Search

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

Many pathogens can cause cancer, but cancer itself does not normally act as an infectious agent. However, transmissible cancers have been found in a few cases in nature: in Tasmanian devils, dogs, and several bivalve species. The transmissible cancers in dogs and devils are known to spread through direct physical contact, but the exact route of transmission of bivalve transmissible neoplasia (BTN) has not yet been confirmed. It has been hypothesized that cancer cells from bivalves could be released by diseased animals and spread through the water column to infect/engraft into other animals. To test the feasibility of this proposed mechanism of transmission, we tested the ability of BTN cells from the soft-shell clam (Mya arenaria BTN, or MarBTN) to survive in artificial seawater. We found that MarBTN cells are highly sensitive to salinity, with acute toxicity at salinity levels lower than those found in the native marine environment. BTN cells also survive longer at lower temperatures, with 50% of cells surviving greater than 12 days in seawater at 10 °C, and more than 19 days at 4 °C. With one clam donor, living cells were observed for more than eight weeks at 4 °C. We also used qPCR of environmental DNA (eDNA) to detect the presence of MarBTN-specific DNA in the environment. We observed release of MarBTN-specific DNA into the water of laboratory aquaria containing highly MarBTN-diseased clams, and we detected MarBTN-specific DNA in seawater samples collected from MarBTN-endemic areas in Maine, although the copy numbers detected in environmental samples were much lower than those found in aquaria. Overall, these data show that MarBTN cells can survive well in seawater, and they are released into the water by diseased animals. These findings support the hypothesis that BTN is spread from animal-to-animal by free cells through seawater.

Eastern oyster (Crassostrea virginica) aquaculture is increasingly playing a significant role in the state of Maine’s (USA) coastal economy. Here, we conducted a qPCR-based survey for Haplosporidium nelsoni, Perkinsus marinus, and Perkinsus chesapeaki in C. virginica (n = 1440) from six Maine sites during the summer–fall of 2016 and 2017. In the absence of reported die-offs, our results indicated the continued presence of the three protozoan parasites in the six sites. The highest H. nelsoni qPCR-prevalence corresponded to Jack’s Point and Prentiss Island ( x ¯ = 40 and 48% respectively), both located in the Damariscotta River Estuary. Jack’s Point, Prentiss Island, New Meadows River, and Weskeag River recorded the highest qPCR-prevalence for P. marinus (32–39%). While the P. marinus qPCR-prevalence differed slightly for the years 2016 and 2017, P. chesapeaki qPCR-prevalence in 2016 was markedly lower than 2017 (60% at all sites for each of the years, respectively). Mean qPCR-prevalence values for P. chesapeaki over the two-year study were ≥40% for samples from Jack’s Point (49%), Prentiss Island (44%), and New Meadows River (40%). This study highlights that large and sustained surveys for parasitic diseases are fundamental for decision making toward the management of the shellfish aquaculture industry, especially for having a baseline in the case that die-offs occur.

Shellfish are known as a potential source of Toxoplasma gondii (responsible for toxoplasmosis), and Cryptosporidium parvum, which is one of the major causes of gastroenteritis in the world. Here we performed a comprehensive qPCR-based monthly survey for T. gondii and C. parvum during 2016 and 2017 in oysters (Crassostrea virginica) (n = 1440) from all six sites along the coast of Maine (USA). Pooled samples (mantle, gills, and rectum) from individual oysters were used for DNA extraction and qPCR. Our study resulted in detections of qPCR positives oysters for T. gondii and C. parvum at each of the six sites sampled (in 31% and 10% of total oysters, respectively). The prevalence of T. gondii was low in 2016, and in September 2017 several sites peaked in prevalence with 100% of the samples testing positive. The prevalence of C. parvum was very low except in one estuarine location (Jack’s Point) in June 2016 (58%), and in October of 2016, when both prevalence and density of C. parvum at most of the sampling sites were among the highest values detected. Statistical analysis of environmental data did not identify clear drivers of retention, but there were some notable statistically significant patterns including current direction and nitrate along with the T. gondii prevalence. The major C. parvum retention event (in October 2016) corresponded with the month of highest dissolved oxygen measurements as well as a shift in the current direction revealed by nearby instrumentation. This study may guide future research to locate any contributing parasite reservoirs and evaluate the potential risk to human consumption.

