Pathogens

Mucormycosis is among the most lethal fungal infections in clinical medicine, with crude mortality rates between 50 and 90% that have improved little over decades of antifungal development. Alveolar macrophages constitute the primary cellular defense against inhaled Mucorales spores, yet quantitative comparative data on phagocytic outcomes across the taxonomic breadth of this fungal order are absent from the literature. We co-incubated 62 Mucorales strains drawn from nine phylogenetic groups and 12 countries with MH-S murine alveolar macrophages and with Dictyostelium discoideum amoeba, at multiplicities of infection (MOI) of 1 and 5 and at incubation times of 1.5 and 3 hours. Automated phagocytic index quantification was combined with spore morphometric measurements and geographic metadata. Macrophage phagocytic indices differed highly significantly among the nine phylogenetic groups across all experimental conditions (Kruskal-Wallis H = 394-780; p = 10^-163 to 10^-74). Group 1 (Absidia and Cunninghamella species) showed the lowest phagocytic index; Groups 5 (Rhizopus arrhizus) and 6 (Gilbertella persicaria and Mycotypha species) showed the highest. Larger spore area correlated negatively with macrophage phagocytic index. In the central comparative finding, Spearman rank correlations between macrophage and D. discoideum phagocytic indices were near zero across all four conditions (r = -0.07 to +0.11; all p > 0.8), demonstrating that protozoan phagocytosis does not predict the mammalian macrophage phagocytic hierarchy. Kinetic analysis further revealed, for the first time in any Mucorales or Mucormycotina species, evidence of vomocytosis in MH-S macrophages challenged with Group 8 (Mucor clade II) spores at MOI 1, and in D. discoideum across four groups at MOI 5 after 3 hours. These findings establish a quantitative, phylogenetically resolved framework for Mucorales innate immune recognition, demonstrate that the evolutionary training ground hypothesis does not hold at the order level for Mucorales, and identify vomocytosis as a previously unrecognised immune evasion mechanism in Mucormycotina.

Barmah Forest Virus (BFV), an arthropod-born virus transmitted by mosquitoes, is of significant public health concern in Australia and regions in the Pacific. Recent climate change and globalization raise the potential for BFV to extend its geographic distribution. Despite the rising importance of BFV, its vector dynamics remain poorly understood, particularly concerning the vector competence of different mosquito species. This study aims to investigate the vector competence of BFV across various mosquito species, beyond those endemic to the Australasian region, especially focusing on global relevant vector species. No transmission was observed for Culex quinquefasciatus and Cx. torrentium as well as Anopheles stephensi. In contrast, both investigated Aedes species, Ae. aegypti as well as Ae. albopictus, exhibited BFV-positive saliva across all four temperature profiles (18{degrees}C, 21{degrees}C, 24{degrees}C or 27{degrees}C) examined. These two invasive mosquito species must therefore be classified as potential vectors for BFV, indicating the potential risk of BFV transmission outside of Australia.

Japanese encephalitis virus (JEV) is an emerging mosquito-borne flavivirus with potential for geographic expansion, yet the risk of establishment in North America remains poorly characterized. We assessed vector competence of three North American Culex species (Cx. pipiens, Cx. quinquefasciatus, and Cx. tarsalis) for the JEV Nakayama strain, isolated from human brain in 1934 in Japan, across five constant temperatures (15, 20, 25, 30, and 33{degrees}C) at 4, 7, and 14 days post-feeding, quantifying infection, dissemination, and transmission rates. Vector competence was low but non-zero across all species. Cx. pipiens showed higher infection rates than the other species, whereas Cx. quinquefasciatus and Cx. tarsalis were minimally susceptible under these experimental conditions. Temperature had limited effects on infection and no detectable effects on dissemination or transmission. These findings suggest limited transmission potential of JEV Nakayama in North America, with Cx. pipiens as a relatively permissive vector.

