Ketamine can be produced by Pochonia chlamydosporia: an old molecule and a new anthelmintic?

BACKGROUND: Infection by nematodes is a problem for human health, livestock, and agriculture, as it causes deficits in host health, increases production costs, and incurs a reduced food supply. The control of these parasites is usually done using anthelmintics, which, in most cases, have not been fully effective. Therefore, the search for new molecules with anthelmintic potential is necessary. METHODS: In the present study, we isolated and characterized molecules from the nematophagous fungus Pochonia chlamydosporia and tested these compounds on three nematodes: Caenorhabditis elegans; Ancylostoma ceylanicum; and Ascaris suum. RESULTS: The ethyl acetate extract showed nematicidal activity on the nematode model C. elegans. We identified the major substance present in two sub-fractions of this extract as ketamine. Then, we tested this compound on C. elegans and the parasites A. ceylanicum and A. suum using hamsters and mice as hosts, respectively. We did not find a difference between the animal groups when considering the number of worms recovered from the intestines of animals treated with ketamine (6 mg) and albendazole (P > 0.05). The parasite burden of larvae recovered from the lungs of mice treated with ketamine was similar to those treated with ivermectin. CONCLUSIONS: The results presented here demonstrate the nematicidal activity of ketamine in vitro and in vivo, thus confirming the nematicidal potential of the molecule present in the fungus P. chlamydosporia may consist of a new method of controlling parasites.

Structure of the protective nematode protease complex H-gal-GP and its conservation across roundworm parasites.

Roundworm parasite infections are a major cause of human and livestock disease worldwide and a threat to global food security. Disease control currently relies on anthelmintic drugs to which roundworms are becoming increasingly resistant. An alternative approach is control by vaccination and 'hidden antigens', components of the worm gut not encountered by the infected host, have been exploited to produce Barbervax, the first commercial vaccine for a gut dwelling nematode of any host. Here we present the structure of H-gal-GP, a hidden antigen from Haemonchus contortus, the Barber's Pole worm, and a major component of Barbervax. We demonstrate its novel architecture, subunit composition and topology, flexibility and heterogeneity using cryo-electron microscopy, mass spectrometry, and modelling. Importantly, we demonstrate that complexes with the same architecture are present in other Strongylid roundworm parasites including human hookworm. This suggests a common ancestry and the potential for development of a unified hidden antigen vaccine.

Urotrema macrotestis and Urotrema scabridum (Digenea: Urotrematidae) parasitizing bats (Mammalia: Chiroptera) in Brazil.

Urotrema scabridum Braun 1900 and Urotrema macrotestis Mané-Garzón and Telias 1965 are reported from the small intestine of Eumops glaucinus (Wagner, 1843). The species were differentiated by the body width, the size and position of acetabulum, the size of testis, the caecal termination, and the distribution of vitellarium. The present study expands the distribution and the hosts of both species in Minas Gerais State and reports U. macrotestis parasitizing bats for the first time.

PCR-RFLP screening of polymorphisms associated with benzimidazole resistance in Necator americanus and Ascaris lumbricoides from different geographical regions in Brazil.

Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infections in the world. Periodic treatment with mass drug administration (MDA) in endemic areas is the recommended action put forth by the World Health Organization. However, MDA can cause the selection of subpopulations that possess the genetic ability to overcome the mechanism of drug action. In fact, beta-tubulin gene mutations (codons 167, 198 and 200) are correlated with benzimidazole resistance in nematodes of veterinary importance. It is possible that these SNPs also have strong correlation with treatment resistance in the human geohelminths A. lumbricoides, Trichuris trichiura and hookworms. Here, we aimed to investigate the presence of some of these canonical molecular markers associated with parasite resistance to benzimidazole in N. americanus and A. lumbricoides collected from six Brazilian states. Nested-PCR and PCR-RFLP were used to detect mutations at codons 167 and 198 in 601 individual eggs of A. lumbricoides collected from 62 human stool samples; however, no mutations were found. Codons 198 and 200 were tested in 552 N. americanus eggs collected from 48 patients using the same methodology, which presented a relative frequency of 1.4% and 1.1%, respectively. The presence of these SNPs in N. americanus eggs is an important finding, indicating that with high benzimidazole drug pressure there is potential for benzimidazole resistance to be selected in this hookworm. However, at these low frequencies it does not indicate that there is at present any benzimidazole resistance problem. This is the first systematic study performed in South America, and the study yielded a landscape of the genetic variants in the beta-tubulin gene and anthelmintic resistance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs.

Fishborne Zoonotic Trematodes Transmitted by Melanoides tuberculata Snails, Peru.

We investigated the transmission of the fishborne trematodes Centrocestus formosanus and Haplorchis pumilio by Melanoides tuberculata snails in Peru. We report on results of experimental, morphological, and molecular approaches and discuss the potential risk for future human cases, given the existence of food habits in the country involving the ingestion of raw fish.

Evaluation of biochemical, hematological and parasitological parameters of protein-deficient hamsters infected with Ancylostoma ceylanicum.

BACKGROUND: Hookworms infect millions of people worldwide and can cause severe clinical symptoms in their hosts. Prospective cohort studies in Brazil show high rates of hookworm reinfection in malnourished children compared to well-nourished children, despite previous treatment. Additionally, soil-transmitted helminth (STH) infections can worsen the nutritional status of affected populations. Therefore, this study aims to clarify the effects of host malnutrition during Ancylostoma ceylanicum infection and how this infection affects host physiological parameters using a hamster model. METHODOLOGY/PRINCIPAL FINDINGS: Hamsters were divided into four experimental groups: normal diet or low-protein diet (also referred to as "malnourished") and A. ceylanicum infection or no infection. More severe pathogenesis was observed in the infected malnourished group, as demonstrated by significant decreases in the hemoglobin concentration, erythrocyte number and packed-cell volume compared to the non-infected malnourished group. Greater numbers of adult parasites and eggs were observed in the malnourished group compared to the control group; however, the oviposition rate was lower in the malnourished group. In general, greater values of total lipids were observed in malnourished animals compared to control animals, including lipids excreted in the stool. CONCLUSIONS: In this work, we have demonstrated that animals fed an isocaloric low-protein diet presented more severe pathogenesis when infected with A. ceylanicum. The increased lipid concentration in the liver and blood is related to the conversion of the excess carbohydrate into fatty acids that increase the concentration of triglycerides in general. Triglycerides were excreted in the feces, indicating that infection associated with malnutrition caused a greater loss of these molecules for this group of animals and confirming the hypothesis that both nutrition and infection are responsible for the malabsorption syndrome. Taken together, the results found in this work confirm the hypothesis that the nutritional condition of the host greatly influences the course of the infection.

Entamoeba histolytica: gene expression analysis of cells invading tissues.

Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis.

Evaluation of humoral and cellular immune response of BALB/c mice immunized with a recombinant fragment of MSP1a from Anaplasma marginale using carbon nanotubes as a carrier molecule.

Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia Anaplasma marginale. Surface proteins (MSPs) of A. marginale are important in the interaction of the pathogen with the host and constitute potential vaccine targets against this pathogen. Currently, there is no commercial inactivated vaccine against bovine anaplasmosis that can generate a protective immune response that effectively prevents the development of clinical disease. The objective of this study was to evaluate the humoral and cellular immune responses of BALB/c mice immunized with the recombinant fragment of rMSP1a from A. marginale using carbon nanotubes as a carrier molecule. The fragment of rMSP1a comprising the N-terminal region of the protein was expressed in Escherichia coli BL21, purified by nickel affinity chromatography and covalently linked to multiwalled carbon nanotubes (MWNTs). After this functionalization, thirty BALB/c mice were divided into five groups, G1 (rMSP1a), G2 (MWNT+rMSP1a), G3 (MWNT), G4 (adjuvant) and G5 (unimmunized). The mice were immunized subcutaneously at days 0, 21 and 42. Blood samples were collected on day 11 after immunization. The spleens were collected, and the splenocytes were cultured for cell proliferation assays and cell immunophenotyping. Mice immunized with rMSP1a (G1 and G2) produced high levels of anti-rMSP1a IgG, demonstrating that the functionalization to carbon nanotubes did not interfere with protein immunogenicity. Immunization with MWNT+rMSP1a significantly induced higher percentages of CD4(+)CD44(+) and CD4(+)CD62L(+) lymphocytes, high levels of TNF-α, and a higher proliferative rate of splenocytes compared to mice immunized with rMSP1a alone (G1 group). Therefore, additional experiments using cattle should be performed to determine the efficacy, safety, immunogenicity and protection induced by rMSP1a associated with MWNT.

Genome of the human hookworm Necator americanus.

The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworm's invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases.

Molecular detection and identification of hemoparasites in pampas deer (Ozotoceros bezoarticus Linnaeus, 1758) from the Pantanal Brazil.

