EPINEST, an agent-based model to simulate epidemic dynamics in large-scale poultry production and distribution networks.

The rapid intensification of poultry production raises important concerns about the associated risks of zoonotic infections. Here, we introduce EPINEST (EPIdemic NEtwork Simulation in poultry Transportation systems): an agent-based modelling framework designed to simulate pathogen transmission within realistic poultry production and distribution networks. We provide example applications to broiler production in Bangladesh, but the modular structure of the model allows for easy parameterization to suit specific countries and system configurations. Moreover, the framework enables the replication of a wide range of eco-epidemiological scenarios by incorporating diverse pathogen life-history traits, modes of transmission and interactions between multiple strains and/or pathogens. EPINEST was developed in the context of an interdisciplinary multi-centre study conducted in Bangladesh, India, Vietnam and Sri Lanka, and will facilitate the investigation of the spreading patterns of various health hazards such as avian influenza, Campylobacter, Salmonella and antimicrobial resistance in these countries. Furthermore, this modelling framework holds potential for broader application in veterinary epidemiology and One Health research, extending its relevance beyond poultry to encompass other livestock species and disease systems.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Introduction: Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. Methods: High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Results: Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. Discussion: MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Resistome profiling reveals transmission dynamics of antimicrobial resistance genes from poultry litter to soil and plant.

Poultry farming is a major livelihood in South and Southeast Asian economies where it is undergoing rapid intensification to meet the growing human demand for dietary protein. Intensification of poultry production systems is commonly supported by increased antimicrobial drug use, risking greater selection and dissemination of antimicrobial resistance genes (ARGs). Transmission of ARGs through food chains is an emerging threat. Here, we investigated transmission of ARGs from chicken (broiler and layer) litter to soil and Sorghum bicolor (L.) Moench plants based on field and pot experiments. The results demonstrate ARGs transmission from poultry litter to plant systems under field as well as experimental pot conditions. The most common ARGs could be tracked for transmission from litter to soil to plants were identified as detected were cmx, ErmX, ErmF, lnuB, TEM-98 and TEM-99, while common microorganisms included Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, and Vibrio cholerae. Using next generation sequencing and digital PCR assays we detected ARGs transmitted from poultry litter in both the roots and stems of S. bicolor (L.) Moench plants. Poultry litter is frequently used as a fertiliser because of its high nitrogen content; our studies show that ARGs can transmit from litter to plants and illustrates the risks posed to the environment by antimicrobial treatment of poultry. This knowledge is useful for formulating intervention strategies that can reduce or prevent ARGs transmission from one value chain to another, improving understanding of impacts on human and environmental health. The research outcome will help in further understanding the transmission and risks posed by ARGs from poultry to environmental and human/animal health.

Antibiotic usage practices and its drivers in commercial chicken production in Bangladesh.

Irrational and inappropriate use of antibiotics in commercial chicken production can contribute to the development of antimicrobial resistance. We aimed to assess antibiotic usage in commercial chicken production in Bangladesh, and identify factors associated with this practice. We conducted a large-scale cross-sectional study to collect information on antibiotic usage in commercial chickens from January to May 2021. Structured interviews were conducted with 288 broiler, 288 layer and 192 Sonali (locally-produced cross-bred) farmers in 20 sub-districts across Bangladesh. The frequency of antibiotic usage, the types of antibiotics and purpose of usage were estimated for each production type. Adjusted odds ratios (aOR) were calculated to measure the association between antibiotic usage and factors related to the characteristics of the farms and farmers using multivariable logistic regression models. The proportion of farms, irrespective of their production type, reporting usage of antibiotics in the 24 hours preceding the interview was 41% (n = 314, 95% CI: 37-44%). Forty-five percent (n = 344, 41-48%) reported antibiotic usage in the last 72 hours, 86% (n = 658, 83-88%) in the last 14 days, and almost all farms, 98% (n = 753, 97-99%), had used antibiotics since the start of their production cycle. Use of antibiotics in the 24 hours preceding an interview was more frequently reported in broiler (OR 1.91, 95% CI: 1.36-2.69) and Sonali (OR 1.94, 95% CI: 1.33-2.33) than layer farms. Oxytetracycline (23-31%, depending on production type), doxycycline (18-25%), ciprofloxacin (16-26%) and amoxicillin (16-44%) were the most frequently used antibiotics. Antibiotics were reported to be used for both treatment and prophylactic purposes on most farms (57-67%). Usage of antibiotics in the 24h preceding an interview was significantly associated with the occurrence of any illnesses in chickens (aOR broiler: 41.22 [95% CI:13.63-124.62], layer: aOR 36.45[9.52-139.43], Sonali: aOR 28.47[4.97-162.97]). Antibiotic usage was mainly advised by veterinary practitioners (45-71%, depending on production type), followed by feed dealers (21-40%) and farmers (7-13%). Improvement of chicken health through good farming practices along with changes in key stakeholders (feed dealers and practitioners) attitudes towards antibiotic recommendations to farmers, may help to reduce the levels of antibiotic usage and thus contribute to mitigate antimicrobial resistance.

Measures, Gaps, and Mitigation Strategies in Bangladesh's COVID-19 Response.

The Coronavirus Disease 2019 (COVID-19) spread rapidly from China to most other countries around the world in early 2020 killing millions of people. To prevent virus spread, world governments implemented a variety of response measures. This paper's objectives were to discuss the country's adopted measures to combat the virus through June 2020, identify gaps in the measures' effectiveness, and offer possible mitigations to those gaps. The measures taken included screening device deployment across international air and land ports, flight suspensions and closures from COVID-19 affected countries, and declaration and extension of a national public holiday (equivalent to lockdowns in other countries). Identified gaps were test kit, PPE, ICU beds, and ventilator shortages, limited public awareness, and insufficient coordination and collaboration among national and international partners. Proper and timely risk mapping, preparedness, communication, coordination, and collaboration among governments and organizations, and public awareness and engagement would have provided sufficient COVID-19 mitigation in Bangladesh.

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm.

A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens.

Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs).

Community engagement: The key to tackling Antimicrobial Resistance (AMR) across a One Health context?

Antimicrobial resistance (AMR) is a One Health problem underpinned by complex drivers and behaviours. This is particularly so in low - and middle-income countries (LMICs), where social and systemic factors fuel (mis)use and drive AMR. Behavioural change around antimicrobial use could safeguard both existing and future treatments. However, changing behaviour necessitates engaging with people to understand their experiences. This publication describes a knowledge-exchange cluster of six LMIC-based projects who co-designed and answered a series of research questions around the usage of Community Engagement (CE) within AMR. Findings suggest that CE can facilitate AMR behaviour change, specifically in LMICs, because it is a contextualised approach which supports communities to develop locally meaningful solutions. However, current CE interventions focus on human aspects, and demand-side drivers, of AMR. Our cluster suggests that broader attention should be paid to AMR as a One Health issue. The popularity of mixed methods approaches within existing CE for AMR interventions suggests there is interdisciplinary interest in the uptake of CE. Unfortunately, the specificity and context-dependency of CE can make it difficult to evaluate and scale. Nevertheless, we suggest that in synthesising learnings from CE, we can develop a collective understanding of its scope to tackle AMR across contexts.   .

Controlling the causative agents of coccidiosis in domestic chickens; an eye on the past and considerations for the future.

Coccidiosis is a potentially severe enteritis caused by species of obligate intracellular parasites of the genus Eimeria. These parasites cause significant economic losses to the poultry industry, predominantly due to compromised efficiency of production as well as the cost of control. These losses were recently estimated to cost chicken producers approximately £10.4 billion worldwide annually. High levels of Eimeria infection cause clinical coccidiosis which is a significant threat to poultry welfare, and a pre-disposing contributory factor for necrotic enteritis. Control of Eimeria parasites and coccidiosis is therefore an important endeavour; multiple approaches have been developed and these are often deployed together. This review summarises current trends in strategies for control of Eimeria, focusing on three main areas: good husbandry, chemoprophylaxis and vaccination. There is currently no "perfect solution" and there are advantages and limitations to all existing methods. Therefore, the aim of this review is to present current control strategies and suggest how these may develop in the future.

Do All Coccidia Follow the Same Trafficking Rules?

The Coccidia are a subclass of the Apicomplexa and include several genera of protozoan parasites that cause important diseases in humans and animals, with Toxoplasma gondii becoming the 'model organism' for research into the coccidian molecular and cellular processes. The amenability to the cultivation of T. gondii tachyzoites and the wide availability of molecular tools for this parasite have revealed many mechanisms related to their cellular trafficking and roles of parasite secretory organelles, which are critical in parasite-host interaction. Nevertheless, the extrapolation of the T. gondii mechanisms described in tachyzoites to other coccidian parasites should be done carefully. In this review, we considered published data from Eimeria parasites, a coccidian genus comprising thousands of species whose infections have important consequences in livestock and poultry. These studies suggest that the Coccidia possess both shared and diversified mechanisms of protein trafficking and secretion potentially linked to their lifecycles. Whereas trafficking and secretion appear to be well conversed prior to and during host-cell invasion, important differences emerge once endogenous development commences. Therefore, further studies to validate the mechanisms described in T. gondii tachyzoites should be performed across a broader range of coccidians (including T. gondii sporozoites). In addition, further genus-specific research regarding important disease-causing Coccidia is needed to unveil the individual molecular mechanisms of pathogenesis related to their specific lifecycles and hosts.

Kinetics of the Cellular and Transcriptomic Response to Eimeria maxima in Relatively Resistant and Susceptible Chicken Lines.

Eimeria maxima is a common cause of coccidiosis in chickens, a disease that has a huge economic impact on poultry production. Knowledge of immunity to E. maxima and the specific mechanisms that contribute to differing levels of resistance observed between chicken breeds and between congenic lines derived from a single breed of chickens is required. This study aimed to define differences in the kinetics of the immune response of two inbred lines of White Leghorn chickens that exhibit differential resistance (line C.B12) or susceptibility (line 15I) to infection by E. maxima. Line C.B12 and 15I chickens were infected with E. maxima and transcriptome analysis of jejunal tissue was performed at 2, 4, 6 and 8 days post-infection (dpi). RNA-Seq analysis revealed differences in the rapidity and magnitude of cytokine transcription responses post-infection between the two lines. In particular, IFN-γ and IL-10 transcript expression increased in the jejunum earlier in line C.B12 (at 4 dpi) compared to line 15I (at 6 dpi). Line C.B12 chickens exhibited increases of IFNG and IL10 mRNA in the jejunum at 4 dpi, whereas in line 15I transcription was delayed but increased to a greater extent. RT-qPCR and ELISAs confirmed the results of the transcriptomic study. Higher serum IL-10 correlated strongly with higher E. maxima replication in line 15I compared to line C.B12 chickens. Overall, the findings suggest early induction of the IFN-γ and IL-10 responses, as well as immune-related genes including IL21 at 4 dpi identified by RNA-Seq, may be key to resistance to E. maxima.

Adopting an intersectoral One Health approach in India: Time for One Health Committees.

Following the several episodes of zoonotic disease outbreaks and the more recent COVID-19 pandemic, the Indian policy initiatives are committed to institutionalize One Health (OH) approaches and promote intersectoral, transdisciplinary collaboration and cooperation. The OH principle needs to be visualized beyond the scope of zoonoses. While conservation, ecological and veterinary professions are getting increasingly engaged with OH, most of the medical/clinical and social sciences professions are only peripherally aware of its nuances. The OH initiatives, by their essentially multidisciplinary nature, entail working across ministries and navigating tacit institutional hierarchies and allocating leadership roles. The logical operational step will be the constitution of One Health Committees (OHC) at the State and district levels. Here, we outline the key foundational principles of OHC and hope that the framework for implementation shall be deliberated through wider consultations and piloted and adopted in a phased manner.

Spotlight on avian pathology: Eimeria and the disease coccidiosis.