Human noroviruses (HuNoVs) are the most common cause of non-bacterial foodborne illnesses worldwide. HuNoVs are notoriously difficult to culture, and current cell culture systems, such as human intestinal enteroids have limitations in efficiency and scalability, which has limited their applications in food safety studies. Recently, it has been shown that transformed salivary gland cell lines (including NS-SV-TT-DC) allow for replication and passaging of norovirus GII.4. Herein, we examined whether we can independently observe viral replication in salivary gland cells and if other strains of human norovirus can replicate in these cells. For this purpose, we first evaluated the replication of several norovirus strains in NS-SV-TT-DC cells by either infecting them with vesicle-cloaked viruses or total viruses in stool filtrates. Low levels of viral replication was observed and vesicle isolation did not improve viral replication. Next, we screened for replication of 50 norovirus samples from 9 genotypes, and 14 isolates demonstrated over a two-fold viral replication. We observed that the initial viral load in the samples does not have a significant effect on replication efficiency. We were also able to passage several isolates. Subsequently, we examined whether interferon inhibitors such as Ruxolitinib, TPCA-1, and BX795 would improve viral replication and observed negligible effects. Finally we tested multiple supplements including MgCl2, bile salt, ceramide, and TAK-779. Most supplements had little or no effect on HuNoV replication in NS-SV-TT-DC cells. Bile salt and TAK-779 showed limited, genotype-specific benefits. Altogether these results indicate that salivary gland cells allow limited replication of certain HuNoV isolates.

Traditional veterinary pathology investigations on wild animals generally conclude a primary cause of death based on a combination of observed clinical signs, gross and microscopic lesions, microbial culture and PCR. However, these methods may fail to capture the broader microbial community of the host. We employed metatranscriptomic sequencing to characterise the potential pathobiome of necropsied marine turtles, comparing these findings with corresponding veterinary pathology reports. Tissue samples were collected from 76 individuals spanning three species of marine turtles -- the green turtle (Chelonia mydas), the hawksbill turtle (Eretmochelys imbricata), and the loggerhead turtle (Caretta caretta) -- across the coastline of New South Wales, Australia. Reported causes of mortality ranged from infectious disease to trauma. We identified a diverse range of parasites within the genera Neospirochis, Hapalotrema, Paraorgymatobothrium, Eimeria and Callitetrarhynchus, and bacteria including the genera Aeromonas, Clostridium, and Vibrio. Novel viruses of the families Astroviridae, Caliciviridae, Hepaciviridae, Hepeviridae, Nyamivirdae, Orthomyxoviridae, Picobirnaviridae and Rhabdoviridae were also identified. In cases where infectious disease was noted in the pathology report, we frequently observed high pathogen read counts consistent with the listed primary diagnoses. However, we also detected a range of incidental infections involving eukaryotic parasites, bacteria, and viruses. The diversity of infectious agents detected in marine turtles suggests that the observed disease outcomes are unlikely to be driven by a single causative agent, but rather by complex interactions between host health, the pathobiome, and environmental stressors. We discuss the potential implications for turtle health and conservation.

Japanese encephalitis virus is a leading cause of encephalitis in humans and agricultural animals across Southern and Eastern Asia. It is primarily transmitted by mosquitoes of the Culex genus, notably Culex tritaeniorhynchus. In the Philippines, JEV is reported to account for 7% to 18% of clinical cases of meningitis and encephalitis. Therefore, updated and comprehensive data on circulating JEV strains in the country are crucial for developing effective mitigation strategies. This study aimed to optimize an assay for JEV detection using one-step reverse transcription polymerase chain reaction (RT-PCR). Additionally, the presence of JEV in mosquitoes collected from eleven cities and municipalities in Laguna was assessed, and the mosquito carriers were identified based on morphological characteristics. Results demonstrated that the optimized one-step RT-PCR assay successfully detected JEV at various dilutions, with a limit of detection (LoD) of 0.115 ng/L. The assay exhibited 100% specificity, with no amplification observed for any viruses other than JEV. Sequence analysis of the positive control, JEV SA14-14-2 RNA, revealed 96.96% to 98.41% similarity to JEV reference sequences in the NCBI database. Among the 5,522 female mosquitoes collected, Culex tritaeniorhynchus and Culex gelidus were the most prevalent species. The highest mosquito species diversity and the most even distribution were found in the three barangays of Paete, but Siniloan, Laguna had the highest vector density for the three Culex species, Culex tritaeniorhynchus, Culex gelidus, and Culex fuscocephala. RT-PCR analysis of 189 pooled mosquito samples revealed no detectable JEV genetic material. This study is likely the first investigation of JEV presence in mosquito samples from Laguna, Philippines. These findings contribute to proactive JEV surveillance, enhancing our understanding of the virus's presence and distribution. The results underscore the importance of targeted prevention and control measures to mitigate JEV transmission in high-risk areas.

