Parasite cryopreservation by vitrification

Parasitic protozoa and helminths and parasitic/vector insects each have distinct requirements for cryopreservation. Most parasitic protozoa respond to cryopreservation stresses similarly to other single cell suspensions, but few species are currently routinely cryopreserved by protocols specifically designed for vitrification. With slow equilibrium cooling, some protozoa osmotically dehydrated by solutes concentrated in the residual unfrozen fraction will survive by vitrifying. Several species of helminths, together with insect embryos cannot be cryopreserved by slow cooling protocols and have an absolute requirement for vitrification. Studies incorporating slow cooling and stepped cooling of both protozoa and helminths, particularly the intraerythrocytic stages of malaria and the schistosomula larvae of Schistosoma mansoni have aided in the design of vitrification protocols for parasites. For helminths, the most widely used cryopreservation protocol, originally successful for cryopreserving S. mansoni schistosomula, consists of the addition of ethanediol in two steps, followed by rapid cooling (approximately 5100 degrees C min(-1)) to -196 degrees C. This technique exploits the temperature-dependent differential in permeability of the cryoprotectant additive (CPA) to first permeate into the organism at 37 degrees C followed by a dehydration-mediated internal CPA increase in concentration resulting from incubation in a second higher CPA concentration at 0 degree C. Samples are rapidly warmed/diluted (approximately 14,000 degrees C min(-1)) to recover the organisms from liquid nitrogen storage. Variations on this technique have also been successful in cryopreserving the larvae and adult worms of filariae, muscle stage larvae of Trichinella spp., the infective stages of gastro-intestinal nematode parasites and insect embryos. Other protocols where the dehydration step precedes CPA addition have been used to cryopreserve entomogenous nematode larvae by vitrification. Techniques that utilize high concentrations of CPA cocktails and slower cooling, developed for the vitrification of mammalian embryos, have been applied to the cryopreservation of parasitic protozoa, but with limited success to date. Where cryopreservation by classical slow cooling methods is possible, vitrification has enhanced the levels of survival obtained. And vitrification has enabled the successful cryopreservation of those parasitic species not able to be cryopreserved by traditional methods. Since a limited number of parasitic organisms has been cryopreserved using vitrification protocols, there is considerable scope for further improvement in the cryopreservation techniques used for many parasitic species.     

Sensitivity of parasites to free radical damage by antiparasitic drugs

Over the last few years a remarkable progress has been made in the understanding of parasites biochemistry, molecular biology, and immunology. This progress is especially encouraging in that emphasis on drug development is shifting from random screening towards a more rational approach. A number of peculiar aspects characteristic of parasites which are not present in other organisms and that might be exploitable for the design of specific agents have been described recently. One of these aspects is their deficiency in defense mechanisms against oxygen toxicity. Catalase is absent in many parasites. Distinct superoxide dismutases have been detected and specific inhibitors of these enzymes have been investigated. Glutathione is absent in some anaerobic protozoa. Peroxidase and reductase activities dependent on a glutathione-spermidine cofactor termed trypanothione have been detected in several trypanosomatids and apparently replace the glutathione peroxidase-glutathione reductase system of other eukaryotic cells. Free radical intermediates have been shown to be involved in the reaction of enzymes present in anaerobic protozoa. In addition, a number of antiparasitic agents have been shown to exert their actions through a free radical metabolism: nitro compounds used against trypanosomatids, anaerobic protozoa and helminths; crystal violet used in blood banks to prevent blood transmission of Chagas' disease; the antimalarial primaquine, chloroquinine, and quinhasou; and quinones active in vitro and in vivo against different parasites.     

Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long‐term

Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. For most microbial strains, cryopreservation in the vapour phase above liquid nitrogen provides the most stable and long‐term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage in the vapour phase above liquid nitrogen on a diverse collection of Fusarium species. In this work, a collection of 374 Fusarium strains from the Fungal Genetics Stock Center, including 24 unique species, were duplicated and sent to the National Laboratory for Genetic Resource Preservation for storage in the vapour phase above liquid nitrogen. After 5 years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells and measuring the rate of growth of each isolate. Additionally, the rate of growth for ～10% of the isolates were compared with the same isolates which had been stored at ?80°C at the Fungal Genetics Stock Center over the same timeframe to determine if cryopreservation in liquid nitrogen vapour provided a comparable method of storage. All National Laboratory for Genetic Resources Preservation isolates grew after being stored at ?165°C for 5 years. In general, the isolates that were stored at ?165°C grew at a faster rate than the isolates stored at ?80°C for the same period. Of the isolates stored at ?165°C, most had greater than 80% cell viability, however, those isolates that had less than 50% cell viability generally also had fewer conidia germinate. These isolates may be at a greater risk for storage over longer times. In conclusion, storage at ?165°C liquid nitrogen provided reliable preservation of a diverse collection of Fusarium spp. over 5 years, and culture viability data indicates that they will remain viable during additional storage for longer periods.     

