1. Investigación

Bonelli’s eagle (Aquila fasciata) is an endangered raptor species in Europe, and trichomonosis is one of the menaces affecting chicks at nest. In this paper, we attempt to describe the oral microbiome of Bonelli’s eagle nestlings and evaluate the influence of several factors, such as captivity breeding, Trichomonas gallinae infection, and the presence of lesions at the oropharynx. The core oral microbiome of Bonelli’s eagle is composed of Firmicutes, Bacteroidota, Fusobacteria and Proteobacteria as the most abundant phyla, and Megamonas and Bacteroides as the most abundant genera. None of the factors analysed showed a significant influence on alfa diversity, but beta diversity was affected for some of them. Captivity breeding exerted a high influence on the composition of the oral microbiome, with significant differences in the four most abundant phyla, with a relative increase of Proteobacteria and a decrease of the other three phyla in comparison with chicks bred at nest. Some genera were more abundant in captivity bred chicks, such as Escherichia-Shigella, Enterococcus, Lactobacillus, Corynebacterium, Clostridium and Staphylococcus, while Bacteroides, Oceanivirga, Peptostreptococcus, Gemella, Veillonella, Mycoplasma, Suttonella, Alloscardovia, Varibaculum and Campylobacter were more abundant in nest raised chicks. T. gallinae infection slightly influenced the composition of the microbiome, but chicks displaying trichomonosis lesions had a higher relative abundance of Bacteroides and Gemella, being the last one an opportunistic pathogen of abscess complications in humans. Raptor’s microbiomes are scarcely studied. This is the first study on the factors that influence the oral microbiome of Bonelli’s eagle.

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 multi-locus sequence typing approach, including the ITS1/5.8S/ITS2 region (ITS), ribosomal RNA small subunit (18S) and Fe-hydrogenase gene (FeHyd). Global prevalence for 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). MLST analysis showed five different MLS types, being ITS-A/18S-VI/FeHyd-A1 and ITS-D/18S-II/Fe-C4 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. Disconrdances between samples cultured in TYM and samples subjected to PCR from oropharyngeal swabs were observed, being swab-ITS-PCR more sensitive.

A juvenile Cinereous Vulture (Aegypius monachus) fledgling was found disorientated on the roof of a building in Madrid City, Spain, in October 2016. A veterinary examination revealed multiple plaques distributed throughout the oropharyngeal cavity. Lesions were located under the tongue and at the choanal slit, hard palate, and esophagus opening and ranged from 2 to 7 mm, coalescing in areas up to 2 cm, with a yellowish color of the surface. Motile trichomonad trophozoites were detected in fresh wet mount smears from the lesions. Sequence analysis of the internal transcribed spacer (ITS)1/5.8S/ITS2 and small subunit ribosomal RNA confirmed that Trichomonas gypaetinii was the etiologic agent. Microbiologic cultures did not reveal any pathogenic bacteria or fungi. The animal recovered successfully after treatment with metronidazole and trimethoprim-sulfamethoxazole and was later released in a suitable habitat. Avian trichomonosis lesions caused by T. gypaetinii have not been reported.

Extensive diversity and even new species have 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 (Columba livia) with clinical signs of avian trichomonosis, a juvenile wood pigeon (Columba palumbus) and an European turtle dove (Streptopelia turtur) without clinical signs, and a nestling of Eurasian eagle owl (Bubo bubo) with gross lesions. 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 differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed three novel sequence types: one for the SSUrRNA region and two for the Fe-hyd gene. Clones from the Eurasian eagle owl and the wood pigeon were identified as Trichomonas gallinae analysing each locus, with an ITS genotype of T. gallinae-1 and T. gallinae-2 groups, respectively, which are commonly reported in wild birds from Europe and America. Clones of trichomonads from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Tetratrichomonas canistomae than with T. gallinae strains at their ITS region, respectively. SSUrRNA sequences grouped clones in a T. gallinae, T. tenax and T. canistomae clade. Further diversity of T. gallinae was detected within the Fe-hyd locus. Morphometric comparison by optical microscopy with clonal cultures of T. gallinae (T. gallinae-1 and T. gallinae-2 ITS genotypes), revealed significant statistical differences on axostyle projection in the clone from European turtle dove, which was 1.6 μm longer (mean 8.2 μm) than the mean values reported for T. gallinae (6.4 μm). Possible new species within the Trichomonas genus were detected in isolates obtained from the racing pigeon and the European turtle dove, but further reports will be needed to confirm their host distribution.

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. A total of 94 clinical cases diagnosed of trichomonosis were selected for the categorization of their lesions at the upper digestive tract. The affected birds were classified into three different categories (mild, moderate and severe) based on the size, the depth and the location of the lesions. Mild grade is found in small and superficial lesions far from the oropharyngeal opening; moderate grade for bigger and deeper lesions, and severe grade for very big and deep lesions that impede swallowing or affect the skull. This revision of lesions will help to understand the pathologic and epidemiological information about avian trichomonosis. Furthermore, it will be helpful for the evaluation, prognosis and possible treatments among veterinarians and related professionals.

Oropharyngeal trichomonosis is a disease that affects wild and domestic birds. Some studies carried out in wildlife recovery centers pointed it out as the main cause of entrance of birds of prey due to infectious diseases. It causes small nodules and ulcers in the crop and other locations of the upper digestive system of the animals. These small lesions can coalesce and form large granulomas, which can provoke the death of the animal by starvation. Trichomonas gallinae is considered the etiological agent of the disease, a flagellated protozoon that is frequently found in the oral cavity of columbiformes, which are considered the main reservoir of the parasite. However, in the last decade, a great progress in the molecular characterization of this and other protozoa has been reached, and the number of genetic variants and even new species within the trichomonads that inhabit the avian oropharynx has expanded. In this review, we outline the latest descriptions of these parasites and their host spectrum; more than 10 genetic variants or new species are included. Although trichomonosis has been described in several groups of birds, the higher impact is usually found on Accipitriformes, Falconiformes and Strigiformes due to their predation or scavenging habits. Psittaciformes and Passeriformes also show clinical signs of the disease, and recently, several epidemic episodes of trichomonosis in fringilids were described across Europe and North America. In addition, chicks of endangered species like the Bonelli´s eagle (Aquila fasciata) are frequently affected by the parasite, as several studies carried out in Spain and nearby countries have proved. In this chapter, we summarize the most important features of the disease, including the biology, the diagnosis and treatment options. Additionally, we will describe the recent scientific advances in the pathology, epidemiology and control of the disease.