The lack of effective anti-JEV therapy possesses significant challenge to control JEV. {beta}-catenin, a key mediator of Wnt signaling pathway regulates different viral replication and host immune responses. However, its role in JEV infection remains to be elucidated. Thus, the current study focused on evaluating iCRT-14, a specific {beta}-catenin inhibitor, against JEV. Treatment with iCRT-14 following JEV infection resulted efficient reduction in viral progeny release, viral RNA and protein levels in Huh7 and HEK293T cells. Further, active and total {beta}-catenin, Cyclin D-1 and GSK3-{beta}, the other key pathway players were also modulated in infected and inhibitor treated cells. Moreover, iCRT-14 showed an IC of 4.56 in Huh7 cell and maximal inhibition at the early stages of the JEV life cycle. Interestingly, the overexpression of {beta}-catenin in both the cells and siRNA-mediated {beta}-catenin knockdown (in Huh7 cells) significantly abrogated JEV replication, as evidenced by decreased viral titers, viral protein expression, and viral as well as total RNA levels. Moreover, the reduction in extracellular (84%) and intracellular (60%) viral titers following iCRT-14 treatment highlights its role in impairing JEV infection. Further, in silico molecular docking and co-immunoprecipitation studies demonstrated interactions between {beta}-catenin and the JEV NS5 and E proteins. Collectively, these findings suggest that optimum level of {beta}-catenin is required for efficient JEV infection, highlighting its potential as a target for designing host-directed control strategies to regulate viral infection.

H5N1 clade 2.3.4.4b avian influenza A viruses pose a significant threat to wild animal populations, domesticated animals, and potentially, the human population. For H5N1s to infect and transmit among mammalian species, mutations for improved utilization of mammalian receptors and enhanced replication at the lower temperatures of the upper respiratory tract need to be acquired. A human H1N1pdm09-like virus was compared to H5N1 genotypes B3.13 and D1.1 for replication at 33{o}C, 37{o}C, and 39{o}C - temperatures consistent with the upper and lower respiratory tract in humans, and dairy cow udder tissue. All H5N1 viruses had increased plaque sizes on MDCK cells at 37{o}C and 39{o}C compared to H1N1pdm09. In primary, differentiated human nasal and bronchial epithelial cultures, all H5N1 viruses show restricted infectious virus production compared to H1N1 at 33{o}C. While H5N1 D1.1 also showed restricted replication at 37{o}C and 39{o}C, the H5N1 B3.13 replicated to nearly equivalent titers as H1N1pdm09. All H5N1 viruses demonstrated similar cell tropism in cells from the upper and lower respiratory tract, infecting more ciliated than non-ciliated cells relative to H1N1pdm09. H1N1, H5N1 B3.13 D1.1 infection induced similar innate immune factors, with nasal epithelial cells producing higher levels compared to bronchial epithelial cells. These data suggest that genotype B3.13 and D1.1 H5N1 viruses show different temperature dependent replication patterns compared to H1N1pdm09.

ABSTRACT Background Cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) may involve parasite dissemination from the primary skin lesion to clinically healthy mucosal sites, a process potentially relevant to the development of mucosal leishmaniasis. Parasite burden and Leishmania RNA Virus 1 (LRV1) have been proposed as factors influencing parasite persistence, dissemination, and treatment response. We investigated whether parasite load and LRV1 detection in skin lesions were associated with Leishmania in clinically healthy nasal mucosa and, in a subset of patients, with therapeutic outcome. We also assessed the relationship between LRV1 and parasite load. Methodology/Principal findings We conducted a prospective observational cohort study. Parasite load and LRV1 were assessed by qPCR in skin lesions and clinically healthy nasal mucosa from patients with localized cutaneous leishmaniasis in Rondonia, Brazilian Amazon. Samples were evaluated before treatment (D0), at the end of treatment (D20), and during follow-up (D90-180). Among 178 patients screened, CL was confirmed in 113. LRV1 was detected in skin lesions in 34.51% of patients and in nasal mucosa in 24.13%. Leishmania DNA was detected in clinically healthy nasal mucosa in 12.38% of patients. LRV1 detection in the skin lesion was associated with detection of Leishmania in the nasal mucosa. Higher parasite loads were observed in more recent lesions. Among patients evaluated for therapeutic outcome, higher parasite load before treatment was associated with treatment failure and was the factor most strongly associated with an unfavorable response in multivariate analysis. Conclusions/Significance LRV1 detection in cutaneous lesions was associated with Leishmania in clinically healthy nasal mucosa, although the significance of this association and its relationship with parasite dissemination require further investigation. In contrast, parasite burden was associated with therapeutic outcome, with higher pre-treatment loads observed in patients who experienced treatment failure. The absence of an association between LRV1 and parasite load suggests that these two parameters may provide distinct information regarding parasite detection at mucosal sites and therapeutic response.

