Comparative transcriptome profiling of virulent and avirulent isolates of Neoparamoeba perurans.

Neoparamoeba perurans, the aetiological agent of amoebic gill disease, remains a persistent threat to Atlantic salmon mariculture operations worldwide. Innovation in methods of AGD control is required yet constrained by a limited understanding of the mechanisms of amoebic gill disease pathogenesis. In the current study, a comparative transcriptome analysis of two N. perurans isolates of contrasting virulence phenotypes is presented using gill-associated, virulent (wild type) isolates, and in vitro cultured, avirulent (clonal) isolates. Differential gene expression analysis identified a total of 21,198 differentially expressed genes between the wild type and clonal isolates, with 5674 of these genes upregulated in wild type N. perurans. Gene set enrichment analysis predicted gene sets enriched in the wild type isolates including, although not limited to, cortical actin cytoskeleton, pseudopodia, phagocytosis, macropinocytic cup, and fatty acid beta-oxidation. Combined, the results from these analyses suggest that upregulated gene expression associated with lipid metabolism, oxidative stress response, protease activity, and cytoskeleton reorganisation is linked to pathogenicity in wild type N. perurans. These findings provide a foundation for future AGD research and the development of novel therapeutic and prophylactic AGD control measures for commercial aquaculture.

Integrated Analysis of Gene Expression and Metabolite Data Reveals Candidate Molecular Markers in Colorectal Carcinoma.

Background: This study investigated potential gene targets and metabolite markers associated with colorectal carcinoma (CRC). Materials & Methods: Gene expression data (GSE110224) related with CRC were obtained from Gene Expression Omnibus, including 17 tumor tissues and 17 normal colon ones. The gene differential analysis, functional analysis, protein-protein interaction (PPI) analysis, and metabolite network construction were performed to identify key genes related to CRC. Moreover, an external dataset was used to validate genes of interest in CRC, and corresponding survival analysis was also conducted. Results: The authors extracted 197 differentially expressed genes (75 upregulated and 122 downregulated genes). Moreover, upregulated genes were closely associated with rheumatoid arthritis and amoebiasis pathways. The downregulated genes were mainly related to bile secretion and proximal tubule bicarbonate reclamation pathway. Combined with PPI network and metabolite prediction, the overlapped nine genes (CXCL1, CXCL8, CXCL10, HDS1782, IL18, PCK1, PTGS2, SERPINB2, TMP1) were found to be critical in CRC. Similar gene expression profiles of nine critical genes were validated by an external dataset, except for SERPINB2. In addition, the expressions of TIMP1, IL1B, and PTGS2 were closely related with prognosis. Finally, the metabolite network analysis revealed that there were close associations between prostaglandin E2 and three pathways (rheumatoid arthritis, amoebiasis, and leishmaniasis). Conclusion: CXCL1/CXCL8/IL1B/PTGS2-prostaglandin E2 axes were the potential signatures involved in CRC progression, which could provide new insights to understand the molecular mechanisms of CRC.

Investigation of the transcriptomic response in Atlantic salmon (Salmo salar) gill exposed to Paramoeba perurans during early onset of disease.

Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans (P. perurans) is a disease affecting Atlantic salmon (Salmo salar). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha (tnf-α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways.

Host-Parasite Interaction of Atlantic salmon (Salmo salar) and the Ectoparasite Neoparamoeba perurans in Amoebic Gill Disease.

Marine farmed Atlantic salmon (Salmo salar) are susceptible to recurrent amoebic gill disease (AGD) caused by the ectoparasite Neoparamoeba perurans over the growout production cycle. The parasite elicits a highly localized response within the gill epithelium resulting in multifocal mucoid patches at the site of parasite attachment. This host-parasite response drives a complex immune reaction, which remains poorly understood. To generate a model for host-parasite interaction during pathogenesis of AGD in Atlantic salmon the local (gill) and systemic transcriptomic response in the host, and the parasite during AGD pathogenesis was explored. A dual RNA-seq approach together with differential gene expression and system-wide statistical analyses of gene and transcription factor networks was employed. A multi-tissue transcriptomic data set was generated from the gill (including both lesioned and non-lesioned tissue), head kidney and spleen tissues naïve and AGD-affected Atlantic salmon sourced from an in vivo AGD challenge trial. Differential gene expression of the salmon host indicates local and systemic upregulation of defense and immune responses. Two transcription factors, znfOZF-like and znf70-like, and their associated gene networks significantly altered with disease state. The majority of genes in these networks are candidates for mediators of the immune response, cellular proliferation and invasion. These include Aurora kinase B-like, rho guanine nucleotide exchange factor 25-like and protein NDNF-like inhibited. Analysis of the N. perurans transcriptome during AGD pathology compared to in vitro cultured N. perurans trophozoites, as a proxy for wild type trophozoites, identified multiple gene candidates for virulence and indicates a potential master regulatory gene system analogous to the two-component PhoP/Q system. Candidate genes identified are associated with invasion of host tissue, evasion of host defense mechanisms and formation of the mucoid lesion. We generated a novel model for host-parasite interaction during AGD pathogenesis through integration of host and parasite functional profiles. Collectively, this dual transcriptomic study provides novel molecular insights into the pathology of AGD and provides alternative theories for future research in a step towards improved management of AGD.

