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Cell culture systems have long been recognised as great resources to mitigate the use of animals in research, offering effective solutions for replacement or reduction with benefits commonly including lower costs, shorter duration and improved reproducibility. The use of in vitro culture methods has been extensively explored for many apicomplexan parasites, supporting significant research advances, but studies with Eimeria are often limited since they still depend on the animal host. In this study we have used 2.5D and 3D culture systems for the first time to evaluate the growth of Eimeria tenella parasites using a panel of cell lines (MDBK, HD11, COLO-680N and HCC4006). Results were compared to growth in 2D monolayers following established protocols. Observations using the fluorescent transgenic strain Et-dYFP showed invasion and development of parasites inside cells suspended in a collagen matrix (2.5D or 3D), supporting the development of asexual stages with the release of first-generation merozoites. Similar findings were observed when Scaffold-free 3D cell spheroids of HD11 cells were infected with sporozoites. No subsequent developmental stages were identified while evaluating these cell lines and further work will be required to improve in vitro culture systems to a point where reduction and replacement of animal use becomes routine.
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Técnicas de Cultivo de Célula , Eimeria tenella , Eimeria tenella/crecimiento & desarrollo , Animales , Técnicas de Cultivo de Célula/métodos , Humanos , Línea Celular , Esporozoítos/crecimiento & desarrollo , Esferoides Celulares/parasitologíaRESUMEN
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.
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Pollos , Coccidiosis , Eimeria tenella , Orgánulos , Enfermedades de las Aves de Corral , Esporozoítos , Animales , Actinas/metabolismo , Pollos/metabolismo , Pollos/parasitología , Eimeria tenella/metabolismo , Eimeria tenella/fisiología , Orgánulos/metabolismo , Orgánulos/fisiología , Esporozoítos/metabolismo , Esporozoítos/fisiología , Coccidiosis/metabolismo , Coccidiosis/parasitología , Coccidiosis/fisiopatología , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/parasitología , Enfermedades de las Aves de Corral/fisiopatologíaRESUMEN
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.
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COVID-19 , Bangladesh/epidemiología , COVID-19/prevención & control , China , Control de Enfermedades Transmisibles , Humanos , SuspensionesRESUMEN
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.
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Eimeria tenella , Parásitos , Animales , Eimeria tenella/metabolismo , Eimeria tenella/ultraestructura , Tomografía con Microscopio Electrónico , Orgánulos/metabolismo , Parásitos/metabolismo , Proteínas Protozoarias/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
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).
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Coccidiosis/veterinaria , Eimeria tenella/inmunología , Enfermedades de las Aves de Corral/parasitología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Animales , Pollos/inmunología , Pollos/parasitología , Coccidiosis/prevención & control , Eimeria tenella/crecimiento & desarrollo , Femenino , Masculino , Enfermedades de las Aves de Corral/prevención & control , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/genética , Saccharomyces cerevisiae/inmunología , Vacunación/métodos , Vacunación/veterinaria , Vacunas de Subunidad/inmunologíaRESUMEN
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.
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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.
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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).
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Coccidiosis , Eimeria , Enfermedades de las Aves de Corral , Vacunas Antiprotozoos , Animales , Pollos , Coccidiosis/prevención & control , Coccidiosis/veterinaria , Eimeria/genética , Humanos , Nigeria , Enfermedades de las Aves de Corral/prevención & controlRESUMEN
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.
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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.
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Antígenos de Protozoos/metabolismo , Eimeria tenella/metabolismo , Proteínas Protozoarias/metabolismo , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Cristalografía por Rayos X , Eimeria tenella/genética , Evolución Molecular , Variación Genética , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Relación Estructura-ActividadRESUMEN
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.