Ticks are obligate hematophagous arthropods and feed exclusively on blood. As blood is nutrient-poor, ticks rely on bacterial endosymbionts to synthesise nutrients, yet the diversity and functional roles of these symbionts in Australian ticks remain largely uncharacterised. This is critical to address as these ticks are of high medical importance in Australia. In this study, shotgun metagenomic sequencing was performed on Bothriocroton concolor, Bothriocroton hydrosauri, Haemaphysalis longicornis and Ixodes holocyclus, enabling the recovery of six complete or partial metagenome-assembled genomes (MAGs). These comprised Coxiella-like endosymbionts (CLE), a facultative Rickettsia symbiont, and two Midichloria mitochondrii strains (Ixholo1 and Ixholo2). Functional annotation of these taxon-specific symbionts revealed the absence of virulence factors and the presence of B-vitamin and/or heme biosynthesis genes, indicative of nutritional mutualism, which is essential for tick hematophagy. The CLEs additionally harbour genes of the shikimate pathway, which modulate blood feeding in ticks by regulating serotonin biosynthesis. Furthermore, functional annotation and pangenomic analysis of Midichloria spp. found evidence that the genus may encompass multiple species, as well as the retention of genes potentially associated with an intramitochondrial lifestyle in M. mitochondrii Ixholo2. Tick microbiomes are dominated by non-pathogenic microorganisms, which are often overshadowed by pathogens. These include the endosymbionts, which can influence host biology and pathogen transmission, and are fundamental for the development of diagnostic tools and taxon-specific tick biocontrols.

Viruses have diverse life history strategies driven by variation in traits such as infectivity, transmission mode, and length and severity of infection that affect their epidemiology and evolution. While well documented among different species, life history and phenotypic variation among variants of the same virus species are less well understood. Foot-and-mouth-disease-virus (FMDV) is an ungulate-infecting picornavirus endemic to many regions, including Sub-Saharan Africa, where it circulates between wildlife and livestock in several serotypes. Recent work suggested that FMDV variants from the three Southern-African Territories serotypes exhibit different life history strategies, with these dynamics potentially causing distinct signatures in viral evolutionary rate, transmission among host species, and movement among regions. To investigate whether any effects of predicted effects occurred in natural settings, and whether these differences were shared with other strains within each serotype, this study used 716 published FMDV sequences (approximately 430bp) from 3 serotypes (SAT1, SAT2, and SAT3) to measure and compare evolutionary rates and transmission between regions and host types in Southern Africa. SAT1 had a slower rate of evolution consistent with a predicted more chronic infection strategy, and SAT2 had higher variability in evolutionary rates and some evidence of transmission from livestock to wildlife, suggesting livestock may play a part in persistence. SAT3 showed an expected intermediate phenotype but was challenging to validate due to small sample size. All SATs showed similar levels of transmission between regions. These results suggest that SAT1, SAT2, and SAT3 exhibit different transmission dynamics and evolutionary signatures, consistent with different life history strategies observed in their representative strains, such as more latency or a multi-host maintenance community.

Dengue virus (DENV) infection causes an acute febrile illness that is usually self-limiting but can become fatal. Thrombocytopenia, or a decrease in circulating platelets, is a major clinical feature of dengue and is strongly associated with the progression to dengue hemorrhagic fever and dengue shock syndrome. In this report, MYH9, a cytoskeletal protein involved in platelets maturation and release, was found to be down-regulated in DENV-infected Huh7 cell lines. The expression of RUNX1, an important transcription factor involved in megakaryopoiesis, was also decreased following DENV-infection. In-silico analysis identified three potential RUNX1 transcription-factor binding site in the promoter-region of MYH9 gene. We therefore investigated whether RUNX1 regulates MYH9 expression during DENV-infection. Luciferase-assay and ChIP assays showed that DENV-infection affect the binding of RUNX1 to the MYH9 promoter, providing insight into the molecular basis underlying the reduced expression of MYH9 expression. Furthermore, the megakaryocyte differentiation markers GATA1, ITGB3, and MYH9 were upregulated in PMA-induced K562-cells. However, DENV infection downregulated these genes and increased the expression of apoptosis marker gene CYCS. These findings suggest that the dengue affects megakaryopoiesis through multiple mechanisms, potentially reducing platelets production while also promoting platelets destruction. Additionally, Fluorescence and electron microscopy revealed that functional defects of MYH9 may affects megakaryopoiesis biogenesis. Overall, this study provides insights into the molecular mechanisms contributing to thrombocytopenia during DENV infection.