Hemoparasites were surveyed in 60 free-living pampas deer Ozotoceros bezoarticus from the central area of the Pantanal, known as Nhecolândia, State of Mato Grosso do Sul, Brazil, through the analysis of nested PCR assays and nucleotide sequencing. Blood samples were tested for Babesia/Theileria, Anaplasma spp., and Trypanosoma spp. using nPCR assays and sequencing of the 18S rRNA, msp4, ITS, and cathepsin L genes. The identity of each sequence was confirmed by comparison with sequences from GenBank using BLAST software. Forty-six (77%) pampas deer were positive for at least one hemoparasite, according to PCR assays. Co-infection occurred in 13 (22%) animals. Based on the sequencing results, 29 (48%) tested positive for A. marginale. Babesia/Theileria were detected in 23 (38%) samples, and according to the sequencing results 52% (12/23) of the samples were similar to T. cervi, 13% (3/23) were similar to Babesia bovis, and 9% (2/23) were similar to B. bigemina. No samples were amplified with the primers for T. vivax, while 11 (18%) were amplified with the ITS primers for T. evansi. The results showed pampas deer to be co-infected with several hemoparasites, including species that may cause serious disease in cattle. Pampas deer is an endangered species in Brazil, and the consequences of these infections to their health are poorly understood.

Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil.

Intraerythrocytic protozoan species of the genera Theileria and Babesia are known to infect both wild and domestic animals, and both are transmitted by hard-ticks of the family Ixodidae. The prevalences of hemoprotozoa and ectoparasites in 15 free-living Mazama gouazoubira, two captive M. gouazoubira and four captive Blastocerus dichotomus from the State of Minas Gerais, Brazil, have been determined through the examination of blood smears and the use of nested polymerase chain reaction (nPCR). The cervid population was inspected for the presence of ticks and any specimens encountered were identified alive under the stereomicroscope. Blood samples were collected from all 21 animals, following which blood smears were prepared, subjected to quick Romanowsky staining and examined under the optical microscope. DNA was extracted with the aid of commercial kits from cervid blood samples and from tick salivary glands. The nPCR assay comprised two amplification reactions: the first was conducted using primers specific for a 1700 bp segment of the 18S rRNA gene of Babesia and Theileria species, whilst the second employed primers designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank. The ticks Amblyomma cajennense, Rhipicephalus microplus and Dermacentor nitens were identified in various of the cervids examined. Of the animals investigated, 71.4% (15/21) were infected with hemoprotozoa, including Theileria cervi (47.6%), Theileria sp. (14.3%), Babesia bovis (4.8%) and Babesia bigemina (4.8%). However, only one of the infected wild cervids exhibited accentuated anaemia (PCV=17%). This is first report concerning the occurrence of Theileria spp. in Brazilian cervids.

Massively parallel sequencing and analysis of the Necator americanus transcriptome.

BACKGROUND: The blood-feeding hookworm Necator americanus infects hundreds of millions of people worldwide. In order to elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of the adult stage of Necator americanus was explored using next-generation sequencing and bioinformatic analyses. METHODOLOGY/PRINCIPAL FINDINGS: A total of 19,997 contigs were assembled from the sequence data; 6,771 of these contigs had known orthologues in the free-living nematode Caenorhabditis elegans, and most of them encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that the C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%); most of these molecules were predicted to be involved in more than one biological pathway. Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. For instance, proteinase inhibitors were inferred to be highly represented in the former species, whereas SCP/Tpx-1/Ag5/PR-1/Sc7 proteins ( = SCP/TAPS or Ancylostoma-secreted proteins) were predominant in the latter. In N. americanus, essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have homologues in the human host. These candidate targets were inferred to be linked to mitochondrial (e.g., processing proteins) or amino acid metabolism (e.g., asparagine t-RNA synthetase). CONCLUSIONS: This study has provided detailed insights into the transcriptome of the adult stage of N. americanus and examines similarities and differences between this species and A. caninum. Future efforts should focus on comparative transcriptomic and proteomic investigations of the other predominant human hookworm, A. duodenale, for both fundamental and applied purposes, including the prevalidation of anti-hookworm drug targets.

Characterization of a Caenorhabditis elegans glc seven-like phosphatase (gsp) orthologue from Haemonchus contortus (Nematoda).