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Coccidiosis, caused by Eimeria species parasites, has long been recognised as an economically significant disease of chickens. As the global chicken population continues to grow, and its contribution to food security intensifies, it is increasingly important to assess the impact of diseases that compromise chicken productivity and welfare. In 1999, Williams published one of the most comprehensive estimates for the cost of coccidiosis in chickens, featuring a compartmentalised model for the costs of prophylaxis, treatment and losses, indicating a total cost in excess of £38 million in the United Kingdom (UK) in 1995. In the 25 years since this analysis the global chicken population has doubled and systems of chicken meat and egg production have advanced through improved nutrition, husbandry and selective breeding of chickens, and wider use of anticoccidial vaccines. Using data from industry representatives including veterinarians, farmers, production and health experts, we have updated the Williams model and estimate that coccidiosis in chickens cost the UK £99.2 million in 2016 (range £73.0-£125.5 million). Applying the model to data from Brazil, Egypt, Guatemala, India, New Zealand, Nigeria and the United States resulted in estimates that, when extrapolated by geographical region, indicate a global cost of ~ £10.4 billion at 2016 prices (£7.7-£13.0 billion), equivalent to £0.16/chicken produced. Understanding the economic costs of livestock diseases can be advantageous, providing baselines to evaluate the impact of different husbandry systems and interventions. The updated cost of coccidiosis in chickens will inform debates on the value of chemoprophylaxis and development of novel anticoccidial vaccines.
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Crianza de Animales Domésticos/economía , Pollos , Coccidiosis/veterinaria , Enfermedades de las Aves de Corral/economía , Animales , Coccidiosis/economíaRESUMEN
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.
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BACKGROUND: Poultry coccidiosis is a parasitic enteric disease with a highly negative impact on chicken production. In-feed chemoprophylaxis remains the primary method of control, but the increasing ineffectiveness of anticoccidial drugs, and potential future restrictions on their use has encouraged the use of commercial live vaccines. Availability of such formulations is constrained by their production, which relies on the use of live chickens. Several experimental approaches have been taken to explore ways to reduce the complexity and cost of current anticoccidial vaccines including the use of live vectors expressing relevant Eimeria proteins. We and others have shown that vaccination with transgenic Eimeria tenella parasites expressing Eimeria maxima Apical Membrane Antigen-1 or Immune Mapped Protein-1 (EmAMA1 and EmIMP1) partially reduces parasite replication after challenge with a low dose of E. maxima oocysts. In the present study, we have reassessed the efficacy of these experimental vaccines using commercial birds reared at high stocking densities and challenged with both low and high doses of E. maxima to evaluate how well they protect chickens against the negative impacts of disease on production parameters. METHODS: Populations of E. tenella parasites expressing EmAMA1 and EmIMP1 were obtained by nucleofection and propagated in chickens. Cobb500 broilers were immunised with increasing doses of transgenic oocysts and challenged two weeks later with E. maxima to quantify the effect of vaccination on parasite replication, local IFN-γ and IL-10 responses (300 oocysts), as well as impacts on intestinal lesions and body weight gain (10,000 oocysts). RESULTS: Vaccination of chickens with E. tenella expressing EmAMA1, or admixtures of E. tenella expressing EmAMA1 or EmIMP1, was safe and induced partial protection against challenge as measured by E. maxima replication and severity of pathology. Higher levels of protection were observed when both antigens were delivered and was associated with a partial modification of local immune responses against E. maxima, which we hypothesise resulted in more rapid immune recognition of the challenge parasites. CONCLUSIONS: This study offers prospects for future development of multivalent anticoccidial vaccines for commercial chickens. Efforts should now be focused on the discovery of additional antigens for incorporation into such vaccines.