High pathogenicity avian influenza (HPAI) H5 clade 2.3.4.4b is the main driver of the ongoing unprecedented global panzootic. The recently emerged HPAI H5N1 clade 2.3.4.4b genotype EA-2024-DI.2.1 has become predominant in Europe, with migratory wild birds, particularly waterfowl, playing a major role in its dissemination. Egypt lies along major Afro-Eurasian migratory flyways, which have historically played an important role in the introduction of emerging H5Nx viruses into the country. In this study, targeted surveillance was conducted on 416 wild birds offered for sale in in live bird markets (LBMs) and roadside trading points in northern Egypt, mainly in Damietta and Port Said. Of these, 118 birds showing mild clinical signs were examined post-mortem and lung and tracheal tissues were collected, while oropharyngeal and cloacal swabs were collected from apparently healthy birds. Avian influenza virus was detected by RT-qPCR in 22 wild birds, all from tissue samples, whereas all swabs from apparently healthy birds were negative. Waterfowl accounted for 16 of the 22 positive birds (72.7%), with Eurasian teal showing the lowest Ct values (21-25). Phylogenetic and whole-genome analyses showed that the sequenced wild-bird viruses clustered within the recently emerged EA-2024-DI.2.1 sub-lineage and were closely related to contemporary European viruses. Compared with the EA-2021-AB genotype currently circulating in Egyptian poultry, the EA-2024-DI.2.1 viruses showed several HA amino acid differences, including A83D, L104M and T195A. These findings provide evidence for the introduction of EA-2024-DI.2.1 into Egypt through migratory wild birds and highlight the importance of continued genomic surveillance at the wild bird domestic poultry interface and antigenic evaluation against vaccines currently used in Egypt.

Leptospirosis is an infectious neglected zoonotic disease caused by pathogenic bacteria of the genus Leptospira. The genus comprises 43 pathogenic species, divided into two clades (P1 and P2), with the potential to cause disease on animals and humans. Despite the major impact of this disease on animal and human health, few quantitative real-time polymerase chain reaction (qPCR) assays have been validated to specifically detect all pathogenic Leptospira species, thwarting diagnosis and epidemiological studies. The gene encoding LipL32, the major leptospiral outer membrane protein, discriminates pathogenic P1 species from P2 and saprophytic. However, with the recent discovery of new species, the current lipL32-based qPCR assay cannot detect all classified P1 species. Furthermore, there are no currently validated molecular methods able to differentiate the presence of P1 and P2 species on clinical samples. Previous analyses have shown that the 23S ribosomal RNA gene displays considerable conservation in P1 and P2 species but sequence divergence in saprophytic species, a promising target for PCR-based detection and discrimination of those two clades. This study optimized and validated an improved lipL32- and 23S-based TaqMan qPCR assay using human and animal clinical samples. These newly optimized and developed assays resulted in a lower limit of detection and increased diagnostic sensitivity, resulting in the detection of all pathogenic species of the genus Leptospira currently described. These assays will improve the detection of leptospires from clinical and environmental samples, providing a valuable epidemiological and clinical tool to support One Health research on this important emerging disease.

Tick-borne encephalitis virus (TBEV) is a zoonotic flavivirus maintained in complex enzootic cycles involving ticks and vertebrate hosts. While vector-mediated transmission has been extensively studied, host-associated mechanisms, including vertical transmission and prolonged viral carriage in natural reservoir populations, remain poorly understood. Here, we investigated TBEV circulation, genetic diversity, and evidence for vertical transmission in bank voles (Clethrionomys glareolus) from a highly endemic region of North-eastern Poland. A total of 258 wild rodents were screened using molecular and serological approaches. TBEV RNA was detected in 14.3% of individuals, whereas seroprevalence was substantially lower (6.6%), revealing limited concordance between viral presence and humoral response under natural conditions. Near-complete genome sequences were obtained from 10 TBEV-positive individuals, representing the first near-complete TBEV genomes generated from wild rodents in Poland. All isolates belonged to the European subtype (TBEV-Eu), and phylogenetic analysis revealed two genetically distinct viral clades co-circulating within a narrow spatiotemporal window. Importantly, TBEV RNA and/or envelope protein were detected in embryos from naturally infected females, providing evidence consistent with vertical transmission in a wild reservoir host. Together, these findings suggest that vertical transmission and complex host infection dynamics may be underappreciated components of TBEV maintenance in natural reservoir populations. Our results highlight the need to integrate vertebrate host dynamics into models of TBEV ecology and support expanded wildlife-based surveillance to better understand and predict zoonotic risk.

