Rodent and human malaria parasites cause dysbiosis in the host gut microbiome, but whether Plasmodium species affecting birds cause dysbiosis in their hosts is currently unknown. Here we used a model of avian malaria infection to test whether parasite infection modulates the bird microbiome. To this aim, bird fecal microbiomes were characterized at different time points after infection of canaries with the avian malaria parasite Plasmodium homocircumflexum. Avian malaria caused no significant changes in the alpha and beta diversity of the microbiome in infected birds. In contrast, we discovered changes in the composition and abundance of several taxa. Co-occurrence networks were used to characterize the assembly of the microbiome and trajectories of microbiome structural states progression were found to be different between infected and uninfected birds. Prediction of functional profiles in bacterial communities using PICRUSt2 showed infection by P. homocircumflexum to be associated with the presence of specific degradation and biosynthesis metabolic pathways, which were not found in healthy birds. Some of the metabolic pathways with decreased abundance in the infected group had significant increase in the later stage of infection. The results showed that avian malaria parasites affect bacterial community assembly in the host gut microbiome. Microbiome modulation by malaria parasites could have deleterious consequences for the host bird. Knowing the intricacies of bird-malaria-microbiota interactions may prove helpful in determining key microbial players and informing interventions to improve animal health.

Upon ingestion from an infected host, tick-borne pathogens (TBPs) have to overcome colonization resistance, a defense mechanism by which tick microbiota prevent microbial invasions. Previous studies have shown that the pathogen Anaplasma phagocytophilum alters the microbiota composition of the nymphs of Ixodes scapularis, but its impact on tick colonization resistance remains unclear. We analyzed tick microbiome genetic data using published Illumina 16S rRNA sequences, assessing microbial diversity within ticks (alpha diversity) through species richness, evenness, and phylogenetic diversity. We compared microbial communities in ticks with and without infection with A. phagocytophilum (beta diversity) using the Bray-Curtis index. We also built co-occurrence networks and used node manipulation to study the impact of A. phagocytophilum on microbial assembly and network robustness, crucial for colonization resistance. We examined network robustness by altering its connectivity, observing changes in the largest connected component (LCC) and the average path length (APL). Our findings revealed that infection with A. phagocytophilum does not significantly alter the overall microbial diversity in ticks. Despite a decrease in the number of nodes and connections within the microbial networks of infected ticks, certain core microbes remained consistently interconnected, suggesting a functional role. The network of infected ticks showed a heightened vulnerability to node removal, with smaller LCC and longer APL, indicating reduced resilience compared to the network of uninfected ticks. Interestingly, adding nodes to the network of infected ticks led to an increase in LCC and a decrease in APL, suggesting a recovery in network robustness, a trend not observed in networks of uninfected ticks. This improvement in network robustness upon node addition hints that infection with A. phagocytophilum might lower ticksʼ resistance to colonization, potentially facilitating further microbial invasions. We conclude that the compromised colonization resistance observed in tick microbiota following infection with A. phagocytophilum may facilitate co-infection in natural tick populations.

Avian malaria infection has been known to affect host microbiota, but the impact of Plasmodium infection on the colonization resistance in bird gut microbiota remains unexplored. This study investigated the dynamics of Plasmodium relictum infection in canaries, aiming to explore the hypothesis that microbiota modulation by P. relictum would reduce colonization resistance. Canaries were infected with P. relictum, while a control group was maintained. The results revealed the presence of P. relictum in the blood of all infected canaries. Analysis of the host microbiota showed no significant differences in alpha diversity metrics between infected and control groups. However, significant differences in beta diversity indicated alterations in the microbial taxa composition of infected birds. Differential abundance analysis identified specific taxa with varying prevalence between infected and control groups at different time points. Network analysis demonstrated a decrease in correlations and revealed that P. relictum infection compromised the bird microbiota’s ability to resist the removal of taxa but did not affect network robustness with the addition of new nodes. These findings suggest that P. relictum infection reduces gut microbiota stability and has an impact on colonization resistance. Understanding these interactions is crucial for developing strategies to enhance colonization resistance and maintain host health in the face of parasitic infections.

Eurasian griffon vultures (Gyps fulvus) are endangered scavenger birds. Haemosporidian parasites infect the blood cells and organs of many avian species globally, using blood-sucking insect vectors, and they negatively affect health of birds and subsequently wildlife biodiversity. Fifty-nine vultures were admitted to the Greek wildlife rehabilitation center “ANIMA” and included in this study. Blood samples were collected, and the microscopy of stained blood smears was performed. Moreover, DNA was extracted, samples were screened for Leucocytozoon, Haemoproteus, and Plasmodium spp. following nested PCR protocols, and positive samples were sequenced. The detected haemosporidians are reported for the first time in Eurasian griffon vultures globally. The overall prevalence was 11.9% (Leucocytozoon spp. 5.1%, Haemoproteus spp. 5.1%, Plasmodium spp. 3.4%); this further corroborates the statement that the vultures’ immunity protects them from blood parasites. Notably, new genetic lineages of Leucocytozoon (GYPFUL02), Haemoproteus (GYPFUL01), and Plasmodium (GYPFUL03) species were recorded for the first time. Furthermore, it was the first molecular isolation of Haemoproteus in Old World vultures and Haemoproteus brachiatus was isolated for the first time from a vulture species. Moreover, this demonstrates the first record of Parahaemoproteus genetic lineages in vultures. Results highlight the genetic diversity of haemosporidians in griffon vultures and the need for additional studies.

Generalist parasites must adapt to diverse host environments to ensure their survival and transmission. These adaptations can involve fixed genetic responses, transcriptional plasticity, or epigenetic mechanisms. The avian malaria parasite Plasmodium homocircumflexum offers an ideal model for studying transcriptional variation across hosts. We experimentally inoculated P. homocircumflexum into different bird species, bypassing the vector, to assess whether gene expression remains stable across hosts, resets in response to new environments, or reflects epigenetic inheritance. We tested two alternative hypotheses: (i) universal gene expression profile (“one key fits all”), where parasite expression remains consistent across hosts. Our outcomes revealed that gene expression differed significantly depending on the host species and time postinfection, rejecting this hypothesis. (ii) Transcriptional plasticity, where gene expression is determined by the recipient host. Contrary to this hypothesis, we observed that gene expression was primarily influenced by the donor at 8 d postinfection (dpi), whereas gene expression was more aligned with the recipient host at 16 dpi. We also explored two mechanisms to explain these patterns: (i) epigenetic inheritance, whereby early transcription reflects the donor environment but adjusts over time, and (ii) genetic differentiation selecting for specific haplotypes. Our data support mechanism (i): 2,647 differentially expressed genes (DEGs) were associated with the donor at 8 dpi, while only 271 DEGs were linked to the recipient at 16 dpi. Single Nucleotide Polymorphism analyses revealed low genetic differentiation, rejecting mechanism (ii). These findings suggest that P. homocircumflexum undergoes a shift from donor-dependent to recipient-dependent gene expression, likely driven by epigenetic regulation and transcriptional plasticity.