Sequence subtyping of Trichomonas gallinae from Bonelli's eagle (Aquila fasciata) during four years (2014-2017) reveals that MLS type is associated with lesions.

Avian trichomonosis is a parasitic disease that affects wild birds. The objective of this work was to determine the importance of avian trichomonosis in Bonelli's eagles to improve conservation measures in this population. One hundred and eighty-eight birds were studied: 181 chicks, two juveniles, one subadult and four adults. The birds were externally examined and gross lesions at the oropharynx registered. Samples from the oropharyngeal cavity were obtained for Trichomonas spp. detection by culture and PCR, and positive samples were subjected to a multilocus sequence typing approach, including the ITS1/5.8S/ITS2 region (ITS), ribosomal RNA small subunit (18S) and Fe-hydrogenase gene (FeHyd). Global prevalence of T. gallinae infection was 37.8% in total, 45.5% in nestlings. Thirty-three percent of the birds developed lesions that ranged from mild (n = 41) to moderate (n = 14) or severe (n = 7). Multilocus sequence typing analysis showed five different MLS types, ITS-A/18S-VI/FeHyd-A1 and ITS-D/18S-II/Fe-C4 being the most frequent. An association between ITS-A/18S-VI/FeHyd-A1 and moderate or severe lesions was observed, but birds with type ITS-A/18S-VI/FeHyd-A2 also developed lesions. On the contrary, birds with MLS type ITS-D/18S-II/FeHyd-C4 displayed only a low proportion of mild lesions. Chicks raised in nests were at higher risk for T. gallinae infection and development of lesions than chicks raised in captivity. Discordances between samples cultured in TYM and samples subjected to PCR from oropharyngeal swabs were observed, swab-ITS-PCR being more sensitive.RESEARCH HIGHLIGHTS 45.5% of Bonelli's eagles in the nest carried T. gallinae and 39.4% showed lesions.PCR from oral swabs showed higher sensitivity than culture in TYM for detection of T. gallinae.MLS types ITS-A/18S-VI/Fe-A1 (and A2) are a risk factor for the development of lesions.

Oral trichomonosis: Description and severity of lesions in birds in Spain.

Avian trichomonosis is a parasitic disease caused by the flagellated protozoan Trichomonas gallinae. Columbiformes are the reservoir host of the parasite, with high levels of infection, but also other domestic and wild birds from a variety of orders are susceptible to the infection and development of gross lesions. In this paper we describe the type and severity of lesions in wild birds in four avian orders (Accipitriformes, Falconiformes, Strigiformes and Columbiformes). A total of 94 clinical cases diagnosed of trichomonosis were selected for the categorization of their lesions in the upper digestive tract. The affected birds were classified into three different categories (mild, moderate and severe) based on size (in relation to the tracheal opening), depth and location of the lesions. Mild cases are those with small and superficial lesions far from the oropharyngeal opening; moderate cases possess larger and deeper lesions, and severe cases very large and deep lesions that impede swallowing or affect the skull. Mild lesions were found in 10.6 % of cases; moderate lesions were observed in 18.1 % of the birds and severe lesions in 71.3 %. Treatment outcomes in birds with either mild or moderate lesions were favorable, while severe lesions were related to poor body score, leading to death or euthanasia in most cases. A relationship between severe lesions and avian order was found, with a higher percentage of birds with this type in Falconiformes, Columbiformes and Strigiformes. Multifocal lesions were more frequent in Columbiformes and Falconiformes. In Strigiformes, 93.3 % of birds showed lesions in the upper jaw. This study seeks to further understanding of avian trichomonosis and to provide information that will be useful to veterinarians and related professionals for assessment, prognosis and treatment choice for these birds.

Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur.

Genetic characterization of oropharyngeal trichomonad isolates from wild birds indicates that genotype is associated with host species, diet and presence of pathognomonic lesions.

Oropharyngeal trichomonad isolates of wild birds from Spain were studied. A total of 1688 samples (1214 of predator birds and 474 of prey species) from wildlife recovery centres and scientific bird-ringing campaigns were analysed from 2011 to 2013. The overall infection prevalence was 20.3% (11.4% in predator birds and 43.3% in prey species). Pathognomonic lesions were present in 26% of the infected birds (57.3% in predator birds and 4.9% in prey species). The most commonly parasitized species were the goshawk (Accipiter gentilis, 74.5%) and the rock pigeon (Columba livia, 79.4%). Host species in which the parasite has not been previously analysed by polymerase chain reaction and sequencing in Spain are also reported: Columba palumbus, Streptopelia turtur, Pica pica, A. 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. Sequence analysis of the ITS1/5.8S/ITS2 region revealed five different genotypes and also some mixed infections. A relationship between genotype and host species was observed, but only two genotypes (ITS-OBT-Tg-1and ITS-OBT-Tg-2) were widely distributed. Genotype ITS-OBT-Tg-1 was most frequently found in predator birds and statistically associated with pathognomonic lesions. Non-strict ornithophagous species were at higher risk to develop disease than ornithophagous ones. Genotypes ITS-OBT-Tcl-1 and ITS-OBT-Tcl-2 are new reports, and ITS-OBT-Tvl-5 is reported for the first time in Spain. They showed higher genetic homology to Trichomonas canistomae and Trichomonas vaginalis than to Trichomonas gallinae, indicating the possibility of new species within this genus.

High prevalence of Blastocystis sp. in pigs reared under intensive growing systems: frequency of ribotypes and associated risk factors.

Three hundred and ninety-five pig fecal samples were analyzed looking for Blastocystis sp. using optical microscopy and PCR. A global prevalence of 46.8% has been observed in this study, although relative values of prevalence differ notably according to the strata examined, ranging from 9.3% in sows to 75% in weaners. Statistic analysis of the data included several risk factors such as different management systems, date of sample collection, fecal consistency, age and sex of the animals. The presence of the parasite was statistically associated to the variables "age" and "date of sample collection", being more prevalent in weaners and grower pigs and warm season. Random fragment-length polymorphism (RFLP-PCR) analysis of positive PCR samples revealed a high homology in the digestion pattern, appearing as two ribotypes. The results were further confirmed by sequencing of ten randomly selected samples, showing that the samples obtained in this study were included in two genotypes: genotype I previously named by Noël et al. [Noël, C., Dufernez, F., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Ho, L.-Ch., Singh, M., Wintjens, R., Sogin, M.L., Capron, M., Pierce, R., Zenner, L., Viscogliosi, E., 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J. Clin. Microbiol. 43, 348-355], in which Blastocystis sp. sequences from humans, pigs and cattle were included, and genotype II, which only included Blastocystis hominis sequences obtained from human and other primates. This is the first report including Blastocystis sequences from swine origin in genotype II.