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.

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.

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 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.

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.

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.

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.

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.

Identification of specific male and female genes in adult Ancylostoma caninum.

The hookworm Ancylostoma canium represents a serious health problem, not only for animals but also for humans. These blood-feeding parasites produce various proteolytic enzymes in order to digest the host hemoglobin. The female worm ingests more blood than does the male. It is not known whether this difference is accompanied by expression of sex-specific proteinases. The identification of new genes related either to the developmental process of maturation of each sex or to the proteinases secreted by these worms could provide researchers with new tools to be used in control programs for this important parasite. The differential-display technique was used to compare the gene expression patterns of adult male and female worms in order to find specific genes that could be used as new targets in the control strategies for this parasite.

Update of the gene discovery program in Schistosoma mansoni with the expressed sequence tag approach

Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of theses genes (81 per cent) had not previously been descibed in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor.