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Schistosoma mansoni eggs retained in host tissues induce innate cytokine release, contributing to the induction of Type-2 immune responses and granuloma formation, important to restrain cytotoxic antigens, but leading to fibrosis. Interleukin(IL)-33 participates in experimental models of inflammation and chemically induced fibrosis, but its role in S. mansoni-induced fibrosis is still unknown. To explore the role of the IL-33/suppressor of the tumorigenicity 2 (ST2) pathway, serum and liver cytokine levels, liver histopathology, and collagen deposition were comparatively evaluated in S. mansoni-infected wild-type (WT) and IL-33-receptor knockout (ST2−/−) BALB/c mice. Our data show similar egg counts and hydroxyproline in the livers of infected WT and ST2−/− mice; however, the extracellular matrix in ST2−/− granulomas was loose and disorganised. Pro-fibrotic cytokines, such as IL-13 and IL-17, and the tissue-repairing IL-22 were significantly lower in ST2−/− mice, especially in chronic schistosomiasis. ST2−/− mice also showed decreased α-smooth muscle actin (α-SMA) expression in granuloma cells, in addition to reduced Col III and Col VI mRNA levels and reticular fibres. Therefore, IL-33/ST2 signalling is essential for tissue repairing and myofibroblast activation during S. mansoni infection. Its disruption results in inappropriate granuloma organisation, partly due to the reduced type III and VI collagen and reticular fibre formation.

For some nematodes, alterations that leads to a resistance genotype may be accompanied by other modifications, causing changes in the biology of the parasite, although the exact mechanisms of this relationship are still not very clear. These alterations can have deleterious effects on their survival or even potentiate their pathogenicity. In this study a phenotypic characterization was carried out to compare two Ancylostoma ceylanicum isolates, a wild type one, kept in the laboratory and an albendazole selected resistant isolate (AceyBZR2). Differences in some analyzed parameters, between the two strains, were registered, as patency period, number and size of the recovered worms, including differences in the body structures. The AceyBZR2 isolate showed to be less adapted to the host, leading to a smaller number of recovered worms. However, no difference on the female egg content was observed between the two isolates. Concerning blood evaluation, no differences were found between the wild type and AceyBZR2 isolates, related to hemoglobin and hematocrit levels. However, animals in the group infected with the wild type isolate had lower serum iron concentrations than animals in the AceyBZR2 group. The possibility that the AceyBZR2 isolate might be resistant to other drugs was evaluated and it was demonstrated that it does not present cross-resistance to ivermectin and nitazoxanide. However, when animals infected with the AceyBZR2 were treated with another drug from the benzimidazoles group (mebendazole), the cross-resistance effect was observed. Morphometric analyses were performed comparing female and male adult worms from the two isolates. The results presented here allow a better understanding of the parasite-host relationship and may constitute a useful basis for establishing future control strategies for soil-transmitted helminths.

Wild rodent species are naturally infected by Schistosoma mansoni; however, the genetic characterization of the parasite, its parasitological features, and its role in human schistosomiasis are poorly understood. In this study, we isolated and characterized Schistosoma from naturally infected Holochilus sciureus, called HS strain, collected from a schistosomiasis endemic region in Maranhão State, Brazil. To isolate the parasite, miracidia obtained from the livers of H. sciureus were used to infect Biomphalaria glabrata of sympatric (called SB) and allopatric (called BH) strains, and the produced cercariae were subcutaneously inoculated into hamsters and/or BALB/c mice. Parasitological kinetics in experimentally infected hosts were evaluated, and the 16S and cox 1 regions of mtDNA from isolated worms were amplified and sequenced. Only miracidia obtained from infected mice, but not from hamsters, were capable of infecting B. glabrata, allowing maintenance of the isolated parasite. Cox1 and 16S mtDNA sequences showed 100% similarity with S. mansoni, and phylogenetic analysis showed that the HS strain of S. mansoni forms an assemblage with isolates from America and Kenya, confirming the conspecificity. Experimental infection of B. glabrata SB with S. mansoni HS resulted in two peaks of cercariae shedding at 45- and 70-days post-infection (dpi), and caused higher mortality than in B. glabrata BH. The worm recovery rate in mice was approximately 13%, and the peak of egg elimination occurred at 10th week post-infection. Therefore, S. mansoni obtained from H. sciureus was successfully isolated, genetically characterized, and maintained in mice, allowing further study of this schistosome strain.