Encephalomyelitis Caused by Balamuthia mandrillaris in a Woman With Breast Cancer: A Case Report and Review of the Literature.

Balamuthia mandrillaris is one cause of a rare and severe brain infection called granulomatous amoebic encephalitis (GAE), which has a mortality rate of >90%. Diagnosis of Balamuthia GAE is difficult because symptoms are non-specific. Here, we report a case of Balamuthia amoebic encephalomyelitis (encephalitis and myelitis) in a woman with breast cancer. She sustained trauma near a garbage dump 2 years ago and subsequently developed a skin lesion with a Mycobacterium abscessus infection. She experienced dizziness, lethargy, nausea and vomiting, inability to walk, and deterioration of consciousness. Next-generation sequencing of cerebrospinal fluid (CSF) samples revealed B. mandrillaris, and MRI of both brain and spinal cord showed abnormal signals. T-cell receptor (TCR) sequencing of the CSF identified the Top1 TCR. A combination of amphotericin B, flucytosine, fluconazole, sulfamethoxazole, trimethoprim, clarithromycin, pentamidine, and miltefosine was administrated, but she deteriorated gradually and died on day 27 post-admission.

The expression of TLR2 and TLR4 in the kidneys and heart of mice infected with Acanthamoeba spp.

BACKGROUND: Acanthamoeba spp. are cosmopolitan protozoans that cause infections in the brain, as well as extracerebral infections in the cornea, lungs and skin. Little is known about the mechanisms of the immunological response to these parasites in organs which are not their main biotope. Therefore, the purpose of this study was to determine the expression of TLR2 and TLR4 in the kidneys and heart of Acanthamoeba spp.-infected mice, with respect to the host's immunological status. METHODS: The mice were grouped into four groups: immunocompetent control mice; immunosuppressed control mice; immunocompetent Acanthamoeba spp.-infected mice; and immunosuppressed Acanthamoeba spp. infected mice. In the study, we used the amoebae T16 genotype which was isolated from a patient. The TLRs expressions in the kidneys and heart of mice were assessed by quantitative real-time polymerase chain reaction. Moreover, we visualized TLR2 and TLR4 proteins in the organs by immunohistochemical staining. RESULTS: In the kidneys, we observed a higher TLR2 expression in immunosuppressed mice at 24 days post-Acanthamoeba spp. infection (dpi) compared to the uninfected mice. There were no statistically significant differences in TLR4 expression in the kidneys between the immunocompetent and immunosuppressed mice, both of infected and uninfected mice. In the heart, we observed a difference in TLR2 expression in immunocompetent mice at 24 dpi compared to immunocompetent mice at 8 dpi. The immunocompetent Acanthamoeba spp.-infected mice had higher TLR4 expression at 8 dpi compared to the immunocompetent uninfected mice. CONCLUSIONS: Our results indicate that TLR2 is involved in response to Acanthamoeba spp. infection in the kidneys, whereas in the heart, both studied TLRs are involved.

Genome-wide association mapping and accuracy of predictions for amoebic gill disease in Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is a parasitic disease caused by the amoeba Paramoeba perurans, which colonizes the gill tissues and causes distress for the host. AGD can cause high morbidity and mortalities in salmonid and non-salmonid fish species. To understand the genetic basis of AGD and improve health status of farmed A. salmon, a population of ~ 6,100 individuals belonging to 150 full-sib families was monitored for development of AGD in the sea of Ireland. The population was followed for two rounds of AGD infections, and fish were gill scored to identify severity of disease in first (N = 3,663) and the second (N = 3,511) infection with freshwater treatment after the first gill-scoring. A subset of this gill-scored population (N = 1,141) from 119 full-sib families were genotyped with 57,184 SNPs using custom-made Affymetrix SNP-chip. GWAS analyses were performed which resulted in five significantly associated SNP variants distributed over chromosome 1, 2 and 5. Three candidate genes; c4, tnxb and slc44a4 were found within QTL region of chromosome 2. The tnxb and c4 genes are known to be a part of innate immune system, and may play a role in resistance to AGD. The gain in prediction accuracy obtained by involving genomic information was 9-17% higher than using traditional pedigree information.