A full-length complementary DNA (cDNA; designated Hc-stp-1) encoding a serine/threonine phosphatase (Hc-STP-1) was isolated from Haemonchus contortus, a strongylid nematode parasite of small ruminants. Hc-stp-1 was shown to be transcribed in males of both adults and fourth-stage larvae, but not in females, early larval stages or eggs. The full-length gene (2854 bp) contained ten exons and nine introns, and encoded a cDNA of 951 bp. Comparisons of the conceptually translated protein (316 amino acids, estimated at approximately 35 kDa) with serine/threonine phosphatases (STPs) from other organisms revealed the presence of the conserved motif LRGNHE. Structural analysis, by comparative modelling, confirmed strict conservation of residues and features involved in catalytic activity, and variation in the ligand-binding interface. Phylogenetic analysis of amino acid sequence data revealed that Hc-STP-1 clustered with STPs from other nematodes (including Caenorhabditis elegans, Trichostrongylus vitrinus, Oesophagostomum dentatum, Ascaris suum and Brugia malayi) to the exclusion of STPs from other organisms. The protein was inferred to be most closely related to the PP1 class of STPs of C. elegans, within a group containing STPs encoded, amongst others, by the genes gsp-3 and gsp-4 in this free-living nematode. The functions of proteins GSP-3 and GSP-4 are known to be central to spermatogenesis and other male-specific processes in C. elegans. The findings from the present and previous studies support the proposal that Hc-stp-1 and its product play a significant role in reproductive and/or developmental processes in maturing or adult male H. contortus.

Male-enriched transcription of genes encoding ASPs and Kunitz-type protease inhibitors in Ancylostoma species.

Various transcripts coding for proteins considered to be central to parasite-host interactions were identified previously as male-enriched in the hookworm Ancylostoma braziliense. Among these genes were an ASP-5-like homologue and a Kunitz-type protease inhibitor. The present study extends this previous work to investigate similar molecules in other hookworms (Ancylostomatidae). Specifically, partial cDNA sequences encoding three different ASP molecules and two different Kunitz-type protease inhibitors were isolated, and the differential transcription between adult male and female worms was compared by conventional and quantitative reverse transcription (RT)-PCR for three species, A. braziliense, Ancylostoma caninum and Ancylostoma ceylanicum. In accordance with previous findings, male-enriched transcription was observed for all molecules explored. Based on this information, it is hypothesized that adult males are responsible for producing proteins essential to the survival of hookworms inside the host and for supporting developmental and reproductive processes in female worms.

Improved insights into the transcriptomes of the human hookworm Necator americanus--fundamental and biotechnological implications.

Hookworms of humans are blood-feeding parasitic nematodes of major socio-economic significance in a wide range of countries. They cause a neglected tropical disease (NTD) called "hookworm disease" (=necatoriasis and/or ancylostomiasis). Necator americanus is the most widely distributed hookworm of humans and is a leading cause of iron deficiency anaemia, which can cause physical and mental retardation and deaths in children as well as adverse maternal-foetal outcomes. Currently, there is a significant focus on the development of new approaches for the prevention and control of hookworms in humans. Technological advances are underpinning the discovery of drug and vaccine targets through insights into the molecular biology and genomics of these parasites and their relationship with the human host. In spite of the widespread socio-economic impacts of human necatoriasis, molecular datasets for N. americanus are scant, limiting progress in molecular research. The present article explores all currently available EST datasets for adult and larval stages of N. americanus using a semi-automated bioinformatic pipeline. In the current repertoire of molecules now available, some have been or are being considered as candidate vaccines against N. americanus. Among others, the most abundant sets of molecules relate to the pathogenesis-related protein (PRP) superfamily, comprising various members, such as the Ancylostoma-secreted or activation-associated proteins (ASPs) and the kunitz-type proteins, both of which are inferred to play key roles in the interplay between N. americanus and the human host. Understanding the molecular biology of these and other novel molecules discovered could have important implications for finding new ways of disrupting the pathways that they are involved in, and should facilitate the identification of new drug and vaccine targets. Also, the bioinformatic prediction of the essentiality of genes and gene products as well as molecular network connectivity of nematode-specific genes, together with sequencing by 454 technology, are likely to assist in the genomic discovery efforts in the very near future, to also underpin fundamental, molecular research of hookworms.

Extensive and complex sequence diversity in mitochondrial cytochrome c oxidase subunit 1 within Necator americanus from Colombia revealed by SSCP-coupled sequencing.