Coccidiosis, caused by Eimeria species parasites, remains a major threat to poultry production, undermining economic performance and compromising welfare. The recent characterization of three new Eimeria species that infect chickens has highlighted that many gaps remain in our knowledge of the biology and epidemiology of these parasites. Concerns about the use of anticoccidial drugs, widespread parasite drug resistance, the need for vaccines that can be used across broiler as well as layer and breeder sectors, and consumer preferences for "clean" farming, all point to the need for novel control strategies. New research tools including vaccine delivery vectors, high throughput sequencing, parasite transgenesis and sensitive molecular assays that can accurately assess parasite development in vitro and in vivo are all proving helpful in the ongoing quest for improved cost-effective, scalable strategies for future control of coccidiosis.

The structure of a major surface antigen SAG19 from Eimeria tenella unifies the Eimeria SAG family.

In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αßα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.

Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus).

More than 68 billion chickens were produced globally in 2018, emphasising their major contribution to the production of protein for human consumption and the importance of their pathogens. Protozoan Eimeria spp. are the most economically significant parasites of chickens, incurring global costs of more than UK £10.4 billion per annum. Seven Eimeria spp. have long been recognised to infect chickens, with three additional cryptic operational taxonomic units (OTUs) first described more than 10 years ago. As the world's farmers attempt to reduce reliance on routine use of antimicrobials in livestock production, replacing drugs that target a wide range of microbes with precise species- and sometimes strain-specific vaccines, the breakthrough of cryptic genetic types can pose serious problems. Consideration of biological characteristics including oocyst morphology, pathology caused during infection and pre-patent periods, combined with gene-coding sequences predicted from draft genome sequence assemblies, suggest that all three of these cryptic Eimeria OTUs possess sufficient genetic and biological diversity to be considered as new and distinct species. The ability of these OTUs to compromise chicken bodyweight gain and escape immunity induced by current commercially available anticoccidial vaccines indicates that they could pose a notable threat to chicken health, welfare, and productivity. We suggest the names Eimeria lata n. sp., Eimeria nagambie n. sp. and Eimeria zaria n. sp. for OTUs x, y and z, respectively, reflecting their appearance (x) or the origins of the first isolates of these novel species (y, z).

Impact of Eimeria tenella Oocyst Dose on Parasite Replication, Lesion Score and Cytokine Transcription in the Caeca in Three Breeds of Commercial Layer Chickens.

Eimeria species parasites infect the gastrointestinal tract of chickens, causing disease and impacting on production. The poultry industry relies on anticoccidial drugs and live vaccines to control Eimeria and there is a need for novel, scalable alternatives. Understanding the outcomes of experimental infection in commercial chickens is valuable for assessment of novel interventions. We examined the impact of different infectious doses of Eimeria tenella (one low dose, three high doses) in three commercial layer chicken lines, evaluating lesion score, parasite replication and cytokine response in the caeca. Groups of eight to ten chickens were housed together and infected with 250, 4,000, 8,000 or 12,000 sporulated oocysts at 21 days of age. Five days post-infection caeca were assessed for lesions and to quantify parasite replication by qPCR and cytokine transcription by RT-qPCR. Comparison of the three high doses revealed no significant variation between them in observed lesions or parasite replication with all being significantly higher than the low dose infection. Transcription of IFN-γ and IL-10 increased in all infected chickens relative to unchallenged controls, with no significant differences associated with dose magnitude (p > 0.05). No significant differences were detected in lesion score, parasite replication or caecal cytokine expression between the three lines of chickens. We therefore propose 4,000 E. tenella oocysts is a sufficient dose to reliably induce lesions in commercial layer chickens, and that estimates of parasite replication can be derived by qPCR from these same birds. However, more accurate quantification of Eimeria replication requires a separate low dose challenge group. Optimisation of challenge dose in an appropriate chicken line is essential to maximize the value of in vivo efficacy studies. For coccidiosis, this approach can reduce the numbers of chickens required for statistically significant studies and reduce experimental severity.

Revisiting the Economic Impacts of Eimeria and Its Control in European Intensive Broiler Systems With a Recursive Modeling Approach.

