and confounding among variables were
explored by stratification of the samples and estimation of OR in each stratum and estimation of
Mantel-Haenszel test (ORMH). Birds infected by more than one genotype (n=6) and birds with nonfrequent genotypes (genotypes ITS-OBT-Tcl-1, ITS-OBT-Tcl-2, ITS-OBT-Tvl-5, n=5) were not
employed in the stratification.
RESULTS
OCURRENCE OF Trichomonas sp.
All the samples were collected from 2011 to 2013, except two samples from goshawks obtained in
2007 that were included in the analysis because they showed a distinct genotype. We processed
cultures from 1688 birds from 53 different species. Table 1 shows detailed information, including
number of samples, percentage of positive cultures and number of birds that displayed gross lesions.
A total of 1214 of the samples were from predators: 17 species of the order Accipitriformes, 7 of the
order Strigiformes and 5 of the order Falconiformes. Within this group we have included samples of
lesser black-backed gull (Larus fuscus, n=1), white stork (Ciconia ciconia, n=1), grey heron (Ardea
cinerea, n=1) and little bittern (Ixobrychus minutus, n=1), as they are able to predate columbid chicks.
Four hundred and seventy four samples were obtained from different prey species, including 13
species of the order Passeriformes, 4 of the order Columbiformes, 2 of the order Psittaciformes, 1 of
the order Galliformes and 1 of the order Gruiformes.
We found 20.3% of the samples positive to culture, 11.4% considering predator birds and 43.3%
among prey species (60.7% if only columbid hosts are considered). No positive birds were found in
9
finch species. Gross lesions compatible with trichomonosis were present in 26% of the analyzed
birds, 57.3% of predator birds and 4.9% of prey species. The highest percentage of parasitized
predator birds was observed in the goshawk (74.5%); regarding to prey species, the rock pigeon
showed the highest percentage (79.4%), both species without birds displaying gross lesions.
PHYLOGENETIC ANALYSIS
Five genotypes have been found in this study among the 84 sequences from the isolates, two of
them have been frequently reported in the literature (ITS-OBT-Tg-1,
ITS-OBT-Tg-2, Sansano-
Maestre et al., 2009; Chi et al., 2013), one of them previously reported in two papers (genotype ITSOBT-Tvl-5, Grabensteiner et al., 2010, Kelly-Clark et al., 2013), and two new genotypes not
previously reported (ITS-OBT-Tcl-1, ITS-OBT-Tcl-2, Tables 2 and 3, Supplementary table 1, Fig. 3
and 4).
The phylogenetic trees constructed support the cluster of oral bird trichomonads isolates found in the
literature into six clades (Fig. 3 and 4): one grouping T. gallinae isolates (2 sub-clades), one highly
similar to T. canistomae, one highly similar to T. tenax, one grouping sequences KC529665 and
FN433473, one including only sequence EU215359, and the last one grouping sequences highly
similar to T. vaginalis (3 sub-clades). Sequences from our isolates belong to different genetic groups
included in 3 of the clades: 2 sequences in the T. gallinae clade, 2 sequences in the T. canistomaelike clade and 1 sequence in the T. vaginalis-like clade.
Genotype ITS-OBT-Tg-2 (GenBank No. KF993680-89, 91-92 and KJ776740, 43) was found in
predator birds and columbids, including mainly birds without gross lesions but also a minor amount of
birds with clinical signs (5/35). Strains of T. gallinae from columbids published by various authors
were in this group: Felleisen (1997) (U86614), Sansano-Maestre et al. (2009) (EU881912), Gerhold et
al. (2008) (EU215362-64) and Grabensteiner et al. (2010) (FN433475). Also, an isolate from a canary
(Serinus canaria) of Grabensteiner et al. (2010) (FN433477) was classified in this genotype.
Genotype ITS-OBT-Tg-1 (GenBank No. KF993690, 93-04 and KJ776739, 41-42) was reported
mostly in predator birds with lesions (n=27/33 predator birds), but also 2 isolates with this genotype
were obtained from asymptomatic predator birds. Additionally, the sequences obtained from a
10
passerine with lesions (magpie – Pica pica) and a psittacine with clinical signs (budgerigar –
Melopsittacus undulatus) were included in this clade. This group contained sequences of T. gallinae
from symptomatic birds including columbids of Gerhold et al. (2008) (EU215369), Grabensteiner et al.
(2011) (FN433476) and Girard et al. (2014) (KC215387), predator birds of Gerhod et al. (2008)
(EU215368) and Sansano-Maestre et al. (2009) (EU881913, 16) and the emerging strain of finches
across Europe of Robinson et al. (2011) (GQ150752) and Ganas et al. (2013) (HG008050) (Table 3).
Three newly described genotypes in our country are reported here: genotypes ITS-OBT-Tcl-1, ITSOBT-Tcl-2 and ITS-OBT-Tvl-5 (GenBank accession No. KF933705, KF933706 and KF933707,
respectively). Remarkably, this is the first description of genotypes ITS-OBT-Tcl-1 and ITS-OBT-Tcl-2
in the literature. All of them were isolated from birds without macroscopic lesions. Genotype ITS-OBTTcl-1 was obtained from European turtle doves, genotype ITS-OBT-Tcl-2 from goshawk nestlings and
genotype ITS-OBT-Tvl-5 from an Egyptian vulture (Neophron percnopterus).
Genotypes ITS-OBT-Tcl-1 and ITS-OBT-Tcl-2 are highly similar (they differ only in 3 nucleotides,
positions: 48, 230 and 305), and clustered together in a clade with T. canistomae (AY244652). These
genotypes had 97% identity with the T. canistomae strain (E-values of 1e-144 and 1e-134,
respectively).
Genotype ITS-OBT-Tvl-5 showed 100% homology with a sequence obtained from a bearded vulture
(Gypaetus barbatus) of Grabensteiner et al. (2010) (FN433478) and bald eagle (Haliaeetus
leucocephalus) of Kelly-Clark et al. (2013) (KF214774). The maximum number of nucleotide changes
between the five genotypes found in this study was 33 (ITS-OBT-Tg-1 vs. ITS-OBT-Tvl-5), and the
mininum variation was 3 (ITS-OBT-Tcl-1 vs. ITS-OBT-Tcl-2).
RISK FACTORS ASSOCIATED TO LESIONS
Genotype, type of host and diet
Genotypes ITS-OBT-Tg-1 and ITS-OBT-Tg-2 were the most frequent among the isolates and there
was an association between genotype and type of host, being genotype ITS-OBT-Tg-1 associated to
11
predator birds in general and genotype ITS-OBT-Tg-2 associated to prey species (OR=4, 95% CI=
1.4-12).