Characterising the mechanisms underlying genetic resistance to amoebic gill disease in Atlantic salmon using RNA sequencing.

BACKGROUND: Gill health is one of the main concerns for Atlantic salmon aquaculture, and Amoebic Gill Disease (AGD), attributable to infection by the amoeba Neoparamoeba perurans, is a frequent cause of morbidity. In the absence of preventive measures, increasing genetic resistance of salmon to AGD via selective breeding can reduce the incidence of the disease and mitigate gill damage. Understanding the mechanisms leading to AGD resistance and the underlying causative genomic features can aid in this effort, while also providing critical information for the development of other control strategies. AGD resistance is considered to be moderately heritable, and several putative QTL have been identified. The aim of the current study was to improve understanding of the mechanisms underlying AGD resistance, and to identify putative causative genomic factors underlying the QTL. To achieve this, RNA was extracted from the gill and head kidney of AGD resistant and susceptible animals following a challenge with N. perurans, and sequenced. RESULTS: Comparison between resistant and susceptible animals primarily highlighted differences mainly in the local immune response in the gill, involving red blood cell genes and genes related to immune function and cell adhesion. Differentially expressed immune genes pointed to a contrast in Th2 and Th17 responses, which is consistent with the increased heritability observed after successive challenges with the amoeba. Five QTL-region candidate genes showed differential expression, including a gene connected to interferon responses (GVINP1), a gene involved in systemic inflammation (MAP4K4), and a positive regulator of apoptosis (TRIM39). Analyses of allele-specific expression highlighted a gene in the QTL region on chromosome 17, cellular repressor of E1A-stimulated genes 1 (CREG1), showing allelic differential expression suggestive of a cis-acting regulatory variant. CONCLUSIONS: In summary, this study provides new insights into the mechanisms of resistance to AGD in Atlantic salmon, and highlights candidate genes for further functional studies that can further elucidate the genomic mechanisms leading to resistance and contribute to enhancing salmon health via improved genomic selection.

Acanthamoeba profilin elicits allergic airway inflammation in mice.

BACKGROUND: In previous studies, we suggested that Acanthamoeba is a new aero-allergen and that patients who showed positive results for the skin-prick test response to Acanthamoeba cross-reacted with several pollen allergens. Additionally, patients with common antibodies reacted to the 13-15 kDa Acanthamoeba unknown allergen. OBJECTIVE: We examined whether profilin of Acanthamoeba is a human airway allergic agent because of its molecular weight. METHODS: We expressed recombinant Ac-PF (rAc-PF) protein using an Escherichia coli expression system and evaluated whether Ac-PF is an airway allergic agent using an allergic airway inflammation animal model. RESULTS: Airway hyperresponsiveness was increased in rAc-PF-inoculated mice. The number of eosinophils and levels of Th2 cytokines, interleukin (IL)-4, IL-5, and IL-13 were increased in the bronchial alveolar lavage fluid of rAc-PF-treated mice. The lungs of the rAc-PF-treated mice group showed enhanced mucin production and metaplasia of lung epithelial cells and goblet cells. CONCLUSION: In this study, we demonstrated that rAc-PF may be an allergen in Acanthamoeba, but further studies needed to identify the mechanisms of allergenic reactions induced by Ac-PF.

A case report of granulomatous amoebic encephalitis by Group 1 Acanthamoeba genotype T18 diagnosed by the combination of morphological examination and genetic analysis.