Background: Klebsiella pneumoniae is a key causative agent of multidrug resistant nosocomial infections. Bacteriophages offer a promising alternative treatment, but the diversity of Klebsiella species requires the characterisation of a broad range of phages, particularly the poorly characterised jumbo phages. Materials and Methods: Phage BMCPR_005026 was isolated from sewage against Klebsiella pneumoniae. Whole genome sequencing, transmission electron microscopy and host range analysis against a diverse set of 40 clinically relevant Klebsiella strains were used to characterise the phage. Results: The virion had a myovirus morphology and genomic analysis placed it within an undefined family in Caudoviricetes where it forms a new species within the genus Alcyoneusvirus. BMCPR_005026 could form plaques on 3 of the 40 Klebsiella strains tested (7.5%) and formed halos. Conclusion: BMCPR_005026 represents a new species of phage within the genus Alcyoneusvirus with activity against 3 Klebsiella KL-types.

Sicyoidochytrium minutum DNA virus (SmDNAV) was isolated several years ago from a protist host of family Thraustochytriaceae of the class Labyrinthulomycetes. This virus shared little similarity to other viruses in gene content and protein sequences, albeit seemingly belonging to the phylum Nucleocytoviricota. By extensive searches in genomic and metagenomic sequence databases, we identified numerous long contigs related to the SmDNAV genome and analyzed proteins shared by these putative viruses. Phylogenetic analyses place these viruses within the class Megaviricetes, outside of all established orders, and as a sister group to the clade combining families Mamonoviridae and Manesviridae. Homologs of SmDNAV proteins were found in association (either integrated or co-sequenced) with other Labyrinthulomycetes and Rhodophyta protists from diverse marine and freshwater environments. Consequently, we propose SmDNAV as the prototype member of a new order, provisionally named Ariadnavirales, within class Megaviricetes, phylum Nucleocytoviricota. Members of Ariadnavirales have lost most of the genes encoding components of the replication and transcription systems that are otherwise conserved in nucleocytoviricots, suggestive of transition to genome replication and expression dependent on the host nucleus.

Parasitic infections pose multifaceted threats to farmed fish, extending beyond direct pathogenicity to facilitate infections of bacteria, viruses, and microparasites. This synergistic interaction often leads to co-infections that significantly exacerbate disease outbreaks and mortality, presenting a severe challenge to aquaculture sustainability. Recently, a novel trypanosomiasis caused by the Trypanosoma carassii spectrum has emerged in cage-cultured Larimichthys crocea along the southeast coast of China, resulting in widespread prevalence and high mortality rates. Although this pathogen is hypothesized to originate from freshwater fish, its transmission route in marine environments has remained elusive. In this study, we investigated potential vectors and intermediate hosts of T. carassii spectrum, including leeches and monogenean in natural marine settings, and simulated transmission pathways using an established laboratory model involving T. carassii spectrum, Micropterus salmoides and the leech Poecilobdella manillensis. First, our field surveys in the coast of Ningde, Fujian Province, revealed a nearly 100% co-infection rate of T. carassii spectrum and the monogenean Neobenedenia girellae in diseased juvenile L. crocea. PCR analysis detected T. carassii spectrum traces in some N. girellae specimens, and subsequent experiments confirmed that N. girellae ingests the trypanosome while feeding on host blood. Furthermore, bacterial co-pathogens, such as Vibrio harveyi, were also detected within N. girellae. We also document two fatal leech infestations: Zeylanicobdella arugamensis in hybrid groupers (Epinephelus moara [female] X Epinephelus lanceolatus [male]) in Zhangpu, and Limnotrachelobdella okae in E. lanceolatus and E. fuscoguttatus in Raoping. These leeches tested negative for trypanosomes but carried pathogenic bacteria that co-infected the host fish; nonetheless, they are established vectors for trypanosome transmission. In a laboratory cohabitation model simulating T. carassii spectrum transmission, infected M. salmoides were housed with healthy conspecifics under three conditions: Group A (with the leech P. manillensis), Group B (no leeches), and Group C (no leeches, with physical separation between infected and healthy fish). After 14 days, blood smear microscopy and PCR analysis revealed infection rates in healthy fish of 58.33% in Group A, 40.00% in Group B, and 0% in Group C. Conclusively, T. carassii spectrum can be transmitted via leeches (with higher efficiency) and may also spread through direct contact under high-density aquaculture conditions, whereas N. girellae may act as an incidental vector, further research is warranted to clarify transmission dynamics in natural marine ecosystems. Additionally, our findings highlight the role of ectoparasites, including N. girellae and leeches, as potential reservoirs and vectors for bacterial pathogens of fish. In high-density intensive aquaculture, this vectorial capacity transforms parasites from primary pathogens into key drivers of polymicrobial disease outbreaks.