OR values indicated that infection by genotype ITS-OBT-Tg-1 is a risk factor for the development of
gross lesions in comparison with the other genotypes (ITS-OBT-Tg-2, ITS-OBT-Tcl-1, ITS-OBT-Tcl-2,
ITS-OBT-Tvl-5; OR=41.9, 95% CI=13.8-127.5) However, the diet (ornithophagous/non-strict
ornithophagous) of the birds and the type of host (predator bird/prey) were also risk factors to develop
symptomatic infections. Considering only birds positive to Trichomonas sp. culture, predator birds
were at higher risk to develop lesions than prey species (OR=4.3, 95% CI=1.5-12.8). In the same
manner, the association between the diet (ornithophagous/non-strict ornithophagous) of predator
birds (considering the goshawk, the Eurasian sparrowhawk, Accipiter nisus and the peregrine falcon,
Falco peregrinus as strict ornithophagous) and the development of gross lesions in birds positive to
Trichomonas sp. culture, indicated that non-strict ornithophagous species were at higher risk to
develop gross lesions than ornithophagous birds (OR=14.9, 95% CI 3.7-60.3).
Influence of genotype
Since variables diet and host type were associated with the genotype found, ORMH was calculated to
more precisely estimate the association between genotype ITS-OBT-Tg-1 and the presence of gross
lesions. The analysis indicated that after removal the influence of confounding variables (type of host
and diet), there were still higher risk to develop symptomatic trichomonosis if genotype ITS-OBT-Tg-1
was present (ORMH=35.4, 95% CI=11.7-107.3, host type stratification and ORMH=37.6, 95% CI=10.5134.4, diet in predator birds stratification). The influence of the genotype was also observed when
separate strata (prey /predator bird) were analyzed (ORprey=10.5, 95% CI=1.2-91.9; ORpredator bird=59.4,
95% CI=15.4-228.6); therefore, being predator bird increased the risk to develop gross lesions if
genotype ITS-OBT-Tg-1 is present. The influence of the genotype could be only observed in non-strict
ornithophagous birds, possibly due to the scarce numbers of birds displaying lesions in
ornithophagous species (n=2) (ORornithophagous=3.3, CI=0.8-14.6; ORnon-stric-ornitophagous=195, 95%
CI=116.2-327.2).
Influence of type of host
12
Although there was higher risk to develop lesions in predator birds than in prey species in each of the
strata (genotype ITS-OBT-Tg-1/ ITS-OBT-Tg-2) analyzed separately (ORgenotype
CI=6.3-7.5; OR
genotype ITS-OBT-Tg-2=1.2,
ITS-OBT-Tg-1
=6.9, 95%
95% CI=1.1-1.4), the results were not consistent when ORMH
test was used. The influence of type of host in the development of gross lesions was not as strong as
the genotype of the isolate (ORMH=2.5, 95% CI=0.6-10.1, genotype stratification).
Influence of diet in predator bird species
Non-strict ornithophagous birds were at more risk to develop gross lesion than ornithophagous birds
although the effect was less intense when the effect of the variable genotype was removed by
stratification (ORMH=8.3, 95% CI=1.2-58, genotype stratification). After stratification by genotype, it
could be observed that the effect of the diet was more intense when genotype ITS-OBT-Tg-1 was
present (ORgenotype ITS-OBT-Tg-1 =90, 95% CI=74.9-108; OR genotype ITS-OBT-Tg-2=1.5, 95% CI=1.1-2.2).
DISCUSSION
No positive cultures were recovered from finch species and no outbreaks or dead birds in this group
were noticed during the course of the study. The fact that birds were from ornithological ringing
campaigns, might be the reason, since mainly healthy individuals were sampled, as they were
capable of performing the migration routes required for each species. Furthermore, the artificial food
supply for small passerines is not an extended practice in Spain as it could be in the United Kingdom.
These facts mean that a lower inter-specific contact and lower prevalence of oral-transmitted
diseases such as avian trichomonosis should be expected. However, we cannot exclude the
presence of the infection in finch birds in our country, as our sample size was small and from a limited
geographic area (n= 132).
The percentage of positive samples obtained in each group (predator birds-11.4% and columbids60.7%) is not very different to the values reported in the study carried out by Sansano-Maestre et
al.(2009) in Spain, being much higher in columbids than in predator birds (19.6%-predator birds and
44.8%-columbids).
We found a low value (4.9%) of prey species with lesions or clinical signs, 2.7% in columbids, (0.37%
in C. livia in the above mentioned study in Spain). In the case of predators, we found 57.3% of the
13
birds with gross lesions, being the common kestrel the most frequently affected (9.4%). SansanoMaestre et al. (2009) reported previously 25% of raptors with gross lesions. These differences in
values could be done by the number of individuals from each species analyzed or the sampling
procedure (many of them came from Recovery Centers and sometimes only samples from suspicious
birds were sent).
The predator bird species with the highest level of infection was the goshawk, in which 74.5% of
nestlings were positive to culture and none of the birds displayed gross lesions. Similar results have
been reported by other authors in Germany, with a prevalence of 65.1% among nestlings but only
4.5% showing pathological lesions (Krone et al., 2005). These high values of infection with low
appearance of gross lesions could be explained if we consider the characteristic ornithophagous diet
of the species, which includes the primary reservoir host of the parasite, columbids. This situation
could lead to an adaptation between host and parasite, leading to milder infections with less clinical
symptoms (Toyne, 1998; Rutz, 2012).
The higher level of parasitism obtained in the rock pigeon (79.4%) indicates its role as reservoir host.
Other studies have shown a lower value (41%) at the same area, although it varied along the year,
reporting maximum levels from December to February (57.1%) and lower from September to
November (35.3%). This finding could be explained by the reproductive stage of the birds, when
courtship rituals and feeding of the young increases the number of infected birds due to an increased
intra-specific contact (Sansano-Maestre et al., 2009).
We have found 5 genotypes in this study (ITS-OBT-Tg-1, ITS-OBT-Tg-2, ITS-OBT-Tcl-1, ITS-OBTTcl-2 and ITS-OBT-Tvl-5). Genotypes ITS-OBT-Tcl-1 and ITS-OBT-Tcl-2 form a distinctive and
different cluster, with no previous isolates that we could find in the literature. This is the first time they
have been reported and therefore, constitute newly discovered genotypes. BLAST analysis indicated
that the highest similarity was obtained with a Trichomonas sp. isolate from a dog (AJ784785) and T.
canistomae (AY244652), in contrast with other T. gallinae isolates. These findings suggest that they
might be classified into an intermediate position or even that they could be new species of this genus.
However, more studies are needed in order to confirm this last suggestion.
14
Genotype ITS-OBT-Tvl-5 is another newly described genotype in Spain, and to our knowledge, the
first detection on the Egyptian vulture. BLAST analysis indicated maximum similarity with a sequence
obtained from a bearded vulture, another scavenger species, published by Grabensteiner et al. in
2010 (FN433478) and from a bald eagle (Kelly-Clark et al., 2013, KF214774). Also, we want to report
that we have received samples from griffon vultures (Gyps fulvus) were cytological fresh smears
showed the presence of live trichomonads, but we did not succeed in the isolation of the parasite so
far because of the bacterial overgrowing flora. We think that these interesting results require further
investigations to determine the genotypes present on vulture and necrophagous species as they
could constitute, again, new species or genus of the parasite.