Cysteine proteinases of parasitic protozoa

Proteinases are involved with many processes in living organisms. In recent years, there has been increasing interest in elucidating the functions the enzymes perform in parasites. These studies have revealed that one class of proteinases, the cysteine proteinases, predominates in many parasitic protozoa. In this article Mick North, Jeremy Mottram and Graham Coombs review what is known about the cysteine proteinases of parasitic protozoa and discuss the approaches being pursued in attempts to design antiparasite drugs based on inhibitors or substrates of these enzymes.     

Cell death and human intestinal protozoa: a brief overview

Protozoan programmed cell death or apoptosis is an important factor in the survival of the parasite and its pathogenicity. The most amazing aspect of protozoan cell death is in its molecular architecture. To date, protozoa lack most of the components of the highly complex cell death machinery studied in multicellular organisms. Hence the unique apoptotic machinery in protozoa can be exploited for the development of therapeutic drugs and diagnostic markers. This review focuses on human intestinal protozoa undergoing cell death and inducing or inhibiting host cell apoptosis. The first part of this review focuses on intestinal protozoa that undergo PCD under various stress conditions. The second part focuses on protozoa that induce or inhibit PCD in their host cell. Although these intestinal parasites differ in their mechanism of infection and intracellular localization, they may activate conserved cell death pathways within themselves and in the host cell. Understanding conserved cell death pathways in the intestinal protozoa and their host-parasite PCD relationship may lead to drug targets which can be used for a broad range of parasitic diseases.     

Regulation of CD8 T cell responses to infection with parasitic protozoa

There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8⁺ T cell response. This review will highlight some of the key studies that established that CD8⁺ T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8⁺ T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC-Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8⁺ T cell responses are initiated and new insights into their phenotypic plasticity     

Two-step freezing procedure for cryopreservation of rumen ciliates,an effective tool for creation of a frozen rumen protozoa bank

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25 degrees C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30 degrees C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2 degrees C/min and 2.5 degrees C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.     

Recent advances in human protozoan parasites of the gastrointestinal tract

An attempt was made in this report to present an update on the recent development on intestinal protozoan infections in humans. Except for a few historical references the review covers the period from 1980 to the time of writing, mid-1985. The emphasis was on the more important parasites and an effort made to cover the latest developments in their biology, epidemiology and pathogenesis. During preparation of this paper I was impressed with the plethora of papers published on some parasites and the paucity of reports on others. There are an increasing number of papers on Cryptosporidium sp. and the interest in the organisms should continue. Furthermore, it will be of interest to follow the association between Blastocystis hominis and disease. These are essentially new protozoan parasites of man, and one wonders how many more intestinal protozoan parasitosis are still waiting to be found. Like the Cryptosporidium sp., it may be a matter of finding the right diagnostic technique to detect the unknown organism.Giardiasis continues to be a cause of diarrhea among various groups especially campers who are drinking untreated water and G. lamblia as well as E. histolytica are being found more frequently in homosexuals with and without AIDS. The ability to predict virulence in strains of E. histolytica by enzyme patterns is intriguing but some skeptics still prefer the older test for virulence by cecal scoring in animals. New animal models are being evaluated and new techniques applied to the study of pathogenic protozoa. In the future the use of new biotechnological methods will most certainly lead to a better understanding of intestinal protozoa as well as of other parasitic organisms.     

Coprozoic Protozoa from Wyoming Mammals

SYNOPSIS. Coprozoic protozoa from elk, bison, bear, moose, coyote, marmot, cattle, horse, sheep and man were kept in feces at 4° C. for 2–6 months. During that time in most of the samples there developed the flagellates Cercomonas sp., Copromonas ruminantium and Monas communis; the amoebae Vahlkampfia sp. and Sappinia diploidea , and ciliates of the Nyctotherus type, and an unidentified smaller species. There was a correlation between numbers of protozoa and bacteria. The same species of protozoa in soil or in soil mixed with boiled feces failed to live. Coprozoic protozoa may require certain essential metabolites from bacteria as do true parasites. The wide variation in appearance of cysts made it practically impossible to identify the protozoa with certainty in that stage. Reliance had to be on motile forms which readily developed in the cold cultures.     

Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture.     

Rumen ciliates: their metabolism and relationships with bacteria and their hosts

To explore the ruminal ecosystem, this paper is an attempt to illustrate the biochemistry and the metabolism of rumen protozoa. Different culture techniques (gnotobiotic, axenic, continuous culture) of ciliate protozoa are reviewed. The ability of ciliates to invade plant tissues, their association and the degradation of plant cell constituents (cellulose, hemicellulose, pectin, glycolipids), and the use of starch and soluble sugars for polysaccharide storage are determined. The interrelationships between ciliate protozoa and bacteria are discussed. These include the enzymic degradation of the engulfed plant particles, the bacterial nitrogen (amino acids, nucleic acids), and vitamins and trace element supplies as nutrient sources for protozoa. The composition of the rumen ciliate population, controlled by phylogenetic factors, geographical distribution, feeding habits and physiological status of the host, by competition between microorganisms and by the diet (nature of the diet, number of meals), is related. The transmission and establishment of the microfauna and its contribution to the microbial nitrogen available for the host are discussed.     

Fast freezing of cow embryos in French straws with an automatic program

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.     

Gastrointestinal parasites of the grasscutter (Thryonomys swinderianus, Temminck 1827) on the Accra Plains of Ghana

The gastrointestinal parasites of the grasscutter were investigated between 1996 and 2006 using 180 grasscutters from the Accra Plains. The aim of the study was to develop strategies for the control of the parasites under captive management. The gastrointestinal tracts of the animals were examined for the presence of both helminth and protozoan parasites with the aid of a hand lens, the direct smear and Willis Flotation techniques. In all, fourteen species of helminths were found including twelve nematodes and two cestodes. Trichuris muris was the most common nematode encountered, whilst Hepatocola hepatica was the least commonly encountered nematode. Three genera of protozoans, viz, Trichomonas spp, Giardia spp and Eimeria spp were also encountered, with Giardia spp and Trichomonas spp being reported for the first time in the grasscutter. Eimeria spp were the most prevalent protozoa. The widespread occurrence of protozoal and helminthic parasites in the grasscutter suggests that routine treatment of grasscutters with coccidiostats, anti-flagellates and anthelminthics such as dimetridazole and albendazole may contribute significantly to improve productivity of animals and reduce the pressure on wild populations.     

Cell invasion by un-palatable parasites

While some intracellular pathogens invade and replicate exclusively in phagocytic host cells, others have evolved mechanisms to stimulate their uptake by cells not equipped with well-developed phagocytic machinery. A common mechanism utilized by bacteria involves the induction of macropinocytosis, or of other F-actin-driven processes which result in engulfment of the pathogen through formation of a plasma membrane-derived vacuole. Interestingly, this type of 'induced phagocytosis' mechanism does not appear to be utilized by protozoan parasites, which are significantly larger than bacteria in size (about 5–10 μm in average length). Intracellular protozoa either restrict themselves to infecting 'professional' phagocytes (one example is the trypanosomatid Leishmania ), or utilize highly unusual mechanisms for gaining access to the intracellular environment. Here we discuss what has been revealed in recent years about the remarkable cell invasion strategies of two highly successful intracellular parasites: Toxoplasma gondii and Trypanosoma cruzi . Toxoplasma utilizes a distinct form of actin/myosin-dependent gliding motility to propel itself into mammalian cells, while T. cruzi invades by subverting a Ca²⁺-regulated lysosomal exocytic pathway.     

The CRISPR/Cas9 system sheds new lights on the biology of protozoan parasites

Applied Microbiology and Biotechnology - The CRISPR/Cas9 system, a natural defence system of bacterial organisms, has recently been used to modify genomes of the most important protozoa parasites....     

