ABSTRACT
The honey bee Apis mellifera is exposed to a variety of biotic and abiotic stressors, such as the highly prevalent microsporidian parasite Nosema (Vairimorpha) ceranae and neonicotinoid insecticides. Both can affect honey bee physiology and microbial gut communities, eventually reducing its lifespan. They can also have a combined effect on the insect's survival. The use of bacterial probiotics has been proposed to improve honey bee health, but their beneficial effect remains an open question. In the present study, western honey bees were experimentally infected with N. ceranae spores, chronically exposed to the neonicotinoid thiamethoxam, and/or supplied daily with the homofermentative bacterium Pediococcus acidilactici MA18/5M thought to improve the honey bees' tolerance to the parasite. Deep shotgun metagenomic sequencing allowed the response of the gut microbiota to be investigated with a taxonomic resolution at the species level. All treatments induced significant changes in honey bee gut bacterial communities. Nosema ceranae infection increased the abundance of Proteus mirabilis, Frischella perrara, and Gilliamella apicola and reduced the abundance of Bifidobacterium asteroides, Fructobacillus fructosus, and Lactobacillus spp. Supplementation with P. acidilactici overturned some of these alterations, bringing back the abundance of some altered species close to the relative abundance found in the controls. Surprisingly, the exposure to thiamethoxam also restored the relative abundance of some species modulated by N. ceranae. This study shows that stressors and probiotics may have an antagonistic impact on honey bee gut bacterial communities and that P. acidilactici may have a protective effect against the dysbiosis induced by an infection with N. ceranae.
ABSTRACT
Anncaliia algerae belongs to microsporidia, a group of obligate intracellular parasites related to fungi. These parasites are largely spread in water and food-webs and can infect a wide variety of hosts ranging from invertebrates to vertebrates including humans. In humans, microsporidian infections are mainly opportunistic as immunocompetent hosts can clear parasites naturally. Recent studies however have reported persistent microsporidian infections and have highlighted them as a risk factor in colon cancer. This may be a direct result of cell infection or may be an indirect effect of the infectious microenvironment and the host's response. In both cases, this raises the question of the effects of microsporidian infection at the host and host-cell levels. We aimed to address the question of human host intracellular response to microsporidian infection through a transcriptomic kinetic study of human foreskin fibroblasts (HFF) infected with A.algerae, a human infecting microsporidia with an exceptionally wide host range. We focused solely on host response studying both coding and small non-coding miRNA expression. Our study revealed a generalized down-regulation of cell miRNAs throughout infection with up to 547 different miRNAs downregulated at some timepoints and also transcriptomic dysregulations that could facilitate parasite development with immune and lipid metabolism genes modulation. We also hypothesize possible small nucleic acid expropriation explaining the miRNA downregulation. This work contributes to a better understanding of the dialogue that can occur between an intracellular parasite and its host at the cellular level, and can guide future studies on microsporidian infection biology to unravel the mode of action of these minimalist parasites at the tissue or host levels.We have also generated a kinetic and comprehensive transcriptomic data set of an infectious process that can help support comparative studies in the broader field of parasitology. Lastly, these results may warrant for caution regarding microsporidian exposure and persistent infections.
ABSTRACT
Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae. While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae.
Subject(s)
Fungal Proteins , Microsporidia , Animals , Fungal Proteins/genetics , Fungal Proteins/metabolism , Microsporidia/genetics , Microsporidia/metabolism , Cytoplasm/metabolism , Organelles/metabolismABSTRACT
Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.
Subject(s)
Biological Products , Dexrazoxane , Microsporidia , Microsporidiosis , Animals , Caenorhabditis elegans , Cell Proliferation , HumansABSTRACT
Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.