The OR results considering genotype and lesions agree with the results published by SansanoMaestre et al. (2009) and Chi et al. (2013), who found genotype ITS-OBT-Tg-1 in birds with gross
lesions and more prevalent among predator birds. There are many isolates from other studies that
showed 100% homology to our genotype ITS-OBT-Tg-1, but in many cases the authors did not report
if the birds displayed gross lesions or evident clinical signs. However, some of them were implied in
epidemic episodes of avian trichomonosis, like GQ150752 (Robinson et al., 2010, United Kingdom),
HG008050 (Ganas et al., 2013, Austria and Slovenia), isolated from finches with clinical signs and
band-tailed pigeon mortality (Girard, et al., 2014, KC215387). Others, were recovered from birds with
gross lesions like a rock pigeon (Grabensteiner et al., 2010, FN433476), a barn owl (Tyto alba)
(Sansano-Maestre et al., 2009, EU881913), a mourning dove (Zenaida macroura) (Gerhold et al.,
2008, EU215369) and a broad-winged hawk (Buteo platypterus) (99% homology; Gerhold, et al.
(2008) EU215368). These findings show that this genotype is found among many species, including
raptors, columbids and other avian species like corvids and finches over the world. Actually, this is the
most frequently isolated genotype from birds with clinical signs and seems to be much more related to
the presence of gross lesions than the others, as previous studies have reflected (Sansano-Maestre
et al., 2009; Chi et al., 2013).
Regarding the relationship between the presence of gross lesions and the diet of predator birds, OR
and ORMH results indicated that birds with non-strict ornithophagous diet are at higher risk of
developing gross lesions than strict ornithophagous species like the goshawk. We would like to
15
highlight here the case of the common kestrels, in which we have found an important percentage of
animals with lesions (9.4%) being genotype ITS-OBT-Tg-1 the most frequent (21/25). This species is
characterized by its generalist predator diet, including mainly insects, lizards, small mammals and
birds (Navarro-López et al., 2014). Their habitat tolerance, being capable of reproduce in urban
environments, has put this raptor species at risk to T. gallinae infection, due to the contact and
proximity to columbid preys at these places. In contrast, we have found the case of the goshawk,
were no gross lesions were found and the main genotype was ITS-OBT-Tg-2 (7/12). This raptor feeds
mainly on birds of columbid species and lives in forestal areas. The absence of lesions may indicate
the evolutionary history of the raptor and its diet, feeding on columbid prey which mainly harboured
genotype ITS-OBT-Tg-2 of T. gallinae, with an adaptation to the parasite and the establishment of
non pathogenic infections. In this case, it could be possible that ornithopagous species develop a
significant immune response acquired through their diet that will protect them against highly virulent
strains (Stabler, 1948). On the other hand, the low percentage of birds with gross lesions could be a
consequence of the prevalence of non-pathogenic genotypes in columbids. Taken the statistical
results, we can conclude that genotype and diet of predator birds are risk factors for clinical
trichomonosis, but the influence of the genotype is much higher than the influence of the diet.
Genotype ITS-OBT-Tg-2 was observed mainly in columbids and raptors without gross lesions, but
also in five sick animals. Taking into consideration the sequences reported in this study and by other
authors, this genotype seems to be more associated to columbids or other prey species than to
predator birds. Indeed, a previous research in Spain found the same genotype only in columbid and
raptor hosts without clinical signs (A-genotype of Sansano-Maestre et al., 2009). Furthermore, last
studies carried out in England have also reported this variant among raptors (7.7%) and columbids
(48.8%) with asymptomatic infections (Chi et al., 2013).
The isolates from birds with macroscopical lesions (n=5) showing genotype ITS-OBT-Tg-2 were:
GenBank accession No. KF993682 from a wood pigeon (C. palumbus) of Madrid; KF993686 from a
booted-eagle (A. pennata) recovered in a district of Valencia city and KF993687 from a long eared
owl (A.otus) found predating on a canary cage in Murcia; KF993690 from a peregrine falcon located
at Madrid city and KJ776743 from Eurasian collared dove from Madrid. The long eared owl showed
initial lesions at the oropharynx compatible with avian trichomonosis and other infectious agents
16
(candidiasis, i.e.), but other pathogens were not investigated at the time of sampling. All of them were
from birds recovered in urban areas or with initial lesions that could have acquired the infection by
predation on infected columbids or, in the case of KF993682 and KJ776743, being one of this
reservoir hosts. This fact could be the reason for detecting genotype ITS-OBT-Tg-2, commonly
isolated from columbids, but lesions might also be a consequence of a mixed infection (genotypes
ITS-OBT-Tg-1 / ITS-OBT-Tg-2). Since genotype ITS-OBT-Tg-1 has been associated to the
development of gross lesions in this and other studies and mixed infections have been reported,
clonal culture methods at recently obtained isolates are necessary to clarify the relevance of mixed
infections (Sansano-Maestre et al., 2009, Grabensteiner et al., 2010; Chi et al., 2013). The clinical
isolates could suffer unexpected selection process due to different culture medium requirements
and/or in-vitro multiplication rates of the variants during the protocol of isolation and axenization of the
parasite (vide supra).
Recently a study has investigated the pH conditions of the oral cavity of Cooper´s hawk and its
influence on the persistence of the infection (Urban and Mannan, 2014). They found that nestlings
had a suitable pH for the survival of the parasite in comparison with adult and fledgling birds, and
showed a higher incidence of infection by Trichomonas sp. In the same sense, each genotype could
require different growing conditions for each isolate, either in the animal or in different culture media.
The association between genotype and host could be explained by the different requirements for
growing of each genotype. Also, the selection of a particular genotype over the others after several
passages in has been previously documented in the literature (Grabensteiner et al., 2010).
We have detected six mixed patterns of sequences (ITS-OBT-Tg-1/ITS-OBT-Tg-2), four in raptors
and two in columbids, with and without gross lesions. We could verify in some isolates that DNA
extraction and PCR of recently obtained isolates without subculture showed a mixed pattern, but after
subculture six or higher displayed only one genotype (data not shown). Only clonal cultures will clarify
this issue.
In this study, we also report new avian hosts for the parasite, with sequences of the ITS1/5.8S/ITS2
region not published at Genbank. These species were: two columbids (C. palumbus and S. turtur),
17
one corvid (P. pica) and 13 raptors (A. gentilis, A. nisus, A. otus, B. bubo, B. buteo, Circus
aeruginosus, Circus cyaneus, Falco naumanni, Falco peregrinus, N. percnopterus, Otus scops,
Pernis apivorus, Strix aluco). These data reflect the extensive presence of the parasite among
different species of raptors, including birds with a predominant insectivore diet, like the European
honey-buzzard (P. apivorus) and common scops-owl (Otus scops), but that can feed on columbids
prior or during the breeding season (Roberts and Coleman, 2001).
More studies are needed to determine the association between pathology and genetic variant, using
multi-locus genetic markers and single copy genes. Also, there is a need to study new bird species in
order to determine the diversity of oral trichomonads present, as different genotypes have been
described on different hosts (genotypes ITS-OBT-Tcl-1, ITS-OBT-Tcl-2 and ITS-OBT-Tvl-5) and a
novel genus has been recently reported (Girard et al., 2014). This new knowledge will help us to
understand the epidemiology of the disease in order to control and limit the mortality outbreaks found
in wildlife.