BACKGROUND: The diagnosis of granulomatous amoebic encephalitis is challenging for clinicians because it is a rare and lethal disease. Previous reports have indicated that Acanthamoeba with some specific genotypes tend to cause the majority of human infections. We report a case of granulomatous amoebic encephalitis caused by Acanthamoeba spp. with genotype T18 in an immunodeficient patient in Japan after allogenic bone marrow transplantation, along with the morphological characteristics and genetic analysis. CASE PRESENTATION: A 52-year old man, who had undergone allogenic bone marrow transplantation, suffered from rapid-growing brain masses in addition to pneumonia and died within 1 month from the onset of the symptoms including fever, headache and disorientation. Infection with Acanthamoeba in the brain and lung was confirmed by histological evaluation; immunohistochemical staining and polymerase chain reaction analysis using autopsy samples also indicated the growth of Acanthamoeba in the brain. Gene sequence analysis indicated that this is the second documented case of infection with Acanthamoeba spp. with genotype T18 in a human host. Postmortem retrospective evaluation of cerebrospinal fluid sample in our case, as well as literature review, indicated that some cases of granulomatous amoebic encephalitis caused by Acanthamoeba may be diagnosable by cerebrospinal fluid examination. CONCLUSION: This case indicates that Acanthamoeba spp. with genotype T18 can also be an important opportunistic pathogen. For pathologists as well as physicians, increased awareness of granulomatous amoebic encephalitis is important for improving the poor prognosis along with the attempt to early diagnosis with cerebrospinal fluid.

Complete genome sequence and bioinformatics analyses of Bacillus thuringiensis strain BM-BT15426.

OBJECTIVES: This study aimed to investigate the genetic characteristics of Bacillus thuringiensis strain BM-BT15426. METHODS: B. thuringiensis strain was identified by sequencing the PCR product (amplifying 16S rRNA gene) using ABI Prism 377 DNA Sequencer. The genome was sequenced using PacBio RS II sequencers and assembled de novo using HGAP. Also, further genome annotation was performed. RESULTS: The genome of B. thuringiensis strain BM-BT15426 has a length of 5,246,329 bp and contains 5409 predicted genes with an average G + C content of 35.40%. Three genes were involved in the "Infectious diseases: Amoebiasis" pathway. A total of 21 virulence factors and 9 antibiotic resistant genes were identified. CONCLUSIONS: The major pathogenic factors of B. thuringiensis strain BM-BT15426 were identified through complete genome sequencing and bioinformatics analyses which contributes to further study on pathogenic mechanism and phenotype of B. thuringiensis.

Cellular gene expression induced by parasite antigens and allergens in neonates from parasite-infected mothers.

Prenatal exposure to parasite antigens or allergens will influence the profile and strength of postnatal immune responses, such contact may tolerize and increase susceptibility to future infections or sensitize to environmental allergens. Exposure in utero to parasite antigens will distinctly alter cellular gene expression in newborns. Gene microarrays were applied to study gene expression in umbilical cord blood cell (UCBC) from parasite-exposed (Para-POS) and non-exposed (Para-NEG) neonates. UCBC were activated with antigens of helminth (Onchocerca volvulus), amoeba (Entamoeba histolytica) or allergens of mite (Dermatophagoides farinae). When UCBC from Para-POS and Para-NEG newborns were exposed to helminth antigens or allergens consistent differences occurred in the expression of genes encoding for MHC class I and II alleles, signal transducers of activation and transcription (STATs), cytokines, chemokines, immunoglobulin heavy and light chains, and molecules associated with immune regulation (SOCS, TLR, TGF), inflammation (TNF, CCR) and apoptosis (CASP). Expression of genes associated with innate immune responses were enhanced in Para-NEG, while in Para-POS, the expression of MHC class II and STAT genes was reduced. Within functional gene networks for cellular growth, proliferation and immune responses, Para-NEG neonates presented with significantly higher expression values than Para-POS. In Para-NEG newborns, the gene cluster and pathway analyses suggested that gene expression profiles may predispose for the development of immunological, hematological and dermatological disorders upon postnatal helminth parasite infection or allergen exposure. Thus, prenatal parasite contact will sensitize without generating aberrant inflammatory immune responses, and increased pro-inflammatory but decreased regulatory gene expression profiles will be present in those neonates lacking prenatal parasite antigen encounter.

Horizontal gene transfer of a Chlamydial tRNA-guanine transglycosylase gene to eukaryotic microbes.

tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes.

Diagnosing Balamuthia mandrillaris Encephalitis With Metagenomic Deep Sequencing.

