{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T05:06:21Z","timestamp":1772168781835,"version":"3.50.1"},"update-to":[{"DOI":"10.12688\/f1000research.123102.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2024,10,1]],"date-time":"2024-10-01T00:00:00Z","timestamp":1727740800000}}],"reference-count":15,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2024,10,1]],"date-time":"2024-10-01T00:00:00Z","timestamp":1727740800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000849","name":"National Centre for the Replacement, Refinement and Reduction of Animals in Research","doi-asserted-by":"publisher","award":["NC\/W000881\/1"],"award-info":[{"award-number":["NC\/W000881\/1"]}],"id":[{"id":"10.13039\/501100000849","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>\n                    We have developed an\n                    <ns3:italic>in vitro<\/ns3:italic>\n                    model for the evaluation of potential anticoccidial properties of novel compounds aimed to control chicken coccidiosis, a costly disease for the poultry industry. This disease is caused by protozoan parasites of the genus\n                    <ns3:italic>Eimeria<\/ns3:italic>\n                    (Apicomplexa), and it is mainly controlled by chemoprophylaxis with ionophores and chemical anticoccidials; however, there is an overall agreement about the limitation of these traditional drugs and the need to improve current methods of control. Anticoccidial activities of novel compounds is currently evaluated by expensive experiments that involve large numbers of chickens. The use of our\n                    <ns3:italic>in vitro<\/ns3:italic>\n                    model for the pre-screening of essential oils led to a reduction of 67% of the chickens used in the\n                    <ns3:italic>in vivo<\/ns3:italic>\n                    trials for validation.\n                    <ns3:italic>Eimeria<\/ns3:italic>\n                    parasites can only complete their life cycle in their animal host, therefore chickens are required for their propagation as they cannot be propagated\n                    <ns3:italic>in vitro.<\/ns3:italic>\n                    In this study, we describe how further optimisation of this\n                    <ns3:italic>in vitro<\/ns3:italic>\n                    model by miniaturisation can have an additional impact in reduction of the number of chickens used for the generation of parasite stocks for provision of the\n                    <ns3:italic>in vitro<\/ns3:italic>\n                    model. We have estimated that the use of one chicken could support the evaluation of 10 compounds with a 96-well plate format versus only two compounds with a 24-well plate format, which means an 80% reduction in chicken use. In this study we have proved that the miniaturisation into a 96-well plate format perfectly mimics the invasion and replication observed before in the 24-well plate format. In addition, the 96-well plate format has allowed the simultaneous pre-screening of higher numbers of anticoccidial drugs at different concentrations following streamlined protocols in a more cost-effective way, factors that are beneficial for a wider uptake of the model by other researchers investigating anticoccidial compounds.\n                  <\/ns3:p>","DOI":"10.12688\/f1000research.123102.2","type":"journal-article","created":{"date-parts":[[2024,10,1]],"date-time":"2024-10-01T05:45:10Z","timestamp":1727761510000},"page":"1135","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Reduction of chickens use to perform in vitro pre-screening of novel anticoccidials by miniaturisation and increased throughput of the current Eimeria tenella compound-screening model"],"prefix":"10.12688","volume":"11","author":[{"given":"Sara","family":"Arias-Maroto","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kelsilandia","family":"Aguiar-Martins","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Javier","family":"Regidor-Cerrillo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4986-6783","authenticated-orcid":false,"given":"Luis","family":"Ortega-Mora","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1512-4682","authenticated-orcid":false,"given":"Virginia","family":"Marugan-Hernandez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2024,10,1]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"115","DOI":"10.1186\/s13567-020-00837-2","article-title":"Re-calculating the cost of coccidiosis in chickens.","volume":"51","author":"D Blake","year":"2020","journal-title":"Vet. Res."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"263-278","DOI":"10.1017\/S0031182019001562","article-title":"Life cycle stages, specific organelles and invasion mechanisms of Eimeria species.","volume":"147","author":"A Burrell","year":"2020","journal-title":"Parasitology."