CONCLUSIONS
The results obtained indicate a relationship between the genotype of the parasite and different hosts.
Thus, the most frequent presentation of the genotypes would be: ITS-OBT-Tg-1-predators and ITSOBT-Tg-2-columbids. Genotypes ITS-OBT-Tcl-1, ITS-OBT-Tcl-2 and ITS-OBT-Tvl-5 (ITS-OBT-Tcl-1turtle doves, ITS-OBT-Tcl-2-goshawks and ITS-OBT-Tvl-5-Egyptian vulture) were found in a very low
number of birds, although none of them was detected in any other avian species. Two of the
genotypes (ITS-OBT-Tg-1 and ITS-OBT-Tg-2) are widespread over wild bird populations. Genotype
ITS-OBT-Tg-1 seems to harvest the most pathogenic potential of the parasite, being responsible of
the development of gross lesions that could be lethal. This study brings new data to the epidemiology
of T. gallinae, pointing out the genetic diversity of the parasite and the different pathogenicity of the
genotypes.
ACKNOWLEDGEMENTS
This research has been granted by funds of the Spanish Ministry of Science and Innovation, project
AGL2011-29442; projects PRCEU-UCH 27/10, 41/11, SANTANDER-PRCEU-UCH 13/12 of University
CEU Cardenal Herrera (Valencia, Spain) and Banco Santander, S. A.
18
Mª Carmen Martínez Herrero has been granted by a fellowship (ACIF/2013/055) of the program for
engagement of predoctoral research personnel VALi+D, and wishes to express her gratitude to the
Conselleria of Science and Education of the Valencian Community.
We would also like to thank to all the staff from the Wildlife Recovery Centers and bird ringing
scientists that have collaborated in this study, with special consideration to: “Servicio de Vida
Silvestre” of the Conselleria de Infraestructuras, Territorio y Medio Ambiente of the Valencian
Community; veterinarians Pilar Sanchís Caballer, Jose María Gil Puerto, Pedro María Mójica, Ana
Cristina Miñano, Marcos Núñez Laiseca, Amalia García Talens, Fernando González (GREFA), Rafael
Molina (Torreferrussa), Xavier González; technical personnel Domingo Cruz Aparisi, Daniel García
Mons, Javier Blasco Giménez, Antonio Pérez Torres, José María Antolín and Ana Antolín Vivas; and
bird ringing scientists and researchers Mario León Ortega, Juan Manuel Pérez García, Francisco
Alberto García Castellanos, Pablo Vera García and Francisco Javier García Gans, Arantza Leal
Nebot and the members of SEO-Monticola, who helped during the field work.
REFERENCES
1. Anderson, N.L., Grahn, R.A., Van Hoosear, K., BonDurant, R.H., 2009. Studies of
trichomonad protozoa in free ranging songbirds: Prevalence of Trichomonas gallinae in house
finches (Carpodacus mexicanus) and corvids and a novel trichomonad in mockingbirds
(Mimus polyglottos). Vet. Parasitol. 161, 178-186.
2. Bunbury, N., Jones, C.G., Greenwood, A.G., Bell, D.J., 2007. Trichomonas gallinae in
Mauritian columbids: implications for and endangered endemic. J. Wildl. Dis. 43, 399-407.
3. Bunbury, N., 2011. Trichomonad infection in endemic and introduced columbids in the
Seychelles. J. Wildl. Dis. 47, 730-733.
4. Cepicka, I., Kutisová, K., Tachezy, J., Kulda, J., Flegr, J., 2005. Cryptic species within the
Tetratrichomonas gallinarum species complex revealed by molecular polymorphism. Vet.
Parasitol. 128, 11-21.
5. Chi, J.F., Lawson, B., Durrant, C., Beckmann, K., John, S., Alrefaei, A.F., Kirkbride, K., Bell,
D.J., Cunningham, A.A., Tyler, K.M., 2013. The finch epidemic strain of Trichomonas gallinae
is predominant in British non-passerines. Parasitology. 140, 1234-1245.
6. Cooper, J.E., Petty, S.J., 1988. Trichomoniasis in free-living goshawks (Accipiter gentilis
gentilis) from Great Britain. J. Wildl. Dis. 24, 80-87.
7. Diamond, L.S., 1957. The establishment of various trichomonads of animals and man
in axenic cultures. J. Parasitol. 43, 488-490.
8. Ecco, R., Preis, I.S., Vilela, D.A.R., Luppi, M.M., Malta, M.C.C., Beckstead, R.B.,
Stimmelmayer, R., Gerhold, R.W., 2012. Molecular confirmation of Trichomonas gallinae and
19
other parabasalids from Brazil using the 5.8S and ITS-1 rRNA regions. Vet. Parasitol. 190,
36-42.
9. Felleisen, R.S., 1997. Comparative sequences analysis of 5.8S rRNA genes and internal
trascribed spacer (ITS) regions of trichomonadid protozoa. Parasitology. 115, 111-119.
10. Forzán, M.J., Vanderstichel, R., Melekhovets, Y.F., McBurney, S., 2010. Trichomoniasis in
finches from the Canadian Maritime provinces — An emerging disease. Can. Vet. J. 51, 391–
396.
11. Ganas, P., Jaskulska, B., Lawson, B., Zadravec, M., Hess, M., Bilic, I., 2013. Multi-locus
sequence typing confirms the clonality of Trichomonas gallinae isolates circulating in
European
finches.
Parasitology.
pp
1-10.
DOI: http://dx.doi.org/10.1017/S0031182013002023.
12. Gaspar da Silva, D., Barton, E., Bunbury, N., Lunness, P., Bell, D.J., Tyler, K.M., 2007.
Molecular identity and heterogeneity of Trichomonad parasites in a closed avian population.
Infect. Genet. Evol. 7, 433-40.
13. Gerhold, R.W., Yabsley, M.J., Smith, A.J., Ostergaard, E., Mannan, W., Cann, J.D., Fischer,
J.R., 2008. Molecular characterization of the Trichomonas gallinae morphologic complex in
the United States. J. Parasitol. 94, 1335-1341.
14. Girard, Y.A., Rogers, K.H., Gerhold, R., Land, K.M., Lenaghan, S.C., Woods, L.W.,
Haberkern, N., Hopper, M., Cann, J.D., Johnson, C.K., 2014. Trichomonas stableri n. sp., an
agent of trichomonosis in Pacific Coast band-tailed pigeons (Patagioenas fasciata monilis).
Int. J. Parasitol. 3, 32-40.
15. Gourlay, P., Decors, A., Jouet, D., Treilles, M., Lemberger, K., Faure, E., Moinet, M., Chi, J.,
Tyler, K., Cunningham, A., Lawson, B., 2011. Finch trichomonosis spreads to France. Europ.
Sect. Wildlife Dis. Assoc. Bull. 2, 9-10.
16. Grabensteiner, E., Bilic, I., Kolbe, T., Hess, M., 2010. Molecular analysis of clonal
trichomonad isolates indicate the existence of heterogenic species present in different birds
and within the same host. Vet. Parasitol. 172, 53-64.
