Captive Breeding and Trichomonas gallinae Alter the Oral Microbiome of Bonelli’s Eagle Chicks

**Background:** Bonelli's eagle (Aquila fasciata) is an endangered raptor in Europe, with approximately 920–1,100 couples remaining and 65% of the European population in Spain. Oropharyngeal trichomonosis caused by Trichomonas gallinae is a major cause of chick mortality, accounting for up to 22% of mortality causes depending on the year, with up to 87.5% of broods showing oropharyngeal lesions in some years. The oral microbiome plays important roles in health and disease resistance, but factors influencing the microbiome of wild raptors remain poorly understood. This study aimed to describe the oral microbiome composition of Bonelli's eagle chicks and assess the influence of captivity breeding, T. gallinae infection, and oropharyngeal lesions.

**Methods:** Oropharyngeal swabs were collected from 83 chicks (27 captive-bred, 56 wild nestlings) aged 30–47 days during April and May across multiple Spanish provinces, covering 66.3% of breeding pairs. DNA was extracted using the DNeasy Blood and Tissue Extraction Kit. T. gallinae was identified by PCR amplification of the ITS region using primers TFR1 and TFR2, followed by sequencing. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced using Illumina MiSeq paired-end protocol. Bioinformatic analysis used QIIME 2 and R, with taxonomy assigned against the SILVA 138 database. Alpha diversity was measured using the Shannon index with Wilcoxon rank tests; beta diversity was assessed via PCoA with Bray-Curtis and Binary-Jaccard indexes using PERMANOVA with 999 permutations.

**Key Results:** T. gallinae was present in 37 of 83 samples: 7/27 captive chicks (25.9% prevalence) and 30/56 wild nestlings (53.6% prevalence by chicks; 21/38 nests infected, 55.3% prevalence by nests). Sequencing revealed 100% identity with genotypes EU881911 and EU881912. Mild oropharyngeal lesions were present in 1/27 captive chicks (3.7%), while 18/56 wild nestlings (32.1%) showed lesions (15 mild, 3 severe). The analysis yielded 4,107,022 high-quality reads corresponding to 2,081 ASVs across 22 phyla and 221 genera. Dominant phyla were Firmicutes, Proteobacteria, Bacteroidota, Fusobacteriota, and Actinobacteriota. Alpha diversity did not differ between captive (Shannon 2.98 [2.80–3.31]) and wild chicks (2.92 [2.67–3.31]), but beta diversity showed strong differences (Bray-Curtis p<0.001; Binary-Jaccard p<0.001). Firmicutes was more abundant in wild chicks (p=0.037), while Proteobacteria was higher in captive chicks (p<0.001). Bacteroidota and Fusobacteria were higher in wild chicks (p<0.001 and p=0.002, respectively). At genus level, Megamonas dominated wild chicks (40.62% [23.24–54.51]) versus captive chicks (2.04% [0.92–13.92]). Escherichia-Shigella, Enterococcus, Lactobacillus, Corynebacterium, Clostridium, and Staphylococcus were more abundant in captive chicks. T. gallinae infection did not significantly alter alpha or beta diversity in wild chicks. In captive chicks, infected birds showed higher Staphylococcus (p=0.033) and Enterococcus (p=0.015). Chicks with oropharyngeal lesions showed higher Bacteroides (p=0.037) and Gemella (p=0.011). In severely affected birds (n=3), Gemella abundance was 7.15 (5.44–8.97) versus 1.12 (0.43–3.11) in mild/no lesion birds (p=0.026), and Ornithobacterium was 1.49 (0.98–3.03) versus 0.21 (0.06–0.51) (p=0.026).

**Clinical Implications:** Captivity breeding is the strongest factor shaping the oropharyngeal microbiome of Bonelli's eagle chicks, leading to reduced bacterial diversity and altered community structure compared to wild nestlings. The increased abundance of potentially pathogenic genera (Enterococcus, Staphylococcus, Escherichia-Shigella) in captive chicks may reflect dietary homogeneity and human influence. The association between Gemella abundance and severe T. gallinae-related lesions is notable, as Gemella is an opportunistic pathogen linked to abscesses and inflammation in humans. The seven-fold higher abundance of Gemella in severely affected birds suggests a potential synergistic interaction between the parasite and this bacterium in lesion pathogenesis. These findings have direct implications for conservation breeding programs, indicating that captive conditions alter the oral microbiome in ways that may affect disease susceptibility. The study is limited by the small number of severely affected birds (n=3) and the cross-sectional design, which cannot establish causality. Future studies comparing healthy and diseased Accipitriformes are needed to confirm these findings.