Subject(s)
Encephalitozoon cuniculi/chemistry , Fungal Proteins/physiology , Microsporidia/chemistry , Ricin/metabolism , Animals , Cell Line , Computational Biology , Dogs , Encephalitozoon cuniculi/genetics , Encephalitozoon cuniculi/immunology , Humans , Madin Darby Canine Kidney Cells , Microsporidia/genetics , Microsporidia/immunology , Rabbits , Recombinant Proteins/genetics , Spores, Fungal/immunology , Spores, Fungal/isolation & purificationABSTRACT
Plant defense inducers (PDIs) are booming and attractive protection agents designed to immunostimulate the plant to reduce subsequent pathogen colonization. The structure-PDI activity relationships of four flavan-3-ols: Epicatechin (EC), Epigallocatechin (EGC), Epicatechin gallate (ECG), Epigallocatechin gallate (EGCG) and Gallotannic acid (GTA) were investigated in both whole plant and suspension cell systems. ECG, EGCG, and GTA displayed elicitor activities. Their infiltration into tobacco leaves induced hypersensitive reaction-like lesions with topical scopoletin and PR-target transcript accumulations. On the contrary, EC and EGC infiltrations fail to trigger the biochemical changes in tobacco tissues. The tobacco BY-2 cells challenged with ECG, EGCG, or GTA led to alkalinization of the BY-2 extracellular medium while EC and EGC did not trigger any pH variation. This work provides evidence that the esterified gallate pattern is as an essential flavonoid entity to induce plant defense reactions in tobacco. The phytoprotective properties of the esterified gallate-free EC and the esterified gallate-rich GTA were evaluated on the tobacco/Phytophthora parasitica var. nicotianae (Ppn) pathosystem. Tobacco treatment with EC did not induce significant protection against Ppn compared to GTA which shows antimicrobial properties on Ppn and decreases the infection on GTA-infiltrated and -sprayed wild-type leaves. GTA protection was impaired in the transgenic NahG tobacco plants, suggesting that protection was mediated by salicylic acid.
Subject(s)
Flavonoids/chemistry , Flavonoids/pharmacology , Nicotiana/drug effects , Plant Diseases/immunology , Catechin/analogs & derivatives , Catechin/chemistry , Catechin/pharmacology , Molecular Structure , Phytophthora/physiology , Plant Diseases/microbiology , Plant Leaves/drug effects , Plant Leaves/immunology , Plant Leaves/microbiology , Nicotiana/immunology , Nicotiana/microbiologyABSTRACT
Nosema ceranae is an emerging and invasive gut pathogen in Apis mellifera and is considered as a factor contributing to the decline of honeybee populations. Here, we used a combined LC-MS and NMR approach to reveal the metabolomics changes in the hemolymph of honeybees infected by this obligate intracellular parasite. For metabolic profiling, hemolymph samples were collected from both uninfected and N. ceranae-infected bees at two time points, 2â¯days and 10â¯days after the experimental infection of emergent bees. Hemolymph samples were individually analyzed by LC-MS, whereas each NMR spectrum was obtained from a pool of three hemolymphs. Multivariate statistical PLS-DA models clearly showed that the age of bees was the parameter with the strongest effect on the metabolite profiles. Interestingly, a total of 15 biomarkers were accurately identified and were assigned as candidate biomarkers representative of infection alone or combined effect of age and infection. These biomarkers included carbohydrates (α/ß glucose, α/ß fructose and hexosamine), amino acids (histidine and proline), dipeptides (Glu-Thr, Cys-Cys and γ-Glu-Leu/Ile), metabolites involved in lipid metabolism (choline, glycerophosphocholine and O-phosphorylethanolamine) and a polyamine compound (spermidine). Our study demonstrated that this untargeted metabolomics-based approach may be useful for a better understanding of pathophysiological mechanisms of the honeybee infection by N. ceranae.
Subject(s)
Bees/metabolism , Hemolymph/chemistry , Host-Pathogen Interactions , Metabolomics/methods , Nosema/physiology , Animals , Bees/chemistry , Chromatography, High Pressure Liquid/methods , Magnetic Resonance Spectroscopy/methods , Metabolome , Metabolomics/instrumentation , Tandem Mass Spectrometry/methodsABSTRACT
Honeybees ensure a key ecosystem service by pollinating many agricultural crops and wild plants. However, in the past few decades, managed bee colonies have been declining in Europe and North America. Researchers have emphasized both parasites and pesticides as the most important factors. Infection by the parasite Nosema ceranae and exposure to pesticides can contribute to gut dysbiosis, impacting the honeybee physiology. Here, we examined and quantified the effects of N. ceranae, the neonicotinoid thiamethoxam, the phenylpyrazole fipronil and the carboxamide boscalid, alone and in combination, on the honeybee gut microbiota. Chronic exposures to fipronil and thiamethoxam alone or combined with N. ceranae infection significantly decreased honeybee survival whereas the fungicide boscalid had no effect on uninfected bees. Interestingly, increased mortality was observed in N. ceranae-infected bees after exposure to boscalid, with synergistic negative effects. Regarding gut microbiota composition, co-exposure to the parasite and each pesticide led to decreased abundance of Alphaproteobacteria, and increased abundance of Gammaproteobacteria. The parasite also induced an increase of bacterial alpha-diversity (species richness). Our findings demonstrated that exposure of honeybees to N. ceranae and/or pesticides play a significant role in colony health and is associated with the establishment of a dysbiotic gut microbiota.