17. Greiner, E.C., Baxter, W.L., 1974. A localized epizootic of trichomoniasis in mourning doves.
J. Wildl. Dis. Vol. 10, 104-106.
18. Höfle, U., Blanco, J.M., Palma, L., Melo, P., 2000. Trichomoniasis in Bonelli’s Eagle
(Hieraaetus fasciatus) nestlings in south-west Portugal. In: Lumeij, J.T., Remple, J.D., Redig,
P.T., Lierz, M., Cooper, J.E. (Eds.), Raptor Biomedicine III. Zoological Education Network.
Lake Worth, Florida, USA, pp. 45-51.
19. Höfle, U., Gortázar, C., Ortiz, J.A., Knispel, B., Kaleta, E.F., 2004. Outbreak of trichomoniasis
in a woodpigeon (Columba palumbus) wintering roost. Eur. J. Wildl. Res. 50, 73-77.
20. Kelly-Clark, W.K., Forzán, M.J., Desmarchelier, M., Greenwood, S.J., 2013. Detection and
characterization of a Trichomonas isolate from a rehabilitated bald eagle (Haliaeetus
leucocephalus). J. Zoo Wildl. Med. 44, 1123-1126.
21. Kleina, P., Bettim-Bandinelli, J., Bonatto, S.L., Benchimol, M., Bogo, M.R., 2004. Molecular
phylogeny of Trichomonadidae family inferred from ITS-1, 5.8S rRNA and ITS-2 sequences.
Int. J. Parasitol. 34, 963-970.
22. Krone, O., Altenkamp, R., Kenntner, N., 2005. Prevalence of Trichomonas gallinae in northern
goshawks from the Berlin area of northeastern Germany. J. Wildl. Dis. 41, 304-309.
20
23. Kutisova, K., Kulda, J., Cepicka, I., Flegr, J., Koudela, B., Teras, J., Tachezy J., 2005.
Tetratrichomonads from the oral cavity and respiratory tract of humans. Parasitol. 131, 30919.
24. Lawson, B., Cunningham, A.A., Chantrey, J., Hughes, L.A., John, S.K., Bunbury, N., Bell,
D.J., Tyler, K.M., 2011a. A clonal strain of Trichomonas gallinae is the aetiologic agent of an
emerging avian epidemic disease. Infect. Genet. Evol. 11, 1638-1645.
25. Lawson, B., Robinson, R.A., Neimanis, A., Handeland, K., Isomursu, M., Agren, E.O.,
Hamnes, I.S., Tyler, K.M., Chantrey, J., Hughes, L.A., Pennycott, T.W., Simpson, V.R.,
John, S.K., Peck, K.M., Toms, M.P., Bennett, M., Kirkwood, J.K., Cunningham, A.A. 2011b.
Evidence of spread of the emerging infectious disease, finch trichomonosis, by migrating
birds. Ecohealth. 8, 143-153.
26. Lennon, R.J., Dunn, J.C., Stockdale, J.E., Goodman, S.J., Morris, A.J., Hamer, K.C., 2013.
Trichomonad parasite infection in four species of Columbidae in the UK. Parasitology. 140,
1368-1376.
27. Molina-López, R.A., Casal, J., Darwich, L., 2011. Causes of Morbidity in Wild Raptor
Populations Admitted at a Wildlife Rehabilitation Centre in Spain from 1995-2007: A Long
Term Retrospective Study. PLoS One. Sept, volume 6, e24603.
28. Narcisi, E.M., Sevoian, M., Honigberg, B.M., 1991. Pathologic changes in pigeons infected
with a virulent Trichomonas gallinae strain (Eiberg). Avian Dis. 35, 55-61.
29. Navarro-López, J., Vergara, P., Fargallo, J.A., 2014. Trophic niche width, offspring condition
and immunity in a raptor species. Oecologia. 174, 1215-24.
30. Neimanis, A.S., Handeland, K., Isomursu, M., Agren, E., Mattsson, R., Hamnes, I.S., Bergsjø,
B., Hirvelä-Koski, V., 2010. First report of epizootic trichomoniasis in wild finches (family
Fringillidae) in southern Fennoscandia. Avian Dis. 54, 136-141.
31. Real, J., Mañosa, S., Muñoz, E., 2000. Trichomoniasis in a Bonelli’s Eagle Population in
Spain. J. Wildl. Dis. 36, 64-70.
32. Roberts, S.J., Coleman, M., 2001. Some Observations on the Diet of European Honeybuzzards in Britain. British Birds. 94, 433-436.
33. Robinson, R.A., Lawson, B., Toms, M.P., Peck, K.M., Kirkwood, J.K., Chantrey, J.,
Clatworthy, I.R., Evans, A.D., Hughes, L.A., Hutchinson, O.C., John, S.K., Pennycott, T.W.,
Perkins, M.W., Rowley, P.S., Simpson, V.R., Tyler, K.M., Cunningham, A.A., 2010. Emerging
infectious disease leads to rapid population declines of common British birds. PLoS One. Aug
18, 5, e12215.
34. Rutz, C., 2012. Brood sex ratio varies with diet composition in a generalist raptor. Biol. J. Linn.
Soc. 105, 937–951.
35. Rutz, C., Whittingham, M.J., Newton, I., 2006. Age-dependent diet choice in an avian top
predator. Proc. Biol. Sci. 273, 579-586.
36. Sansano-Maestre, J., Garijo-Toledo, M.M., Gómez-Muñoz, M.T., 2009. Prevalence and
genotyping of Trichomonas gallinae in pigeons and birds of prey. Avian Pathol. 38, 201-207.
37. Stabler, R.M., 1948. Protection in pigeons against virulent Trichomonas gallinae acquired by
infection with milder strains. J. Parasitol. 34, 150-153.
38. Stimmelmayr, R., Stefani, L.M., Thrall, M.A., Landers, K., Revan, F., Miller, A., Beckstead, R.,
Gerhold, R., 2012. Trichomonosis in free-ranging Eurasian collared doves (Streptopelia
21
decaocto) and African collared dove hybrids (Streptopelia risoria) in the Caribbean and
description of ITS-1 region genotypes. Avian Dis. 56, 441-445.
39. Swinnerton, K.J., Greenwood, A.G., Chapman, R.E., Jones, C.G., 2005. The incidence of the
parasitic disease trichomoniasis and its treatment in reintroduced and wild Pink Pigeons
Columba mayeri. Ibis. 147, 772-782.
40. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:
Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance,
and Maximum Parsimony Methods. Mol. Biol. Evol. 28, 2731-2739.
41. Toyne, E.P., 1998. Breeding season diet of the Goshawk Accipiter gentilis in Wales. Ibis.
140, 569–579.
42. Urban, E.H., Mannan, W., 2014. The potential role of oral pH in the persistence of
Trichomonas gallinae in Cooper’s hawks (Accipiter cooperii). J. Wild. Dis. 50, 1. DOI:
10.7589/2012-12-322
43. Villanúa, D., Höfle, U., Pérez-Rodríguez, L., Gortázar, C., 2006. Trichomonas gallinae in
wintering common wood pigeons Columba palumbus in Spain. Ibis. 148, 641-648.