The Truman Show for protozoan parasites: A review of in vitro cultivation platforms

Protozoan parasites are responsible for severe disease and suffering in humans worldwide. Apart from disease transmission via insect vectors and contaminated soil, food, or water, transmission may occur congenitally or by way of blood transfusion and organ transplantation. Several recent outbreaks associated with fresh produce and potable water emphasize the need for vigilance and monitoring of protozoan parasites that cause severe disease in humans globally. Apart from the tropical parasite Plasmodium spp., other protozoa causing debilitating and fatal diseases such as Trypanosoma spp. and Naegleria fowleri need to be studied in more detail. Climate change and socioeconomic issues such as migration continue to be major drivers for the spread of these neglected tropical diseases beyond endemic zones. Due to the complex life cycles of protozoa involving multiple hosts, vectors, and stringent growth conditions, studying these parasites has been challenging. While in vivo models may provide insights into host–parasite interaction, the ethical aspects of laboratory animal use and the challenge of ready availability of parasite life stages underline the need for in vitro models as valid alternatives for culturing and maintaining protozoan parasites. To our knowledge, this review is the first of its kind to highlight available in vitro models for protozoa causing highly infectious diseases. In recent years, several research efforts using new technologies such as 3D organoid and spheroid systems for protozoan parasites have been introduced that provide valuable tools to advance complex culturing models and offer new opportunities toward the advancement of parasite in vitro studies. In vitro models aid scientists and healthcare providers in gaining insights into parasite infection biology, ultimately enabling the use of novel strategies for preventing and treating these diseases.     

Hematozoa in endemic subspecies of common kestrel in the Cape Verde Islands

We examined 130 Common Kestrel (Falco tinnunculus) representing two endemic subspecies and nine resident island populations on the Cape Verde archipelago between 1996 and 1999 to study diversity, prevalence, and intensity of hematozoa. Hematozoan diversity was very low; we detected only Plasmodium fallax, a species that is rarely found in Falconoformes, and, possibly, Haemoproteus brachiatus. Moreover, prevalence of Plasmodium fallax was low (1.5%) with a mean intensity of infection of 0.05 protozoa/10(-3) erythrocytes. Only one bird (0.8%) was infected with a gametocyte that was most likely Haemoproteus brachiatus; the intensity in this infected bird was 1.5 protozoa/10(-3) erythrocytes. A single parasite or two parasites were observed in blood smears in four additional birds, but identification to genus was not possible. This is the first record of blood parasites in birds on the Cape Verde Archipelago. The low prevalence of these parasites might be because of arid and less-favorable conditions for the pathogen's vectors. The sedentary nature and high level of isolation of the island kestrel populations are also factors that could decrease the probability of infection.     

Parasites and human evolution

Our understanding of human evolutionary and population history can be advanced by ecological and evolutionary studies of our parasites. Many parasites flourish only in the presence of very specific human behaviors and in specific habitats, are wholly dependent on us, and have evolved with us for thousands or millions of years. Therefore, by asking when and how we first acquired those parasites, under which environmental and cultural conditions we are the most susceptible, and how the parasites have evolved and adapted to us and we in response to them, we can gain considerable insight into our own evolutionary history. 1 , 2 As examples, the tapeworm life cycle is dependent on our consumption of meat,³ the divergence of body and head lice may have been subsequent to the development of clothing, 4 , 5 and malaria hyperendemicity may be associated with agriculture. 6 Thus, the evolutionary and population histories of these parasites are likely intertwined with critical aspects of human biology and culture. Here I review the mechanics of these and multiple other parasite proxies for human evolutionary history and discuss how they currently complement our fossil, archeological, molecular, linguistic, historical, and ethnographic records. I also highlight potential future applications of this promising model for the field of evolutionary anthropology.     

Observations on Bacterial Feeding by the Rumen Ciliate Isotricha prostoma

SYNOPSIS. Starvation of Isotricha prostoma for 72–96 hours decreased the cellular amylopectin granules and facilitated the microscopic search for bacterial feeding. I. prostoma selected and ingested only certain rods from among many types of rumen bacteria. In order to isolate the bacteria important as a food source for Isotricha , the starved protozoa were allowed to feed on mixed rumen bacteria, washed, and the crushed protozoan contents quickly cultured for bacteria. Several strains of bacteria were isolated in pure culture. Three of the rod strains isolated were rapidly ingested by I. prostoma when fed to the ciliate. In a monobacterial culture I. prostoma divided once before succumbing.     

Epidemiology of Dientamoeba fragilis Jepps and Dobell, 1918. 3. Further studies on Enterobius transmission through eggs]

In two new attempts to infect human subjects with pin-worms that were infested, at that time, with protozoa Dientamoeba fragilis, presence of these protoza in the intestinal canal was observed in one case for more than 84 days, and in another case for more than 31 days after contamination. The duration of latent periods fixed for Enterobius vermicularis was almost identical (51 and 50 days). New attempts implement and confirm the results of the first successful attempts to transfer the infection (Ockert, 1972b) as well as the hypothesis of the epidemiological interrelation between both parasites (Burrows and Swerdlow 1956).