Tick-borne disease risk reflects the combined influence of vector abundance, pathogen prevalence, human exposure, and seasonal activity, yet these components are rarely integrated at the county level. We developed species-specific ecological risk scores for three medically important tick species (Acari: Ixodidae): Amblyomma americanum (L.), Dermacentor variabilis (Say), and Ixodes scapularis Say, across all 102 Illinois counties using data from 2019 to 2022. County-level predicted tick abundance and pathogen prevalence were scaled and combined using equal weights to generate primary vector-level risk scores. Robustness to the equal-weight assumption was evaluated by systematically varying the relative weights assigned to abundance and pathogen prevalence, while a separate sensitivity analysis incorporated reported tick-bite incidence where available. Associations with human disease were evaluated using negative binomial regression of tick-borne disease case counts on the corresponding risk scores, with county population included as an offset. Monthly phenology indicators were constructed by integrating human disease cases, reported tick bites, and sampled tick abundance and were evaluated in relation to temperature and precipitation. Seasonal risk was estimated by combining county-level vector risk with species-specific monthly phenology, generating monthly risk surfaces that were deployed through an interactive R Shiny application. Higher risk scores were significantly associated with greater disease case rates for each tick species. County rankings were highly stable under moderate changes in abundance and prevalence weighting. Seasonal activity differed among species, with A. americanum and D. variabilis showing concentrated warm-season risk and I. scapularis exhibiting a broader seasonal profile. These county-level seasonal risk maps identify where and when relative tick-borne disease risk is elevated in Illinois, supporting targeted prevention, surveillance, and clinical awareness.

Understanding the vertebrate hosts exploited by Culicoides biting midges is fundamental to assessing the transmission risk of livestock arboviruses. Blood meal analysis provides direct evidence of host-vector interactions under natural conditions; however, molecular data on Culicoides host feeding are lacking in Ireland. This study aimed to identify the vertebrate blood meal hosts of Culicoides species of veterinary importance and establish host-vector associations relevant to arbovirus transmission. A total of 115 morphologically blood-fed Culicoides females collected from 13 sites across Ireland were analysed using a sequential molecular workflow. Species-specific real-time PCR assays were used to screen for vertebrate hosts, with positive detections confirmed by mitochondrial DNA sequencing. Blood-fed Culicoides were subsequently identified to species level using DNA barcoding, enabling direct linkage between vector species and their vertebrate hosts. Vertebrate DNA was detected in 50 specimens, with cattle and horses accounting for the majority of identified blood meals. Culicoides obsoletus accounted for most cattle (n = 12) and horse (n = 11) blood meals. Culicoides scoticus fed on cattle (n = 6), horses (n = 4) and sheep (n = 1), while C. dewulfi and C. chiopterus fed predominantly on cattle, with occasional horse blood meals. Four cattle blood meals were identified from C. punctatus. No cervid-derived blood meals were detected. This study provides the first molecular characterisation of Culicoides blood meal hosts in Ireland, confirming host-vector associations between major vector species and livestock hosts of veterinary importance. Although the number of successfully characterised blood meals was modest, reflecting the inherent difficulty of recovering recently blood-fed Culicoides from field collections, the findings establish an important baseline for Ireland. The sequential molecular workflow provides a practical framework for future blood meal investigations and will support improved understanding of host-vector interactions relevant to arbovirus surveillance and risk assessment.