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Pollos/parasitología , Coccidiosis/veterinaria , Eimeria tenella , Vacunas Antiprotozoos , Animales , Antígenos de Protozoos/inmunología , Peso Corporal/efectos de los fármacos , Pollos/inmunología , Coccidiosis/prevención & control , Coccidiosis/terapia , Eimeria/efectos de los fármacos , Eimeria/crecimiento & desarrollo , Eimeria/inmunología , Eimeria tenella/efectos de los fármacos , Eimeria tenella/crecimiento & desarrollo , Eimeria tenella/inmunología , Genes Protozoarios/inmunología , Interferón gamma/efectos de los fármacos , Interleucina-10/metabolismo , Enfermedades de las Aves de Corral/parasitología , Enfermedades de las Aves de Corral/prevención & control , Vacunas Antiprotozoos/biosíntesis , Vacunas Antiprotozoos/uso terapéutico , Transfección , Transgenes/inmunología , Vacunación/métodos , Vacunación/veterinaria , Vacunas Atenuadas/biosíntesis , Vacunas Atenuadas/uso terapéuticoRESUMEN
Eimeria infection impacts upon chicken welfare and economic productivity of the poultry sector. Live coccidiosis vaccines for chickens have been available for almost 70 years, but the requirement to formulate blends of oocysts from multiple Eimeria species makes vaccine production costly and logistically demanding. A multivalent vaccine that does not require chickens for its production and can induce protection against multiple Eimeria species is highly desirable. However, despite the identification and testing of many vaccine candidate antigens, no recombinant coccidiosis vaccine has been developed commercially. Currently, assessment of vaccine efficacy against Eimeria, and the disease coccidiosis, can be done only through in vivo vaccination and challenge experiments but the design of such studies has been highly variable. Lack of a "standard" protocol for assessing vaccine efficacy makes comparative evaluations very difficult, complicating vaccine development, and validation. The formulation and schedule of vaccination, the breed of chicken and choice of husbandry system, the species, strain, magnitude, and timing of delivery of the parasite challenge, and the parameters used to assess vaccine efficacy all influence the outcomes of experimental trials. In natural Eimeria infections, the induction of strong cell mediated immune responses are central to the development of protective immunity against coccidiosis. Antibodies are generally regarded to be of lesser importance. Unfortunately, there are no specific immunological assays that can accurately predict how well a vaccine will protect against coccidiosis (i.e., no "correlates of protection"). Thus, experimental vaccine studies rely on assessing a variety of post-challenge parameters, including assessment of pathognomonic lesions, measurements of parasite replication such as oocyst output or quantification of Eimeria genomes, and/or measurements of productivity such as body weight gain and feed conversion rates. Understanding immune responses to primary and secondary infection can inform on the most appropriate immunological assays. The discovery of new antigens for different Eimeria species and the development of new methods of vaccine antigen delivery necessitates a more considered approach to assessment of novel vaccines with robust, repeatable study design. Careful consideration of performance and welfare factors that are genuinely relevant to chicken producers and vaccine manufacturers is essential.
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Apicomplexans, including species of Eimeria, pose a real threat to the health and wellbeing of animals and humans. Eimeria parasites do not infect humans but cause an important economic impact on livestock, in particular on the poultry industry. Despite its high prevalence and financial costs, little is known about the cell biology of these 'cosmopolitan' parasites found all over the world. In this review, we discuss different aspects of the life cycle and stages of Eimeria species, focusing on cellular structures and organelles typical of the coccidian family as well as genus-specific features, complementing some 'unknowns' with what is described in the closely related coccidian Toxoplasma gondii.
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Pollos , Coccidiosis/veterinaria , Eimeria/fisiología , Estadios del Ciclo de Vida , Enfermedades de las Aves de Corral/parasitología , Animales , Coccidiosis/parasitología , Eimeria/crecimiento & desarrollo , Orgánulos/fisiologíaRESUMEN
Dermanyssus gallinae, the poultry red mite, is a global threat to the commercial egg-laying industry. Control of D. gallinae is difficult, with only a limited number of effective pesticides and non-chemical treatments available. Here, we characterize the candidate vaccine antigen D. gallinae cathepsin D-1 (Dg-CatD-1) and demonstrate that purified refolded recombinant Dg-Cat-D1 (rDg-CatD-1) is an active aspartyl proteinase which digests haemoglobin with a pH optimum of pH 4. Soluble protein extracts from D. gallinae also have haemoglobinase activity, with a pH optimum comparable to the recombinant protein, and both proteinase activities were inhibited by the aspartyl proteinase inhibitor Pepstatin A. Enzyme activity and the ubiquitous localization of Dg-CatD-1 protein in sections of adult female mites is consistent with Dg-CatD-1 being a lysosomal proteinase. Using Dg-CatD-1 as a model vaccine antigen, we compared vaccine delivery methods in laying hens via vaccination with: (i) purified rDg-CatD-1 with Montanide™ ISA 71 VG adjuvant; (ii) recombinant DNA vaccines for expression of rDg-CatD-1 and (iii) transgenic coccidial parasite Eimeria tenella expressing rDg-CatD-1. In two independent trials, only birds vaccinated with rDg-CatD-1 with Montanide™ ISA 71 VG produced a strong and long-lasting serum anti-rDg-Cat-D1 IgY response, which was significantly higher than that in control birds vaccinated with adjuvant only. Furthermore, we showed that egg-laying rates of D. gallinae mites fed on birds vaccinated with rDg-CatD-1 in Montanide™ ISA 71 VG was reduced significantly compared with mites fed on unvaccinated birds. RESEARCH HIGHLIGHTS Dermanyssus gallinae cathepsin D-1 (Dg-CatD-1) digests haemoglobin Vaccination of hens with rDg-CatD-1 in Montanide™ ISA 71 VG results in long-lasting IgY levels Serum anti-rDg-CatD-1 antibodies reduce egg laying in D. gallinae after a single blood meal.