44. Zadravec, M., Marhold, C., Slavec, B., Rojs, O.Z., Racnik, J., 2012. Trichomonosis in finches
in Slovenia. Vet. Rec. 171, 253-254.
22
Table 1. Species analyzed in this study, total number of samples (n), percentage of positive
cultures (P) and birds with gross lesions (L). Diet (ornithophagous-O/non-strict ornithophagousNO) and type of bird (predator/prey) is also shown.
SPECIES
Accipiter gentilis
Accipiter nisus
Aegypius monachus
Aquila adalberti
Aquila chrysaetos
Aquila fasciata
Aquila pennata
Ardea cinerea
Asio flammeus
Asio otus
Athene noctua
Bubo bubo
Buteo buteo
Ciconia ciconia
Circaetus gallicus
Circus aeruginosus
Circus cyaneus
Circus pygargus
Columba livia
Columba palumbus
Coturnix coturnix
Falco columbarius
Falco naumanni
Falco peregrinus
Falco subbuteo
Falco tinnunculus
Gallinula chloropus
Garrulus glandarius
Gyps fulvus
Ixobrychus minutus
Larus fuscus
Melopsittacus undulatus
Milvus migrans
Milvus milvus
Myiopsitta monachus
Neophron percnopterus
Otus scops
Passerine species*
Pernis apivorus
Pica pica
Streptopelia decaocto
Streptopelia turtur
Strix aluco
Tyto alba
TOTAL
n
55
18
30
3
25
21
19
1
2
11
80
57
34
1
3
11
7
35
34
10
1
3
249
24
2
373
1
2
50
1
1
1
11
9
3
2
12
132
4
4
289
3
18
36
1688
P (%)
74.5
27.8
0
0
4
0
21.1
0
0
27.3
0
5.3
8.8
0
0
18.2
28.6
0
79.4
70
0
0
4.4
4.2
0
14.2
0
0
0
0
0
100
0
0
0
50
8.3
0
25
25
57.8
100
16.7
5.6
20.3
L (%)
0
27.8
0
0
8
4.8
21.1
0
0
27.3
0
5.3
11.8
0
0
9.1
0
0
0
60
0
0
0.8
12.5
0
9.4
0
0
2
0
nd
100
9.1
11.1
0
0
8.3
0
0
25
1
0
44.4
8.3
5.3
TYPE OF HOST
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREY
PREY
PREY
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREY
PREDATOR
PREDATOR
PREDATOR
PREDATOR
PREY
PREDATOR
PREDATOR
PREY
PREDATOR
PREDATOR
PREY
PREDATOR
PREDATOR
PREY
PREY
PREDATOR
PREDATOR
DIET
O
O
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
O
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
*Passerine species included:
−
Carduelis carduelis
−
Carduelis chloris
−
Cyanistes caeruleus
−
Emberiza schoeniclus
−
Fringilla coelebs
−
Parus major
−
Passer domesticus
−
Serinus serinus
−
Sturnus unicolor
−
Turdus merula
23
Table 2. Information of the T. gallinae isolates sequenced in this study.
SPECIES
Accipiter gentilis
Aquila pennata
Asio otus
Buteo buteo
Circus aeruginosus
Circus cyaneus
Columba palumbus
Falco naumanni
Falco peregrinus
Falco tinnunculus
Pernis apivorus
Streptopelia decaocto
Accipiter gentilis
Accipiter nisus
Aquila pennata
Asio otus
Bubo bubo
Buteo buteo
Circus cyaneus
Falco peregrinus
Falco tinnunculus
Melopsittacus undulatus
Pica pica
Otus scops
Streptopelia decaocto
LAB CODE
RM3/12
RM10/12
R256/11
R257/11
R43/12
R45/12
R57/12
R134/12
R137/12
RM11/12
RM3/13
R221/11
RM5/13
PM1/12
P178/13
P179/13
R11/13
R59/13
R66/13
RM1/12
R100/11
R24/12
R25/12
R42/12
R237/13
PM2/12
P2/12
P139/12
P321/12
P329/12
P31/13
P33/13
P34/13
P38/13
P94/13
RM7/13
R64/11
RM13/12
R213/11
RM14/12
R16/12
R17/12
RM8/13
RM9/12
RM1/12
RM5/12
RM6/12
RM7/12
RM8/12
R55/11
R72/11
R97/11
R99/11
R125/11
R147/11
R176/11
R179/11
R180/11
R189/11
R49/12
R127/12
R131/12
R191/13
R193/13
R198/13
P112/13
OM1/12
R210/13
P96/11
LESION
NO
NO
NO
NO
NO
NO
NO
YES
YES
NO
NO
NO
NO
YES
NO
NO
NO
NO
NO
YES
NO
NO
NO
NO
NO
YES
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
YES
YES
YES
YES
YES
NO
YES
YES
YES
YES
YES
YES
YES
YES
YES
nd*
nd*
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
NO
GENETIC GROUP
ITS-OBT-Tg-2
ITS-OBT-Tg-1
24
Strix aluco
Tyto alba
Streptopelia turtur
Accipiter gentilis
Neophron percnopterus
Accipiter gentilis
Accipiter nisus
Asio otus
Columba palumbus
Falco tinnunculus
P95/13
RM12/12
R183/13
P243/12
P262/12
P196/13
R30/07
R31/07
R235/13
R44/12
NO
YES
YES
NO
NO
NO
NO
RM15/12
R176/13
PM2/13
P349/12
R186/13
YES
YES
NO
YES
YES
NO
NO
ITS-OBT-Tcl-1
ITS-OBT-Tcl-2
ITS-OBT-Tvl-5
MIXED INFECTION (ITSOBT-Tg-1/ITS-OBT-Tg-2)
* nd: not determined.
25
Table 3. Genotypes of the ITS1/5.8SS/ITS2 region of oral Trichomonas sp. isolated from birds described by other authors and genetic group
codification used in this paper (ITS-OBT-Tx-n). ITS: ITS1/5.8S/ITS2 region. OBT: Oropharyngeal Bird Trichomonad. Tx: Organism homology group
(Simplicimonas spp.; Tcl: T. canistomae-like; Tg: T. gallinae; Ts: T. stableri; Tt: T. tenax; Trichomonadida spp.; Tvl: T. vaginalis-like), and n: subgroup
Genotype Chi
Genotype
Sansano
A*
Genotype
Grabensteiner
Genotype
Gerhold
number (new numbers were used when homology was less than 99%).