Subject(s)
Bees/drug effects , Bees/microbiology , Fungicides, Industrial/adverse effects , Gastrointestinal Microbiome/physiology , Insecticides/adverse effects , Nosema/physiology , Animals , Biphenyl Compounds/adverse effects , Niacinamide/adverse effects , Niacinamide/analogs & derivatives , Pyrazoles/adverse effects , Thiamethoxam/adverse effectsABSTRACT
Honeybees ensure a key ecosystemic service by pollinating many agricultural crops and wild plants. However, since few decades, managed bee colonies have declined worldwide. This phenomenon is considered to be multifactorial, with a strong emphasis on both parasites and pesticides. Infection by the parasite Nosema ceranae and exposure to pesticides can contribute to adverse effects, resulting in a perturbation of the honeybee physiology. We thus hypothesized that probiotic treatment could be promising to treat or prevent these disturbances. The aim of this study was to evaluate the effects of probiotics on N. ceranae-infected and intoxicated honeybees (by the insecticide thiamethoxam and the fungicide boscalid). For this purpose, experiments were conducted with five probiotics. Among them, Pediococcus acidilactici (PA) showed the best protective effect against the parasite and pesticides. PA significantly improved the infected honeybee lifespan as prophylactic and curative treatments (respectively 2.3 fold and 1.7 fold). Furthermore, the exposure to pesticides induced an increase of honeybee mortality compared with the control group (pâ¯<â¯.001) that was restored by the PA treatment. Despite its beneficial effect on honeybee lifespan, the PA administration did not induce changes in the gut bacterial communities (neither in abundance or diversity). N. ceranae and the pesticides were shown to deregulate genes involved in honeybee development (vitellogenin), immunity (serine protease 40, defensin) and detoxification system (glutathione peroxidase-like 2, catalase), and these effects were corrected by the PA treatment. This study highlights the promising use of PA to protect honeybees from both pathogens and pesticides.
Subject(s)
Insecticides , Nosema , Animals , Bees , PediococcusABSTRACT
The invasive microsporidian species, Nosema ceranae, causes nosemosis in honeybees and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the site of infection; the microsporidium can disturb the functioning of this organ and, thus, the bee physiology. Host defense against pathogens is not limited to resistance (i.e. the immune response) but also involves resilience. This process implies that the host can tolerate and repair damage inflicted by the infection- by the pathogen itself or by an excessive host immune response. Enterocyte damage caused by N. ceranae can be compensated by proliferation of intestinal stem cells (ISCs) that are under the control of multiple pathways. In the present study, we investigated the impact of N. ceranae on honeybee epithelium renewal by following the mitotic index of midgut stem cells during a 22-day N. ceranae infection. Fluorescence in situ hybridization (FISH) and immunostaining experiments were performed to follow the parasite proliferation/progression in the intestinal tissue, especially in the ISCs as they are key cells for the midgut homeostasis. We also monitored the transcriptomic profile of 7 genes coding for key proteins involved in pathways implicated in the gut epithelium renewal and homeostasis. We have shown for the first time that N. ceranae can negatively alter the gut epithelium renewal rate and disrupt some signaling pathways involved in the gut homeostasis. This alteration is correlated to a reduced longevity of N. ceranae-infected honeybees and we can assume that honeybee susceptibility to N. ceranae could be due to an impaired ability to repair gut damage.
Subject(s)
Bees/parasitology , Intestinal Mucosa/pathology , Intestinal Mucosa/parasitology , Animals , NosemaABSTRACT
The common and widespread parasite Nosema ceranae is considered a major threat to the Western honey bee at both the individual and colony levels. Several studies demonstrated that infection by this parasite may affect physiology, behavior, and survival of honey bees. N. ceranae infection impairs midgut integrity and alters the energy demand in honey bees. The infection can also significantly suppress the bee immune response and modify pheromone production in worker and queen honey bees leading to precocious foraging. However, the presence of N. ceranae is not systematically associated with colony weakening and honey bee mortality. This variability depends upon parasite or host genetics, nutrition, climate or interactions with other stressors such as environmental contaminants or other parasites.