OBJECTIVE: Identification of a particular cause of meningoencephalitis can be challenging owing to the myriad bacteria, viruses, fungi, and parasites that can produce overlapping clinical phenotypes, frequently delaying diagnosis and therapy. Metagenomic deep sequencing (MDS) approaches to infectious disease diagnostics are known for their ability to identify unusual or novel viruses and thus are well suited for investigating possible etiologies of meningoencephalitis. METHODS: We present the case of a 74-year-old woman with endophthalmitis followed by meningoencephalitis. MDS of her cerebrospinal fluid (CSF) was performed to identify an infectious agent. RESULTS: Sequences aligning to Balamuthia mandrillaris ribosomal RNA genes were identified in the CSF by MDS. Polymerase chain reaction subsequently confirmed the presence of B. mandrillaris in CSF, brain tissue, and vitreous fluid from the patient's infected eye. B. mandrillaris serology and immunohistochemistry for free-living amoebas on the brain biopsy tissue were positive. INTERPRETATION: The diagnosis was made using MDS after the patient had been hospitalized for several weeks and subjected to costly and invasive testing. MDS is a powerful diagnostic tool with the potential for rapid and unbiased pathogen identification leading to early therapeutic targeting.

Which Th pathway is involved during late stage amoebic gill disease?

Amoebic gill disease (AGD) is an emerging disease in North European Atlantic salmon (Salmo salar Linnaeus 1758) aquaculture caused by the amoeba Paramoeba perurans. The host immune response to AGD infection is still not well understood despite past attempts to investigate host-pathogen interactions. With the significant increase in our knowledge of cytokine genes potentially involved in Th responses in recent years, we examined their involvement in this disease using Atlantic salmon post-smolts sampled 3 weeks after exposure to either 500 or 5000 cells/l P. perurans. Gene expression analysis of cytokines potentially involved in the different Th pathways was performed on the first gill arch including the interbranchial lymphoid tissue (ILT). Th1, Th17 and Treg pathways were found to be significantly down regulated, mainly in samples from fish given the higher dose. In contrast, the Th2 pathway was found to be significantly up regulated by both infection doses. Correlation analysis of the gene expression data and the P. perurans load, assessed by real time RT-PCR of the 18S rRNA, was also performed. In humans, Th2 driven responses are characterized by the production of IgE, which in the majority of worm infections results in the generation of a Th2-mediated response and directs the immune system away from a Th1 inflammatory response. The present results seen during late stage AGD suggest that either an immune evasion strategy, similar to the responses driven by helminthic parasites to avoid cell-mediated killing mechanisms, or an allergic reaction caused by the parasite, is occurring.

Effects of single and repeated infections with Neoparamoeba perurans on antibody levels and immune gene expression in Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is the main health problem for the salmon industry in Tasmania, Australia and is now reported in most salmon producing countries. Antibody and gene expression responses to the pathogen, Neoparamoeba perurans, have been studied independently following primary exposure; however, the effects of sequential reinfection, which can often occur during net-pen culture of salmon, remain unclear. The association between the transcription of immunoglobulin (Ig) and their systemic and mucosal antibody levels in regards to AGD is unknown. Herein, we assessed the antibody responses as well as Ig transcription in the gills of Atlantic salmon infected only once and also sequentially with N. perurans. After four successive AGD challenges, no significant differences in plasma or skin mucus levels of IgM were observed between AGD-naïve and challenged fish. However, IgM gene expression in gill lesions of AGD-affected fish increased up to 31 d after infection, while no changes in IgT, TCR and CD8 transcription were observed. Changes at IgM transcription level did not match the lack of antibody response in mucus, which is possibly explained by weak correlations existing between protein and mRNA abundances in cells and tissues. In the second experiment, which investigated Ig responses to AGD at the transcriptional as well as antibody production level in salmon after a single infection, the levels of serum or skin mucus IgM antibody were not affected and no changes in the IgM or IgT transcription were induced.

Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.

Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES) proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25) in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF) inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation.

Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

Case report: Cutaneous amebiasis: the importance of molecular diagnosis of an emerging parasitic disease.

Cutaneous amebiasis is the least common clinical form of human amebiasis in Mexico, sexual amebiasis was only occasionally observed before the late 1980s. However, in the last few decades, most of the documented cases of cutaneous amebiasis from around the world are sexually transmitted. We present two cases of sexually transmitted genital amebiasis. The molecular characterization of the Entamoeba species in the affected tissues underlines the importance of an etiological diagnosis using specific and sensitive techniques that avoid the rapid destruction of tissues and the irreversible sequelae to the anatomy and function of the affected organs. In addition, for those interested in the study of the human-amoebic disease relationship and its epidemiology, the detection of a new, mixed infection in an invasive case of amebiasis reveals new perspectives in the study of the extraordinarily complex host-parasite relationship in amebiasis.