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"77-86","DOI":"10.1016\/j.molbiopara.2008.07.006","article-title":"A toolbox facilitating stable transfection of Eimeria species.","volume":"162","author":"J Clark","year":"2008","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"77","DOI":"10.3390\/ani14010077","article-title":"Thyme, Oregano, and Garlic Essential Oils and Their Main Active Compounds Influence Eimeria tenella Intracellular Development.","volume":"14","author":"M Felici","year":"2023","journal-title":"Animals (Basel)."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"109318","DOI":"10.1016\/j.vetpar.2020.109318","article-title":"Pharmacokinetics and anticoccidial activity of ethanamizuril in broiler chickens.","volume":"289","author":"Y Fu","year":"2021","journal-title":"Vet. Parasitol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"575-584","DOI":"10.1016\/j.psj.2020.11.027","article-title":"Response of broiler chickens fed diets supplemented with a bioactive olive pomace extract from Olea europaea to an experimental coccidial vaccine challenge.","volume":"100","author":"J Herrero-Encinas","year":"2021","journal-title":"Poult. Sci."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"251-260","DOI":"10.1080\/00071668.2020.1837344","article-title":"Agaricus subrufescens and Pleurotus ostreatus mushrooms as alternative additives to antibiotics in diets for broilers challenged with Eimeria spp.","volume":"62","author":"G Lima","year":"2021","journal-title":"Br. Poult. Sci."},{"key":"ref8","doi-asserted-by":"publisher","DOI":"10.5061\/dryad.mw6m90605","article-title":"Reduction of chickens use to perform in vitro pre-screening of novel anticoccidials by miniaturisation and increased throughput of the current Eimeria tenella compound-screening model, Dryad, [Dataset].","author":"V Marugan-Hernandez","year":"2022"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"463","DOI":"10.1186\/s13071-016-1756-2","article-title":"Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system.","volume":"9","author":"V Marugan-Hernandez","year":"2016","journal-title":"Parasit. Vectors."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"579833","DOI":"10.3389\/fcimb.2020.579833","article-title":"The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture.","volume":"10","author":"V Marugan-Hernandez","year":"2020","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"530-534","DOI":"10.1111\/j.1550-7408.1976.tb03834.x","article-title":"Comparative in vitro development of precocious and normal strains of Eimeria tenella (Coccidia).","volume":"23","author":"L McDougald","year":"1976","journal-title":"J. Protozool."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"e81","DOI":"10.1002\/cpmc.81","article-title":"Laboratory Growth and Genetic Manipulation of Eimeria tenella.","volume":"53","author":"I Pastor-Fernandez","year":"2019","journal-title":"Curr. Protoc. Microbiol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"420","DOI":"10.3389\/fvets.2020.00420","article-title":"In vitro Anticoccidial Study of Oregano and Garlic Essential Oils and Effects on Growth Performance, Fecal Oocyst Output, and Intestinal Microbiota in vivo.","volume":"7","author":"E Sidiropoulou","year":"2020","journal-title":"Front. Vet. Sci."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"86-96","DOI":"10.1016\/j.vetpar.2016.12.005","article-title":"Anticoccidial efficacy testing: in vitro Eimeria tenella assays as replacement for animal experiments.","volume":"233","author":"A Thabet","year":"2017","journal-title":"Vet. Parasitol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"229-237","DOI":"10.1053\/tvjl.2000.0563","article-title":"Farm animal welfare: the five freedoms and the free market.","volume":"161","author":"A Webster","year":"2001","journal-title":"Vet. J."}],"container-title":["F1000Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,2,7]],"date-time":"2025-02-07T17:25:11Z","timestamp":1738949111000},"score":1,"resource":{"primary":{"URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,1]]},"references-count":15,"URL":"https:\/\/doi.org\/10.12688\/f1000research.123102.2","relation":{"has-version":[{"id-type":"doi","id":"10.12688\/f1000research.123102.1","asserted-by":"subject"}],"has-review":[{"id-type":"doi","id":"10.5256\/f1000research.172468.r356737","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.172468.r340270","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.172468.r340270","asserted-by":"object"},{"id-type":"doi","id":"10.5256\/f1000research.172468.r356737","asserted-by":"object"}]},"ISSN":["2046-1402"],"issn-type":[{"value":"2046-1402","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,1]]},"assertion":[{"value":"Indexed","URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.172468.r356737, Benjamin Adjei-Mensah, Department of Animal Science, University of Ghana School of Agriculture (Ringgold ID: 356390), Accra, Greater Accra Region, 07 Feb 2025, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2#referee-response-356737","order":0,"name":"referee-response-356737","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.135172.r173894, Joseph D. Turner, Centre for Drugs and Diagnostics, Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, 13 Nov 2023, version 1, 1 approved with reservations, 1 not approved","URL":"https:\/\/f1000research.com\/articles\/11-1135\/v1#referee-response-173894","order":1,"name":"referee-response-173894","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Virginia Marugan-Hernandez<\/b>; \n<i>Posted: 24 Sep 2024<\/i>; We would like to thank Joseph D. Turner for his time and effort reviewing our manuscript. We provide below responses for the different queries and issues raised.  \n<b>Rationale for selection of seeding densities in 96-well plates needs more explanation - was it based on scaling of volume, well area or well volume compared with 24 well plate, for instance?<\/b>  The two densities were derived of the previously optimised number for the 24-well plate format that was conferring a 100% confluence without an excess of cells (Marugan-Hernandez et al., 2020). This has been now detailed in \u2018Optimisation of infection ratios\u2019 section: \u2018\n<i>Based on the amount of 0.3&times;10 <sup>6<\/sup> per well in 500 <\/i>\n<i>\u03bc<\/i>\n<i>l, previously optimised for the 24-well plates format to confer a confluence of 100% without excess of cells (where the surface of each well is four time larger than in the 96-well plate format; 0.3&times;10 <sup>6<\/sup> \/ 4 = 0.075&times;10 <sup>6<\/sup>), we tested 0.1&times;10 <sup>6<\/sup> and 0.05&times;10 <sup>6 <\/sup>cells per well for the 96-well plate format (just above and below to the equivalent figure)<\/i>.\u2019  \n<b>Readers may wish to understand why it is necessary to culture at 41 degrees centigrade for infection of MDBK with sporozoites as this is unusual for mammalian cell culture.<\/b>  The temperature in the chicken intestine is 41\n<sup>o<\/sup>C and this is the optimal temperature for \n<i>Eimeria tenella<\/i> development, if the temperature is lower, the parasite will not replicate. This has been clarified in \u2018Optimisation of infection ratios\u2019 section: \u2018\n<i>This temperature is necessary for parasite development, as chicken Eimeria parasites develop in the intestine of chickens, whose internal temperature is 41 <sup>o<\/sup>C, higher than mammalian hosts. MDBK cells can support this increased temperature for up to six\/seven days, longer than the required time for in vitro parasite replication.<\/i>\u2019  \n<b>A flow chart of the invasion and replication inhibition assays would help clarify timings of when sporozoites and drugs were added \/ removed, as without careful reading this is hard to follow.<\/b>  A flow chart has been included (Figure 1) aimed to clarify the experimental design of the invasion and replication assays. \n<i>\u2018Figure 1. Flow chart of sporozoite i<\/i>\n<i>nvasion and replication inhibition assays. <\/i> \n<b><i>1.<\/i><\/b>\n<i> MDBK cells were seeded into 96-well plates to form a confluent monolayer (0.05x10<sup>6<\/sup> cells\/well); <b>2.<\/b> Sporozoites were purified from oocysts using DE-52 columns (Pastor-Fernandez et al., 2019); <b>3.<\/b> Sporozoites were pre-incubated with anticoccidial drugs at four different concentrations (or without drugs for controls) for 1 hour at 4<sup>o<\/sup>C; <b>4.<\/b> Sporozoites were centrifuged, resuspended in AdDMEM and added to MDBK monolayers to allow invasion. <b>5.<\/b> Infected cell monolayers were lysed and collected; <b>6.<\/b> Genomic DNA was extracted from collected infected cell monolayers; <b>7.<\/b> Real time qPCR was used to evaluate the number of parasite genomes for each sample.\u2019<\/i>  \n<b>I agree the data supports the conclusion that infection and growth rate of Eimeria is similar between optimised 24 and 96 well plate cultures, but a suitable statistical comparison would be needed to confirm this.<\/b>  A statistical comparison using the non-parametric Spearman's rank test has been included. This has shown a high degree of correlation with r=1. All this has been detailed in \u2018Data analysis\u2019 and \u2018Sporozoites of \n<i>E. tenella<\/i> invaded and replicated at equivalent rates when transferred to a 96-well plate format\u2019 sections: \u2018\n<i>Correlation (r) between sporozoite quantification between the 24 and 96-well plate format cultures was determined using the nonparametric Spearman's rank test using<\/i>\n<i> GraphPad Prism version 6.00 (GraphPad Software, La Jolla, California, USA)<\/i>.\u2019 \u2018\n<i>The linear rate of replication showed a very strong correlation between the 96 and the 24-well plate format models (r=1; 95% confidence interval; <\/i>\n<i>Figure <\/i>\n<i>2)<\/i>\u2019  \n<b>Further, there is a lack of statistics to test whether any of the drug compounds significantly inhibited invasion and\/or growth - using PCR quantifications at either time point or the overall growth rate.