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham's F12-5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham's F12-5% FBS- 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham's F12-5% FBS- 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications.

"Dermo" disease caused by the protozoan parasite Perkinsus marinus (Perkinsozoa) is one of the main obstacles to the restoration of oyster populations in the USA. Perkinsus 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 Perkinsus 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 Plasmodium falciparum. Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites. Six compounds with IC50 in the µM range were used to compare the degree of susceptibility in vitro of eight P. marinus strains from the USA and five Perkinsus species from around the world. The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species. Both Perkinsus marinus isolates and Perkinsus 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 Perkinsus proliferation in vitro and characterized Perkinsus phenotypes based on their resistance to six compounds. We also discuss the implications of these findings in the context of oyster management. The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for "Dermo" disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1-2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA(0)) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA(0) mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA(0) mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA(0) mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.

Bivalve molluscs are key components of the estuarine environments as contributors to the trophic chain, and as filter -feeders, for maintaining ecosystem integrity. Further, clams, oysters, and scallops are commercially exploited around the world both as traditional local shellfisheries, and as intensive or semi-intensive farming systems. During the past decades, populations of those species deemed of environmental or commercial interest have been subject to close monitoring given the realization that these can suffer significant decline, sometimes irreversible, due to overharvesting, environmental pollution, or disease. Protozoans of the genera Perkinsus, Haplosporidium, Marteilia, and Bonamia are currently recognized as major threats for natural and farmed bivalve populations. Since their identification, however, the variable publication rates of research studies addressing these parasitic diseases do not always appear to reflect their highly significant environmental and economic impact. Here we analyzed the peer- reviewed literature since the initial description of these parasites with the goal of identifying potential milestone discoveries or achievements that may have driven the intensity of the research in subsequent years, and significantly increased publication rates. Our analysis revealed that after initial description of the parasite as the etiological agent of a given disease, there is a time lag before a maximal number of yearly publications are reached. This has already taken place for most of them and has been followed by a decrease in publication rates over the last decade (20- to 30- year lifetime in the literature). Autocorrelation analyses, however, suggested that advances in parasite purification and culture methodologies positively drive publication rates, most likely because they usually lead to novel molecular tools and resources, promoting mechanistic studies. Understanding these trends should help researchers in prioritizing research efforts for these and other protozoan parasites, together with their development as model systems for further basic and translational research in parasitic diseases.

Galectins are characterized by their binding affinity for ß-galactosides, a unique binding site sequence motif, and wide taxonomic distribution and structural conservation in vertebrates, invertebrates, protista, and fungi. Since their initial description, galectins were considered to bind endogenous (self) glycans and mediate developmental processes and cancer. In the past few years, however, numerous studies have described the diverse effects of galectins on cells involved in both innate and adaptive immune responses, and the mechanistic aspects of their regulatory roles in immune homeostasis. More recently, however, evidence has accumulated to suggest that galectins also bind exogenous (non-self) glycans on the surface of potentially pathogenic microbes, parasites, and fungi, suggesting that galectins can function as pattern recognition receptors (PRRs) in innate immunity. Thus, a perplexing paradox arises by the fact that galectins also recognize lactosamine-containing glycans on the host cell surface during developmental processes and regulation of immune responses. According to the currently accepted model for non-self recognition, PRRs recognize pathogens via highly conserved microbial surface molecules of wide distribution such as LPS or peptidoglycan (pathogen-associated molecular patterns; PAMPs), which are absent in the host. Hence, this would not apply to galectins, which apparently bind similar self/non-self molecular patterns on host and microbial cells. This paradox underscores first, an oversimplification in the use of the PRR/PAMP terminology. Second, and most importantly, it reveals significant gaps in our knowledge about the diversity of the host galectin repertoire, and the subcellular targeting, localization, and secretion. Furthermore, our knowledge about the structural and biophysical aspects of their interactions with the host and microbial carbohydrate moieties is fragmentary, and warrants further investigation.