Subject(s)
Bees/microbiology , Microsporidiosis/veterinary , Nosema/physiology , Animals , Bees/physiology , Behavior, Animal/physiology , Female , Microsporidiosis/pathologyABSTRACT
The intestinal protistan parasite Blastocystis is characterized by an extensive genetic variability with 17 subtypes (ST1-ST17) described to date. Only the whole genome of a human ST7 isolate was previously sequenced. Here we report the draft genome sequence of Blastocystis ST4-WR1 isolated from a laboratory rodent at Singapore.
ABSTRACT
Microsporidian genomes are the leading models to understand the streamlining in response to a pathogenic lifestyle; they are gene-poor and often possess small genomes. In this study, we show a feature of microsporidian genomes that contrasts this pattern of genome reduction. Specifically, genome investigations targeted at Anncaliia algerae, a human pathogen with a genome size of 23 Mb, revealed the presence of a hitherto undetected diversity in transposable elements (TEs). A total of 240 TE families per genome were identified, exceeding that found in many free-living fungi, and searches of microsporidian species revealed that these mobile elements represent a significant portion of their coding repertoire. Their phylogenetic analysis revealed that many cases of ancestry involve recent and bidirectional horizontal transfers with metazoans. The abundance and horizontal transfer origin of microsporidian TEs highlight a novel dimension of genome evolution in these intracellular pathogens, demonstrating that factors beyond reduction are at play in their diversification.
Subject(s)
DNA Transposable Elements , Gene Transfer, Horizontal , Genome, Fungal , Microsporidia/genetics , Mycoses/microbiology , Evolution, Molecular , Humans , Microsporidia/classification , Microsporidia/isolation & purification , Molecular Sequence Data , PhylogenyABSTRACT
Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.
Subject(s)
Bees/microbiology , Host-Pathogen Interactions , Nosema/physiology , Animals , Bees/metabolism , Insect Proteins/metabolism , Protein Interaction Maps , Proteomics/methodsABSTRACT
Intracellular pathogens including bacteria, viruses and protozoa hijack host cell functions to access nutrients and to bypass cellular defenses and immune responses. These strategies have been acquired through selective pressure and allowed pathogens to reach an appropriate cellular niche for their survival and growth. To get new insights on how parasites hijack host cellular functions, we developed a SILAC (Stable Isotope Labeling by Amino Acids in Cell culture) quantitative proteomics workflow. Our study focused on deciphering the cross-talk in a host-parasite association, involving human foreskin fibroblasts (HFF) and the microsporidia Anncaliia algerae, a fungus related parasite with an obligate intracellular lifestyle and a strong host dependency. The host-parasite cross-talk was analyzed at five post-infection times 1, 6, 12 and 24 hours post-infection (hpi) and 8 days post-infection (dpi). A significant up-regulation of four interferon-induced proteins with tetratricopeptide repeats IFIT1, IFIT2, IFIT3 and MX1 was observed at 8 dpi suggesting a type 1 interferon (IFN) host response. Quantitative alteration of host proteins involved in biological functions such as signaling (STAT1, Ras) and reduction of the translation activity (EIF3) confirmed a host type 1 IFN response. Interestingly, the SILAC approach also allowed the detection of 148 A. algerae proteins during the kinetics of infection. Among these proteins many are involved in parasite proliferation, and an over-representation of putative secreted effectors proteins was observed. Finally our survey also suggests that A. algerae could use a transposable element as a lure strategy to escape the host innate immune system.