<\/b>  Statistical analysis have been included to evaluate differences in the \u2018number of zoites\u2019 between the different groups. This has now been detailed in \u2018Data analysis\u2019 and \u2018\n<i>Eimeria tenella<\/i> 96-well plate format \n<i>in vitro<\/i> model is suitable to evaluate inhibition of invasion and development after treatment with traditional anticoccidials\u2019 sections. A new figures (Figure 3) has been added. \n<i>\u2018Comparison of groups evaluating differences in \u2018number of zoites\u2019 after anticoccidial drug treatment vs. controls was performed using non-parametric Kruskal-Wallis test. The analysis was followed by Dunnett\u2019s multiple comparisons as a post-hoc test. Statistically significant differences were established using a p&lt;0.05. All analyses were performed using GraphPad Prism version 6.00.\u2019<\/i> \n<i>\u2018Pre-treatment of sporozoites with different concentrations of AMP, ROB and SAL, three drugs with reported anticoccidial activity, showed a relative dose dependant effect ( Figure 3). There were no significant differences in the number of sporozoites able to invade host cells after the pre-treated groups vs. the controls at either 2 and 24 hpi ( Figure 3, A and B). However, treatment with the highest dose of AMP (50 <\/i>\n<i>\u03bc<\/i>\n<i>g\/ml) show a significant reduction in replication at both 44 and 52 hpi. Similarly, treatment with ROB (50 <\/i>\n<i>\u03bc<\/i>\n<i>g\/ml) exhibited a significant reduction in parasite numbers at 52 hpi ( Figure 3, C and D).\u2019<\/i> \n<i>\u2018<\/i>\n<i>Figure 3. Effects of anticoccidial drugs in intracellular numbers of parasites.<\/i> \n<i>A and B. Graphs shows the number of parasites by qPCR after invasion at 2 and 24 hpi, respectively (before nuclear division has started, see Figure 2); C and D. Graphs shows the number of parasites by qPCR after nuclear replication at 44 and 52 hpi, respectively. X-axis numbers represent the specific drug dilution used in <\/i>\n<i>\u03bc<\/i>\n<i>g\/ml. Asterisks indicate significant differences with the DMEM\/DMSO controls (Dunnett\u2019s multiple comparison test p&lt;0.05).\u2019<\/i>  \n<b>Further, there is no discussion whether these drugs and concentrations tested induce similar or different levels of inhibition in the miniaturised 96-well system compared with the prior standardised 24 well plate system.<\/b>  We have included the comparison of anticoccidial drugs pre-treatment between the 24 and 96-well plate format in the \u2018Discussion\u2019 section: \n<i>\u2018When the same anticoccidial drugs (AMP, ROB and SAL) were evaluated in the 24-well plate format, only concentrations of 5 <\/i>\n<i>\u03bc<\/i>\n<i>g\/ml were tested ( Marugan-Hernandez et al., 2020). Comparing this same concentration in the 96-well plate format, AMP and SAL showed equivalent effects in parasite numbers in invasion and replication for both models. AMP did not show any significant change regarding the controls at any point, whereas SAL did not show differences in invasion, but replication was significantly reduced at 44 hpi (Two-way ANOVA, followed by Dunnett\u2019s post-hoc test). ROB at 5 <\/i>\n<i>\u03bc<\/i>\n<i>g\/ml did not correlate with previous results from the 24-well plate model where strong effects were found in both invasion and replication. In the 96-well plate format, ROB did not show any effect in invasion or replication at 5 <\/i>\n<i>\u03bc<\/i>\n<i>g\/ml. Interestingly, this concentration has shown inhibitory effects in subsequent studies ( Felici et al., 2023) performed in the 96-well plate format, that were equivalent to the 24-well plate format described before. We attributed the lack of significant effects from ROB at 5 <\/i>\n<i>\u03bc<\/i>\n<i>g\/ml in the current study to a loss of effect due to continue use of the drug (where stock has gone several freeze-thaw rounds), which we have observed after using it as inhibition control in further studies.\u2019<\/i> &nbsp;  \n<b>Without statistical tests of the 3 replicates per condition to demonstrate meaningful levels of drug efficacy, and how reproducible between different experiments, and how consistent cf the 24-well original screen, it is not currently possible to conclude the 96-well plate system 'has been successfully miniaturised'.&nbsp;<\/b> \n<b>Please test that Eimeria infectivity \/ growth is not significantly different in 96- v 24-well plate systems.<\/b> \n<b>Please could the authors provide statistical tests to determine which drugs significantly reduce invasion and\/or replication in their miniaturised assay.<\/b> \n<b>Could the authors compare drug efficacy achieved in 24 vs 96 well assays with any or all drug compounds screened?<\/b>  All these points have been now included in the new version of the manuscript. Detailed responses can be seen above.","URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2#referee-comment-12517","order":2,"name":"referee-comment-12517","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.172468.r340270, Xiaokai Song, Nanjing Agricultural University, Nanjing, 23 Dec 2024, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/11-1135\/v2#referee-response-340270","order":3,"name":"referee-response-340270","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was funded by an NC3Rs Skills and Knowledge transfer grant \u2013 reference NC\/W000881\/1.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}