Host-free Culture Research Articles

The transcriptome from asexual to sexual in vitro development of Cystoisospora suis (Apicomplexa: Coccidia)

The apicomplexan parasite Cystoisospora suis is an enteropathogen of suckling piglets with woldwide distribution. As with all coccidian parasites, its lifecycle is characterized by asexual multiplication followed by sexual development with two morphologically distinct cell types that presumably fuse to form a zygote from which the oocyst arises. However, knowledge of the sexual development of C. suis is still limited. To complement previous in vitro studies, we analysed transcriptional profiles at three different time points of development (corresponding to asexual, immature and mature sexual stages) in vitro via RNASeq. Overall, transcription of genes encoding proteins with important roles in gametes biology, oocyst wall biosynthesis, DNA replication and axonema formation as well as proteins with important roles in merozoite biology was identified. A homologue of an oocyst wall tyrosine rich protein of Toxoplasma gondii was expressed in macrogametes and oocysts of C. suis. We evaluated inhibition of sexual development in a host-free culture for C. suis by antiserum specific to this protein to evaluate whether it could be exploited as a candidate for control strategies against C. suis. Based on these data, targets can be defined for future strategies to interrupt parasite transmission during sexual development.

Growth of ‘Candidatus Liberibacter asiaticus’ in a host-free microbial culture is associated with microbial community composition

'Candidatus Liberibacter asiaticus' ('Ca. L. asiaticus'), the suspected causative agent of citrus greening disease, is one of many phloem-restricted plant pathogens that have not been isolated and grown in an axenic culture. In this study, infected Asian citrus psyllids were used to prepare a host-free source of 'Ca. L. asiaticus'. Host-free mixed microbial cultures of 'Ca. L. asiaticus' were grown in the presence of various antibiotic treatments to alter the composition of the microbial communities. Our hypothesis was that the presence of selected antibiotics would enhance or reduce the presence of 'Ca. L. asiaticus' in a host-free culture composed of a mixed bacterial population through changes in the microbial community structure. We determined how 'Ca. L. asiaticus' growth changed with the various treatments. Treatment with vancomycin (50 μg/mL), streptomycin (0.02 μg/mL), or polymyxin B (4 μg/mL) was associated with an increased abundance of 'Ca. L. asiaticus' of 7.35 ± 0.27, 5.56 ± 0.15, or 4.54 ± 0.83 fold, respectively, compared to untreated mixed microbial cultures, while treatment with 100 μg/mL vancomycin; 0.5, 1, or 2 μg/mL streptomycin; or 0.5 μg/mL of polymyxin B was associated with reduced growth. In addition, the growth of 'Ca. L. asiaticus' was associated with the microbial community composition of the mixed microbial cultures. A positive relationship between the presence of the Pseudomonadaceae family and 'Ca. L. asiaticus' growth was observed, while the presence of 'Ca. L. asiaticus' was below the detection limit in cultures that displayed high abundances of Bacillus cereus. Our findings offer strategies for developing effective axenic culture conditions and suggest that enrichment of the Bacillaceae family could serve as a paratransgenic approach to controlling citrus greening disease.

Host-Free Systems for Differentiation of Axenic Leishmania.

This chapter describes, in detail, the method our laboratory developed to differentiate L. donovani promastigotes into amastigotes in a host-free culture. This method is based on previous observations that Leishmania promastigotes can combine two environmental signals, typical to lysosomes, acidic pH (~5.5) and body temperature (37°C), into a signal that induces differentiation. Based on this concept, we have modified medium 199 to make it into an amastigote-specific medium. Shifting promastigotes to this medium, followed by incubation in a CO2 incubator, induced differentiation. Axenic amastigotes reach maturation within 5days, resembling the time it takes in vivo. This chapter provides a complete protocol that should be useful for both Old and New World species of Leishmania.

Microspongium alariae in Alaria esculenta: a widely-distributed non-parasitic brown algal endophyte that shows cell modifications within its host

Abstract Alaria esculenta is an important kelp species in northern Europe, Atlantic Canada and USA and the Arctic, with high economic potential. Microspongium alariae, a brown algal endophyte using A. esculenta as host, is reported for the first time from Scotland (Great Britain) and Brittany (France), suggesting a wide distribution in NW Europe. The alga was found growing epi-endophytically in A. esculenta stipes and was occasionally associated with warts. Isolated Microspongium thalli grew in host-free cultures and formed plurilocular sporangia in a broad range of temperature and irradiance conditions. DNA barcoding using the nuclear ribosomal ITS1, the mitochondrial COI and the plastidial rbcL confirmed the identity of the endophyte as M. alariae. Electron microscopy was used to compare the alga when endophytic in Alaria with a host-free culture. As an endophyte, cell diameter, pyrenoid diameter and cell wall thickness were reduced. In contrast, there were more plasmodesma connections between endophyte cells, possibly to enhance nutrient transport along the endophytic thallus. In the light of this evidence, a parasitic life style is considered unlikely for the species and the adaptive value of endophytism in M. alariae remains to be elucidated.

Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum

Leishmania parasites cycle between the alimentary tract of a sandfly vector as free-living promastigotes and the acidified phagolysosomes of the vertebrate host macrophage as aflagellated amastigotes. The differentiation process can be mimicked in host-free culture by switching promastigotes (e.g. 25°C, neutral pH) to a phagolysosomal-like environment (e.g. 37°C, acidic pH and 5% CO2) for certain, but not all Leishmania species. Axenically grown amastigotes have been shown to share several morphological and biochemical characteristics with macrophage-derived intracellular amastigotes. In this study, we used a DNA oligonucleotide full-genome array to compare global RNA expression profiling of Leishmania infantum axenic amastigotes to intracellular amastigotes derived from infected macrophages. In general, 40% more genes (518 genes vs. 309 genes) were found upregulated in axenic amastigotes compared to intracellular amastigotes. Comparisons in expression profiling between axenic amastigotes and intracellular amastigotes revealed substantial differences in regulated mRNA abundance. Remarkably, among the differentially upregulated transcripts only 12% were common to both amastigote preparations. The major differences between axenic and intracellular amastigotes were observed in metabolic process, especially in fatty acid metabolism, in intracellular transport and membrane vesicular fusion, in proteolysis, in the number and type of protein kinases and RNA binding proteins and in the response to oxidative stress. These findings highlight the importance of the host macrophage in driving the parasite to specific adaptations, which consequently result in highly regulated changes in gene expression.

Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni.

The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism's complex molluscan-mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.