Secondary <i>Perkinsus marinus</i> growth-inhibition screen (IC₅₀).

<p>Biological triplicate cultures were exposed to an 8 -point dose-response curve (10 µM to 0.156 µM). The effect of the drugs on <i>P. marinus</i> proliferation was evaluated as above.</p

Drug discovery against Dermo disease.

<p>(A) Time line for the discovery of drugs against Dermo disease, starting when the etiological agent was described until this study. Most of the discoveries did happen after the development of the culture methodologies for <i>Perkinsus</i> spp. in 1993 and most studies have been carried out in <i>in vitro</i> cultures. (B) Percentage of the compounds active against <i>Perkinsus</i> based on their chemical nature. (C) Percentage of available compounds against Dermo tested in <i>Perkinsus marinus</i> and <i>Perkinsus olseni</i>.</p

Identification of MMV Malaria Box Inhibitors of <i>Perkinsus marinus</i> Using an ATP-Based Bioluminescence Assay

<div><p>“Dermo” disease caused by the protozoan parasite <i>Perkinsus marinus</i> (Perkinsozoa) is one of the main obstacles to the restoration of oyster populations in the USA. <i>Perkinsus</i> spp. are also a concern worldwide because there are limited approaches to intervention against the disease. Based on the phylogenetic affinity between the Perkinsozoa and Apicomplexa, we exposed <i>Perkinsus</i> trophozoites to the Medicines for Malaria Venture Malaria Box, an open access compound library comprised of 200 drug-like and 200 probe-like compounds that are highly active against the erythrocyte stage of <i>Plasmodium falciparum</i>. Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against <i>P. marinus</i> trophozoites. Six compounds with IC₅₀ in the µM range were used to compare the degree of susceptibility <i>in vitro</i> of eight <i>P. marinus</i> strains from the USA and five <i>Perkinsus</i> species from around the world. The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across <i>Perkinsus</i> strains and species. Both <i>Perkinsus marinus</i> isolates and <i>Perkinsus</i> spp. presented different patterns of response to the panel of compounds tested, supporting the concept of strain/species variability. Here, we expanded the range of compounds available for inhibiting <i>Perkinsus</i> proliferation <i>in vitro</i> and characterized <i>Perkinsus</i> phenotypes based on their resistance to six compounds. We also discuss the implications of these findings in the context of oyster management. The <i>Perkinsus</i> system offers the potential for investigating the mechanism of action of the compounds of interest.</p></div

<i>Perkinsus</i> spp. and <i>Perkinsus marinus</i> strains used in the study.

<p><i>Perkinsus marinus</i> PRA240 was used for the primary screen. A total of eight <i>P. marinus</i> strains isolated from oysters from the East and Gulf Coast of the USA and five <i>Perkinsus</i> spp. from around the world were used for the secondary screen. In all the cases cultures were maintained in Dulbecco modified Eagle's: Ham's F12 (1∶2) supplemented with 5% fetal bovine serum.</p><p>*<i>Perkinsus qugwadi</i> and <i>Perkinsus beihaiensis</i> have never been available in culture <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111051#pone.0111051-Blackbourn1" target="_blank">[57]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111051#pone.0111051-Moss1" target="_blank">[58]</a>.</p><p><i>Perkinsus</i> spp. and <i>Perkinsus marinus</i> strains used in the study.</p

List of compounds active against <i>Plasmodium falciparum</i> selected for the MMV Malaria Box (http://www.mmv.org/malariabox) for secondary <i>Perkinsus marinus</i> growth-inhibition screen (IC₅₀) and <i>Perkinsus marinus</i> strain and <i>Perkinsus</i> species sensitivity.

<p>*<i>Plasmodium falciparum</i> 3D7;</p><p>**<i>Perkinsus marinus</i> PRA240 primary screen.</p><p>List of compounds active against <i>Plasmodium falciparum</i> selected for the MMV Malaria Box (<a href="http://www.mmv.org/malariabox" target="_blank">http://www.mmv.org/malariabox</a>) for secondary <i>Perkinsus marinus</i> growth-inhibition screen (IC₅₀) and <i>Perkinsus marinus</i> strain and <i>Perkinsus</i> species sensitivity.</p

Percentage of inhibition of <i>Perkinsus marinus</i> using the MMV Malaria Box.

<p>Biological triplicate cultures were grown in sterile 96-well plates (100 µl; 2.0×10⁶ cells/ml) and cells were exposed to the MMV Malaria Box (20 µM). The effect of the drugs on <i>P. marinus</i> proliferation was evaluated using the ATPlite assay at day 4 post-exposure to the selected drugs. Readings for each concentration were normalized to the control wells with each solvent (100% activity). A total of 122 (67.0%) compounds resulted in at least 50% inhibition; from these compounds, 13 (7.1%) resulted in at least 90% inhibition (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111051#pone-0111051-g002" target="_blank">Figure 2</a>).</p

Figure 3

<p>Distribution by continent of published papers in the literature reporting in the genera <i>Perkinsus</i>, <i>Haplosporidium</i>, <i>Marteilia</i>, and <i>Bonamia</i> (A). Countries where <i>Perkinsus</i> spp. have been reported by 2013 (B).</p

Search strings for protozoan parasites and bivalve groups.

<p>*Abalones, mussels, cockles, and scallops, which can also be infected by some of these protozoan parasites, were not included in the host search for percent of literature reporting diseases.</p

Figure 1

<p>Number of papers in the literature (SCOPUS Database) reporting in the genera <i>Perkinsus</i>, <i>Haplosporidium</i>, <i>Marteilia</i>, and <i>Bonamia</i>; column in orange correspond to 1993, the year when the methodologies for culturing <i>Perkinsus</i> were published (A). Cumulative papers over the same period of time (B). Percent of the literature reporting each particular protozoan parasite (C).</p

Number of papers published every year (1960–2013) and cumulative papers (inset figures) in the literature (SCOPUS Database) using <i>Plasmodium</i> and <i>Cryptosporidium</i> as search strings.

<p>Columns in red correspond to the years when the genomes were published.</p