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Pollos/inmunología , Infestaciones por Ácaros/veterinaria , Ácaros/inmunología , Enfermedades de las Aves de Corral/prevención & control , Vacunación/veterinaria , Vacunas/administración & dosificación , Adyuvantes Inmunológicos , Animales , Formación de Anticuerpos , Pollos/parasitología , Femenino , Infestaciones por Ácaros/parasitología , Infestaciones por Ácaros/prevención & control , Proteínas RecombinantesRESUMEN
Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host-specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens results in an intestinal disease called coccidiosis, characterized by mucoid or hemorrhagic enteritis, which is associated with impaired feed conversion or mortality in severe cases. Intensive farming practices have increased the significance of coccidiosis since parasite transmission is favored by high-density housing of large numbers of susceptible chickens. Routine chemoprophylaxis and/or vaccination with live parasite vaccines provides effective control of Eimeria, although the emergence of drug resistance and the relative cost and production capacity of current vaccine lines can prove limiting. As pressure to reduce drug use in livestock production intensifies, novel vaccination strategies are needed. Development of effective protocols supporting genetic complementation of Eimeria species has until recently been hampered by their inability to replicate efficiently in vitro. Now, the availability of such protocols has raised the prospect of generating transgenic parasite lines that function as vaccine vectors to express and deliver heterologous antigens. For example, this technology has the potential to streamline the production of live anticoccidial vaccines through the generation of parasite lines that co-express immunoprotective antigens derived from multiple Eimeria species. In this paper we describe detailed protocols for genetic manipulation, laboratory growth, and in vivo propagation of Eimeria tenella parasites, which will encourage future work from other researchers to expand biological understanding of Eimeria through reverse genetics. © 2019 by John Wiley & Sons, Inc.
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Técnicas de Cultivo de Célula/métodos , Coccidiosis/veterinaria , Eimeria tenella/crecimiento & desarrollo , Eimeria tenella/genética , Parasitología/métodos , Enfermedades de las Aves de Corral/parasitología , Transfección/métodos , Animales , Pollos , Coccidiosis/parasitología , Eimeria tenella/fisiologíaRESUMEN
Eimeria tenella can cause the disease coccidiosis in chickens. The direct and often detrimental impact of this parasite on chicken health, welfare, and productivity is well recognized; however, less is known about the secondary effects that infection may have on other gut pathogens. Campylobacter jejuni is the leading cause of human bacterial foodborne disease in many countries and has been demonstrated to exert negative effects on poultry welfare and production in some broiler lines. Previous studies have shown that concurrent Eimeria infection can influence the colonization and replication of bacteria, such as Clostridium perfringens and Salmonella enterica serovar Typhimurium. Through a series of in vivo coinfection experiments, this study evaluated the impact that E. tenella infection had on C. jejuni colonization of chickens, including the influence of variations in parasite dose and sampling time after bacterial challenge. Coinfection with E. tenella resulted in a significant increase in C. jejuni colonization in the cecum in a parasite dose-dependent manner but a significant decrease in C. jejuni colonization in the spleen and liver of chickens. The results were reproducible at 3 and 10 days after bacterial infection. This work highlights that E. tenella not only has a direct impact on the health and well-being of chickens but can have secondary effects on important zoonotic pathogens.
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Infecciones por Campylobacter/microbiología , Campylobacter jejuni/aislamiento & purificación , Pollos/microbiología , Coccidiosis/complicaciones , Coinfección , Eimeria tenella , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/parasitología , Animales , Ciego/microbiología , Coinfección/microbiología , Coinfección/parasitologíaRESUMEN
The poultry red mite, Dermanyssus gallinae, is a major worldwide concern in the egg-laying industry. Here, we report the first draft genome assembly and gene prediction of Dermanyssus gallinae, based on combined PacBio and MinION long-read de novo sequencing. The â¼959-Mb genome is predicted to encode 14,608 protein-coding genes.