Ionophore compounds active against Eimeria species are widely used in intensive broiler systems and have formed the backbone of coccidiosis control for almost 50 years. Producers, however, are under pressure to reduce ionophore use due to consumer concerns over antimicrobial usage in food animals, and antimicrobial resistance. Moreover, current vaccines against Eimeria are commonly considered to be less cost-effective in intensive broiler systems, especially in Europe where attenuated live vaccines are used. An economic assessment of the impact of Eimeria and the disease coccidiosis, including the cost implications of different efficacies of control, is therefore timely to provide evidence for industry and policy development. A mechanistic model of broiler production under varying infection and control states was used to construct a dataset from which system productivity can be measured. Coccidiosis impact increased rapidly as control efficacy decreased. In the total absence of control, median impact was found to maximize at between €2.55 and €2.97 in lost production per meter squared of broiler house over a 33 day growing period. Coccidiosis remains a major risk to intensive broiler systems and the model developed allows investigation of issues related to coccidiosis control, antimicrobial use and the development of antimicrobial resistance.

The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture.

In vitro development of the complete life cycle of Eimeria species has been achieved in primary cultures of avian epithelial cells with low efficiency. The use of immortalized cell lines simplifies procedures but only allows partial development through one round of parasite invasion and intracellular replication. We have assessed the suitability of Madin-Darby Bovine Kidney (MDBK) cells to support qualitative and quantitative studies on sporozoite invasion and intracellular development of Eimeria tenella. Analysis of parasite ultrastructure by transmission electron microscopy and serial block face-scanning electron microscopy proved the suitability of the system to generate good quality schizonts and first-generation merozoites. Parasite protein expression profiles elucidated by mass spectrometry corroborated previous findings occurring during the development of the parasite such as the presence of alternative types of surface antigen at different stages and increased abundance of proteins from secretory organelles during invasion and endogenous development. Quantitative PCR (qPCR) allowed the tracking of development by detecting DNA division, whereas reverse transcription qPCR of sporozoite- and merozoite-specific genes could detect early changes before cell division and after merozoite formation, respectively. These results correlated with the analysis of development using ImageJ semi-automated image analysis of fluorescent parasites, demonstrating the suitability and reproducibility of the MDBK culture system. This systems also allowed the evaluation of the effects on invasion and development when sporozoites were pre-incubated with anticoccidial drugs, showing similar effects to those reported before. We have described through this study a series of methods and assays for the further application of this in vitro culture model to more complex studies of Eimeria including basic research on parasite cell biology and host-parasite interactions and for screening anticoccidial drugs.

Phylogenetic Inference Using Cytochrome C Oxidase Subunit I (COI) in the Poultry Red Mite, Dermanyssus gallinae in the United Kingdom Relative to a European Framework.

The poultry red mite (Dermanyssus gallinae), an obligatory blood feeding ectoparasite, is primarily associated with laying hens where it is estimated to cause losses of ~€231 million per annum to European farmers. Moderate to high infestation levels result in negative impacts on hen welfare, including increased cannibalism, irritation, feather pecking, restlessness, anemia, and mortality. Acaricides are currently the prevailing method of population control for D. gallinae, although resistance against some classes of acaricide has been widely reported. The development of resistance highlights a growing need for research into alternative control methods, including the development of a suitable and effective vaccine. Understanding the genetic structure of D. gallinae populations can support improved management of acaricide resistance and sustainability of future vaccines, but limited data are currently available. The aim of this study was to characterize D. gallinae isolates from Europe, targeting the cytochrome c oxidase subunit 1 (COI) gene to gain an insight into population structure and genetic diversity of currently circulating mites. Dermanyssus gallinae isolates were collected from Albania, Belgium, Croatia, Czech Republic, Denmark, France, Greece, Italy, the Netherlands, Portugal, Romania, Slovenia, Turkey and the United Kingdom. Genomic DNA was extracted from individual adult D. gallinae mites and a 681bp fragment of the COI gene was amplified and sequenced. Phylogenetic analyses of 195 COI sequences confirmed the presence of multiple lineages across Europe with 76 distinct haplotypes split across three main haplogroups and six sub-haplogroups. Importantly there is considerable inter- and intra-country variation across Europe, which could result from the movement of poultry or transfer of contaminated equipment and/or materials and husbandry practices.