IV*
A*
B*
B*
D, E
I
C
V
C
A
Genbank sequences (author)
Organism sequence homology (% of BLAST identity, E-value)
Genetic group
ITS-OBT-Tg-1
AY349182 (Kleina)
T. gallinae AY349182 (Kleina) (100%, 1e-154)
EF208019 (Gaspar da Silva)
T. gallinae AY349182 (Kleina) (100%, 4e-108)
EU215369 (Gerhold)*
T. gallinae EU881911 (Sansano) (100%, 9e-146)
EU290649 (Anderson)*
T. gallinae EU881911 (Sansano) (100%, 1e-159)
FN433476 (Grabensteiner)*
T. gallinae AY349182 (Kleina) (100%, 1e-154)
GQ150752 (Robinson)*
T. gallinae AY349182 (Kleina) (100%, 5e-107)
HG008050 (Ganas)*
T. gallinae AY349182 (Kleina) (100%, 1e-138)
KC215387 (Girard)*
T. gallinae JN007005 (Reimann) (100%, 0.0)
EU215368 (Gerhold)*
T. gallinae KC215387 (Girard) (99%, 1e-170)
EU881911,13*,15,16* (Sansano)
T. gallinae KC215387 (Girard) (99%, 0.0; 100%, 3e-171; 100%, 3e-171; 100%, 3e-171)
EU215363-64 (Gerhold)
T. gallinae U86614 (Felleisen) (100%, 3e-125; 100%, 4e-154)
FN433475 (Grabensteiner)
T. gallinae U86614 (Felleisen) (100%, 4e-154)
EU215362 (Gerhold)
T. gallinae U86614 (Felleisen) (99%, 0.0)
EU881912, 14, 17 (Sansano)
T. gallinae U86614 (Felleisen) (99%, 0.0); EU215362 (Gerhold) (99%, 5e-169; 99%, 5e169)
FN433477 (Grabensteiner)
T. gallinae EU881912 (Sansano) (99%, 9e-151)
U86614 (Felleisen)
T. gallinae EU881912 (Sansano) (99%, 0.0)
ITS-OBT-Tg-2
26
II*
III*
FN433474 (Grabensteiner)*
T. tenax (KF164607) (99%, 2e-147)
ITS-OBT-Ttl-1
KF993705 (this paper)
T. canistomae (AY244652) (97%, 1e-144)
ITS-OBT-Tcl-1
KF993706 (this paper)
T. canistomae (AY244652) (97%, 1e-134)
ITS-OBT-Tcl-2
FN433473 (Grabensteiner)*
T. canistomae (AY244652) (91%, 3e-110 )
KC529665 (Chi)
T. gallinae FN433473 (Grabensteiner) (99%, 4e-150)
ITS-OBTTrichomonas sp.1
ITS-OBTTrichomonas sp.2
I
EU215361 (Gerhold)
T. vaginalis (U86613) (Felleisen) (99%, 0.0)
ITS-OBT-Tvl-1
L
EU215366 (Gerhold)
T. vaginalis (U86613) (Felleisen) (99%, 2e-167 )
ITS-OBT-Tvl-2
H
EU215360 (Gerhold)
T. vaginalis (U86613) (Felleisen) (98%, 8e-172)
ITS-OBT-Tvl-3
J
EU215365 (Gerhold)
T. vaginalis (U86613) (Felleisen) (98%, 2e-168 )
ITS-OBT-Tvl-4
FN433478 (Grabensteiner)
T. vaginalis (FJ813603) (97%, 6e-138)
ITS-OBT-Tvl-5
KF214774 (Kelly-Clark)
Not yet released in GenBank
KF993707 (this paper)
T. vaginalis (FJ813603) (97%, 2e-152)
G
EU215359 (Gerhold)
T. vaginalis (U86613) (Felleisen) (92%, 1e-145)
ITS-OBTTrichomonas sp.3
K*
EU215367 (Gerhold)
T. stableri (KC215389) (100%, 7e-167)
ITS-OBT-Ts-1
JX089392 (Ecco)*
T. stableri (KC215389) (100%, 1e-48)
KC215389 (Girard)*
T. stableri (KC215390) (99%, 0.0)
VI
F
KC215390 (Girard)*
T. stableri (KC215389) (99%, 0.0)
EU215358 (Gerhold)
T. stableri (KC215389) (94%, 4e-104)
JX089388 (Ecco)*
Simplicomonas sp. (HQ334182) (99%, 8e-100)
EU290650 (Anderson)
Histomonas sp. (HQ334185) (83%, 4e-45)
ITS-OBT-Ts-2
ITS-OBTSimplicomonas spp.
ITS-OBTTrichomonadida
spp.
*Associated to infections with gross lesions.
References according to authors mentioned in the table: Anderson: Anderson et al., 2009; Chi: Chi et al., 2013; Ecco: Ecco et al., 2012; Felleisen: Felleisen, 1997; Ganas: Ganas et
al., 2013; Gaspar da Silva: Gaspar da Silva et al., 2007; Gerhold: Gerhold et al., 2008; Girard: Girard et al., 2014; Grabensteiner: Grabensteiner et al., 2010; Kelly-Clark: Kelly-Clark
et al., 2013; Kleina: Kleina et al., 2004; Robinson: Robinson et al., 2010; Sansano-Maestre: Sansano-Maestre et al., 2009.
27
Supplementary table 1. Sequences of the ITS1/5.8S/ITS2 region of T. gallinae isolates of this
study with GenBank accession numbers. Lesion: macroscopic lesion compatible with
tricomonosis,
GENBANK
ACCESSION
NUMBER
GENETIC GROUP
SPECIES
LESION
ISOLATE CODE
KF993680
ITS-OBT-Tg-2
Streptopelia decaocto
NO
P94/13
KF993681
ITS-OBT-Tg-2
Columba palumbus
NO
P178/13
KF993682
ITS-OBT-Tg-2
Columba palumbus
YES
PM1/12
KF993683
ITS-OBT-Tg-2
Falco tinnunculus
NO
R42/12
KF993684
ITS-OBT-Tg-2
Accipiter gentilis
NO
R45/12
KF993685
ITS-OBT-Tg-2
Falco naumanni
NO
R59/13
KF993686
ITS-OBT-Tg-2
Aquila pennata
YES
R134/12
KF993687
ITS-OBT-Tg-2
Asio otus
YES
R137/12
KF993688
ITS-OBT-Tg-2
Circus aeruginosus
NO
R221/11
KF993689
ITS-OBT-Tg-2
Pernis apivorus
NO
R237/13
KF993690
ITS-OBT-Tg-1
Falco peregrinus
YES
RM1/12
KF993691*
ITS-OBT-Tg-2
Accipiter gentilis
NO
RM3/12
KF993692*
ITS-OBT-Tg-2
Buteo buteo
NO
RM3/13
KF993693
ITS-OBT-Tg-1
Pica pica
YES
OM1/12
KF993694
ITS-OBT-Tg-1
Streptopelia decaocto
NO
P95/13
KF993695
ITS-OBT-Tg-1
Melopsittacus undulatus
YES
P112/13
KF993696
ITS-OBT-Tg-1
Bubo bubo
YES
R16/12
KF993697
ITS-OBT-Tg-1
Falco tinnunculus
YES
R127/12
KF993698
ITS-OBT-Tg-1
Tyto alba
YES
R183/13
KF993699
ITS-OBT-Tg-1
Aquila pennata
YES
R213/11
KF993700*
ITS-OBT-Tg-1
Accipiter gentilis
NO
RM7/13
KF993701*
ITS-OBT-Tg-1
Buteo buteo
YES
RM8/13
KF993702
ITS-OBT-Tg-1
Circus cyaneus
NO
RM9/12
KF993703
ITS-OBT-Tg-1
Strix aluco
YES
RM12/12
KF993704
ITS-OBT-Tg-1
Asio otus
YES
RM14/12
KF993705*
ITS-OBT-Tcl-1
Streptopelia turtur
NO
P243/12
KF993706*
ITS-OBT-Tcl-2
Accipiter gentilis
NO
R30/07
KF993707*
ITS-OBT-Tvl-5
Neophron percnopterus
NO
R235/13
KJ776739
ITS-OBT-Tg-1
Accipiter nisus
NO
RM13/12
KJ776740
ITS-OBT-Tg-2
Circus cyaneus
NO
RM5/13
KJ776741
ITS-OBT-Tg-1
Falco tinnunculus
YES
RM2/12
KJ776742
ITS-OBT-Tg-1
Otus scops
YES
R210/13
KJ776743
ITS-OBT-Tg-2
Streptopelia decaocto
YES
PM2/12
*Sequences included in the phylogenetic analysis.