In spite of the human health importance of hookworms, there has been only a small number of studies of genetic variability within Necator americanus, and none in South America. In the present study, we investigated sequence variability in a 395-bp region of the mitochondrial cytochrome c oxidase subunit 1 gene among N. americanus individuals (n=100) from humans of six villages in Colombia, employing a mutation scanning-coupled sequencing approach. Haplotypic diversity within N. americanus varied from 0.95 to 1.0 (0.9743+/-0.0068) and nucleotide diversity from 0.025 to 0.045 (0.0257+/-0.013). Nucleotide variation was reflected in nine amino acid alterations over 132 positions. The network of cox1 haplotypes (n=59) displayed a complex relationship with no apparent association between haplotype and geographical origin in Colombia. The extensive haplotypic variability detected suggests different epidemiological or disease characteristics for distinct population variants of N. americanus.

Identification of gender-regulated genes in Ancylostoma braziliense by real-time RT-PCR.

Ancylostoma braziliense belongs to the family Ancylostomatidae and infects cats and dogs in various parts of the tropical world. It is also a zoonotic parasite causing cutaneous larva migrants in humans. There are very few, either biological or molecular, studies of this species. In this study, differential display was used to identify differentially expressed genes in male and female A. braziliense. Nineteen new sequences were identified and examined by real-time RT-PCR to confirm male-female specificity. Ten were more expressed in males, while two were more expressed in females. Molecules shown to be important in other host-parasite relationships were also found in this study.

Mitochondrial DNA variation of the dog hookworm Ancylostoma caninum in Brazilian populations.

The mitochondrial cytochrome oxidase I gene was partially sequenced for 164 Ancylostoma caninum individuals, originating from five different localities in Brazil, with the aim of describing the genetic diversity and genetic structure of Brazilian hookworm populations. Allelic and nucleotide diversity were moderate (overall h=0.88 and pi=0.016) and were similar among cities. There was moderate genetic differentiation among the populations sampled (approximately Phi(ST)=0.12) and a weak but nonsignificant correlation between geographical and genetic distance. This genetic structure was similar to that observed among populations of the human hookworm, Necator americanus, but distinct from that typically found in trichostrongylid nematode parasites of livestock. Thus, a pattern of different genetic structures among different groups of nematodes is emerging. We also observed a few individuals that had a highly divergent mtDNA sequence (almost 7% sequence divergence from the other sequences). These results in combination with data from other studies suggest that A. caninum populations worldwide consist of a mix of previously differentiated populations, or perhaps even cryptic species. This study contributes to the knowledge of genetic structure and diversity of hookworms, which in turn will be useful in developing methods for their control.

Differential diagnosis of dog hookworms based on PCR-RFLP from the ITS region of their rDNA.

Species of Ancylostoma infecting dogs and sometimes humans are sympatric in many parts of the world. The establishment of a specific molecular diagnostic tool is important, not only to refine information for epidemiological studies, but also to evaluate the efficacy of vaccine programmes and assist in the development of specific drug treatments. The ITS region from 20 specimens of A. braziliense, collected from three separate geographical areas of Brazil, and from 10 specimens of A. caninum, collected from the same area in Brazil were sequenced and analyzed. Alignment of sequences showed that this gene is highly conserved. The intraspecific polymorphism for both species was less then 1%, whereas the interspecific polymorphism was 6.2, 7.3 and 9.4% between A. ceylanicum and A. braziliense; A. caninum and A. ceylanicum and A. ceylanicum and A. braziliense, respectively. Among the three species it was 12.3%. This revealed the ITS region as highly conserved and consequently a good molecular marker for diagnostic studies. In this work, four restriction enzymes were used in a PCR-RFLP using the ITS region of rDNA, to establish a differential diagnosis which discriminates between three Ancylostoma species, A. braziliense, A. caninum and A. ceylanicum. The best pattern was given by the HinfI enzyme, which produced different fragment sizes for each of the three species. Furthermore, the diagnostic tool differentiates DNA extracted directly from faeces of Ancylostoma-infected dogs.

Identification of an expressed gene in Dipylidium caninum.

Recombinant DNA studies have been focused on developing vaccines to different cestodes. But few studies involving Dipylidium caninum molecular biology and genes have been done. Only partial sequences of mitochondrial DNA and ribosomal RNA gene are available in databases. Any molecular work with this parasite, including epidemiology, study of drug-resistant strains, and vaccine development, is hampered by the lack of knowledge of its genome. Thus, the knowledge of specific genes of different developmental stages of D. caninum is crucial to locate potential targets to be used as candidates to develop a vaccine and/or new drugs against this parasite. Here we report, for the first time, the sequencing of a fragment of a D. caninum expressed gene.