Subject(s)
Host-Parasite Interactions , Intracellular Space/parasitology , Microsporidia/physiology , DNA Transposable Elements/genetics , Fibroblasts/cytology , Fibroblasts/parasitology , Fungal Proteins/metabolism , Humans , Intracellular Space/metabolism , Microsporidia/genetics , Microsporidia/metabolism , ProteomeABSTRACT
A unicellular Rhodophyte was identified by sequencing of its 18S rRNA encoding gene as belonging to the Rhodella violacea specie. With the objective to optimise the production of biomass and exopolysaccharide by this strain, effects of irradiance, pH and temperature on its photosynthetic activity were investigated. In a second time a stoichiometric study of the well-known f/2 medium led to its supplementation in N and P to increase biomass and then exopolysaccharide yields when the strain was cultivated in photobioreactors. The use of optimal conditions of culture (irradiance of 420 µE/m(2)/s, pH of 8.3 and temperature of 24 °C) and f/2 supplemented medium led to significant increases of biomass and exopolysaccharide productions. The structural characterisation of the produced exopolysaccharide revealed that it was sulphated and mainly composed of xylose. The different culture conditions and culture media tested had no significant impact on the structure of produced exopolysaccharides.
Subject(s)
Microalgae/metabolism , Photobioreactors , Polysaccharides/biosynthesis , Rhodophyta/metabolism , Biomass , Culture Media/chemistry , Fermentation , Hydrogen-Ion Concentration , Photosynthesis , Phylogeny , RNA, Ribosomal, 18S/metabolism , Temperature , Time Factors , Xylose/chemistryABSTRACT
Genetic diversity of a host species is a key factor to counter infection by parasites. Since two separation events and the beginning of beekeeping, the Western honeybee, Apis mellifera, has diverged in many phylogenetically-related taxa that share common traits but also show specific physiological, behavioural and morphological traits. In this study, we tested the hypothesis that A. mellifera taxa living in a same habitat should respond differently to parasites like Nosema ceranae, a microsporidia living in host's midgut. We used the Poulin and Combes' concept of virulence to compare the susceptibility of three A. mellifera taxa to N. ceranae infection. Three criteria were measured 10 days post-infection (dpi): the host mortality, the host sugar consumption and the development success of the parasite (i.e. number of spores produced). Interestingly, we showed that the observed variation in susceptibility to infection by N. ceranae is not linked to honeybee taxa but results from the variability between colonies, and that those differences are probably linked to genetic variations. The use of these three criteria allows us to conclude that the differences in susceptibility are mediated by a genetic variability in honeybee workers from resistance to tolerance. Finally, we discuss the consequences of our findings for beekeeping management.
Subject(s)
Bees/microbiology , Genetic Predisposition to Disease , Host-Pathogen Interactions , Nosema/physiology , Animals , Bees/classification , Bees/genetics , Cluster Analysis , Evolution, Molecular , Genetic Variation , Microsporidiosis/genetics , Microsporidiosis/microbiologyABSTRACT
Blastocystis is the most common eukaryotic parasite in the intestinal tract of humans. Because of its potential impact in public health, we acquired the first data concerning the prevalence of this parasite and the frequency of the Blastocystis subtypes (STs) in the Lebanese population. In this study, fecal samples from 220 Lebanese symptomatic and asymptomatic patients were collected and a total of 42 patients (19%) were identified as positive for this parasite by direct-light microscopy of smears. Among these, 36 Blastocystis isolates were genotyped using partial small subunit ribosomal RNA gene sequencing. The ST distribution in the present Lebanese population was as follows: ST3 (33.3%), ST2 (33.3%), ST1 (30.6%), and ST4 (2.8%). These data were compared with those available in other Middle Eastern and neighboring countries. Finally, ST1 was significantly more prevalent among symptomatic patients of this Lebanese population.
Subject(s)
Blastocystis Infections/epidemiology , Blastocystis/genetics , Blastocystis/isolation & purification , DNA, Protozoan/isolation & purification , Gastrointestinal Tract/parasitology , Adolescent , Adult , Aged , Aged, 80 and over , Blastocystis/classification , Child , Child, Preschool , DNA, Protozoan/genetics , Feces/parasitology , Female , Genotype , Humans , Infant , Lebanon/epidemiology , Male , Middle Aged , Molecular Epidemiology , Prevalence , Ribosome Subunits, Small/genetics , Sequence Analysis, DNA , Specimen Handling , Young AdultABSTRACT
Because of their limitations, current subtyping methods likely underestimate mixed human intra- and inter-subtype infections with Blastocystis sp. leading to erroneous data in the context of epidemiological studies. We confirmed this hypothesis by the identification of several isolates belonging to three subtypes in a patient considered at high risk of mixed infection through her lifestyle in rural area and long history of travelling.