28
Supplementary table 2. Sequences of the ITS1/5.8S/ITS2 region of Trichomonas sp. of others
authors used for the phylogenetic trees with GenBank accession numbers.
GENBANK
ACCESSION
NUMBER
ORGANISM
HOST SPECIES
LESION
AUTHOR
AY244652
Trichomonas canistomae
Canis familiaris
UNKNOWN
Kutisova et al., 2005
AY245137
Pentatrichomonas
hominis
Homo sapiens
UNKNOWN
Cepicka et al., 2005
EU215359
Trichomonas sp.
Columba passerina
UNKNOWN
Gerhold et al., 2008
EU215361
Trichomonas sp.
Zenaida asiatica
UNKNOWN
Gerhold et al., 2008
EU215360
Trichomonas sp.
Zenaida asiatica
UNKNOWN
Gerhold et al., 2008
EU215365
Trichomonas sp.
Zenaida macroura
UNKNOWN
Gerhold et al., 2008
EU215366
Trichomonas sp.
Accipiter cooperii
UNKNOWN
Gerhold et al., 2008
EU215367
Trichomonas sp.
Patagioenas fasciata
UNKNOWN
Gerhold et al., 2008
EU290650
Trichomonas sp.
Mimus polyglottos
UNKNOWN
Anderson et al., 2009
EU881911
Trichomonas gallinae
Columba livia
NO
Sansano-Maestre et al., 2009
EU881912
Trichomonas gallinae
Columba livia
NO
Sansano-Maestre et al., 2009
FN433473
Trichomonas sp.
Columba livia
UNKNOWN
Grabensteiner et al., 2010
FN433474
Trichomonas sp.
Columba livia
UNKNOWN
Grabensteiner et al., 2010
FN433478
Trichomonas gallinae
Gypaetus barbatus
UNKNOWN
Grabensteiner et al., 2010
KC215389
Trichomonas stableri
Patagioenas fasciata monilis
YES
Girard et al., 2014
KC215390
Trichomonas stableri
Patagioenas fasciata monilis
YES
Girard et al., 2014
KC529665
Trichomonas gallinae
Caloenas nicobarica
NO
Chi et al., 2013
U86613
Trichomonas vaginalis
Homo sapiens
UNKNOWN
Felleisen, R.S. 1997
U86615
Trichomonas tenax
UNKOWN
UNKNOWN
Felleisen, R.S. 1997
29
Fig. 1. Map of Spain showing the origin of the sampled birds. Shaded areas correspond to
areas of coverage of Recovery Centers (admissions). Dots indicate the main Recovery Centers:
1-DEMA (Almendralejo, Badajoz). 2-CERI (Toledo). 3-GREFA (Majadahonda, Madrid). 4-El
Ardal (Albendea, Cuenca). 5-La Alfranca (Pastriz, Zaragoza). 6-Torreferrusa (Santa Perpétua
de Mogoda, Barcelona). 7-La Granja (El Saler, Valencia). 8-Santa Faz (Alicante). 9-El Valle (La
Alberca, Murcia).
30
Fig. 2. From left to right: gross lesions of avian trichomonosis in wood pigeon (Columba
palumbus), common kestrel (Falco tinnunculus) and Eurasian tawny owl (Strix aluco)*. Isolates
P349/12, R164/12 and R198/13 respectively.*Photograph of Amalia García Talens.
31
94
EU881912 Trichomonas gallinae
KF993691 (Agen-1)
KF993692 (Bbut-1)
93
KF993700 (Agen-2)
70
94
EU881911 Trichomonas gallinae
KF993701 (Bbut-2)
AY244652 Trichomonas canistomae
KF993705 (Stur-1)
KF993706 (Agen-3)
U86615 Trichomonas tenax
98 FN433474 Trichomonas sp.
EU215359 Trichomonas sp.
KC529665 Trichomonas gallinae
100 FN433473 Trichomonas sp.
98 KF993707 (Nper-1)
FN433478 Trichomonas sp.
100
KC215389 Trichomonas stableri
91
EU215367 Trichomonas sp.
KC215390 Trichomonas stableri
81 EU215361 Trichomonas sp.
EU215360 Trichomonas sp.
EU215365 Trichomonas sp.
U86613 Trichomonas vaginalis
EU215366 Trichomonas sp.
AY245137 Pentatrichomonas hominis
EU290650 Trichomonadida sp.
0.05
Fig. 3. Phylogenetic tree of the region ITS1/5.8S/ITS2 of T. gallinae by Neighbor-Joining
method. Sequences are indicated with GenBank accession number and those from this study
also include the host species with the initial of the gender and three letters of species name.
Bootstrap values lower than 70 are not shown (n=2000 replicates).
32
EU881912 Trichomonas gallinae
84
KF993691 (Agen-1)
85 KF993692 (Bbut-1)
KF993700 (Agen-2)
83
EU881911 Trichomonas gallinae
KF993701 (Bbut-2)
KF993705 (Stur-1)
KF993706 (Agen-3)
AY244652 Trichomonas canistomae
U86615 Trichomonas tenax
88 FN433474 Trichomonas sp.
EU215359 Trichomonas sp.
KC529665 Trichomonas gallinae
98 FN433473 Trichomonas sp.
97
97 KF993707 (Nper-1)
FN433478 Trichomonas sp.
KC215390 Trichomonas stableri
EU215367 Trichomonas sp.
KC215389 Trichomonas stableri
77 EU215361 Trichomonas sp.
EU215360 Trichomonas sp.
EU215365 Trichomonas sp.
U86613 Trichomonas vaginalis
EU215366 Trichomonas sp.
AY245137 Pentatrichomonas hominis
EU290650 Trichomonadida sp.
0.05
Fig. 4. Phylogenetic tree of the region ITS1/5.8S/ITS2 of T. gallinae by Maximum Likelihood
method. Sequences are indicated with GenBank accession number and those from this study
also include the host species with the initial of the gender and three letters of species name.
Bootstrap values lower than 70 are not shown (n=2000 replicates).
33