Wild rodent species are naturally infected by Schistosoma mansoni; however, the genetic characterization of the parasite, its parasitological features, and its role in human schistosomiasis are poorly understood. In this study, we isolated and characterized Schistosoma from naturally infected Holochilus sciureus, called HS strain, collected from a schistosomiasis endemic region in Maranhão State, Brazil. To isolate the parasite, miracidia obtained from the livers of H. sciureus were used to infect Biomphalaria glabrata of sympatric (called SB) and allopatric (called BH) strains, and the produced cercariae were subcutaneously inoculated into hamsters and/or BALB/c mice. Parasitological kinetics in experimentally infected hosts were evaluated, and the tRNA

Currently, a great proportion of the emerging infectious human diseases are zoonotic, with most of the pathogens originated from wildlife. In this sense, synanthropic animals such as marsupials play important role in the dissemination of pathogens due to their proximity to human dwellings. These hosts are affected by many gastrointestinal parasites, including species with zoonotic potential. The aim of this study was to assess the diversity of gastrointestinal parasites infecting the black-eared opossum D. aurita captured in urban areas of Southeastern, Brazil. In addition, the potential risk for the human population based on the One Health perspective has been discussed. Forty-nine marsupial specimens were captured with Tomahawk live traps and fecal samples were collected. The samples were evaluated by parasitological procedures. Eggs and oocysts were analyzed at different magnifications (400 × and 1000 ×), and their identification, together with adult nematodes, was established on morphological and morphometric data. Forty-three hosts (87.76%) scored positive for at least one gastrointestinal parasite, being 83.67% (41/49) for helminths, and 65.30% (32/49) for protozoa. For Cryptosporidium sp., only 13 samples were evaluated due to insufficient amount of feces obtained of some animals. A prevalence of 23.08% (3/13) was reported for this parasite. PCR analysis revealed Ancylostomatidae eggs to belong to the genus Ancylostoma. Our results demonstrated that multiparasitism is frequently found in these animals and a high percentage of potentially zoonotic parasites are observed, implying that D. aurita may be involved in zoonotic cycles in urban environments.

Definitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized withAncylostoma ceylanicumsaline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.

Hookworms are intestinal parasites that cause major public health problems, especially in developing countries. To differentiate eggs from different hookworm species, it is necessary to use molecular methodologies, since the eggs are morphologically similar. Here, we performed the molecular identification of single hookworm eggs from six Brazilian states. Of the 634 eggs individually analyzed, 98.1% (622/634) represented Necator americanus, and surprisingly, 1.9% (12/634 eggs from the same patient) represented Ancylostoma caninum. DNA analysis of the A. caninum-positive stool sample revealed no contamination with animal feces. This is the first report of the presence of A. caninum eggs in human feces, which may have a direct implication for the epidemiology of hookworm infection caused by this species. This suggests the need for special attention regarding prophylaxis, as different reservoirs, previously not described, may have great relevance for the spread of A. caninum.

Preventive chemotherapy is recommended by the WHO as the main strategy for controlling infections caused by nematodes in humans, aiming to eliminate the morbidity associated with these infections. This strategy consists of routine periodic administration of benzimidazoles, among other drugs. Although these drugs decrease the intensity of infections, they have the potential to exert selection pressure for genotypes bearing mutations associated with drug resistance, which may result in the establishment of resistant worm populations. There is evidence in the literature of resistance to these drugs in nematodes that infect humans, including in the species Trichuris trichiura. Single-nucleotide polymorphisms (SNPs) in the beta-tubulin gene located at codons 167, 198, and 200 are associated with the mechanism of resistance to benzimidazoles in nematodes. Here, we standardized a molecular technique based on an amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to analyze codons 167, 198, and 200 of T. trichiura. The ARMS-PCR methodology was successfully established to evaluate the codons of interest. A total of 420 samples of individual eggs were analyzed from populations obtained from five Brazilian states. A mutation in codon 198 was observed at a frequency of 4.8% (20/420), while for the other two codons, no polymorphism was observed. This is the first report of the presence of this mutation in populations of T. trichiura in Brazil. This fact and the emergence of the problem already observed in other species reinforces the need for regular monitoring of SNPs related to benzimidazole resistance using techniques that are highly sensitive and specific.

Hookworms represent a serious problem for human and animal health in different parts of the world. One of the suggested control strategies for parasitosis caused by members of the Ancylostomatidae family is mass drug aministration with benzimidazole compounds. This strategy has been proven to lead to the establishment of resistant strains in several nematodes related to SNPs at codons 167, 198 and 200 of the beta-tubulin isotype-1 gene. Through bioassay and in vivo analysis, we successfully isolated an albendazole-resistant A. ceylanicum strain by drug selective pressure. We observed a strong correlation between the presence of SNPs at codon 198 and drug resistance. We also described for the first time, in hookworms, the presence of SNP A200L, already described at low frequencies in ruminant nematodes. The results presented here are important for updating the current knowledge about anthelmintic resistance in hookworms. The answers and the new questions raised may provide a basis for the establishment of more effective control strategies.

Bats are parasitized by a wide spectrum of ecto and endoparasites, but their role as a reservoir for some zoonoses is not fully understood. The objective of this work was to evaluate the presence of Leishmania DNA in the blood of bats from 30 municipalities in the state of Minas Gerais, Brazil. We analyzed samples of 120 bats, covering 29 species. The blood samples were used for DNA extraction and submitted to conventional PCR analysis with primers directed to the Leishmania ITS-1 region of the rRNA. In total, 1.67% (2/120 samples) were positive for Leishmania spp., detected in animals from the metropolitan region of Belo Horizonte, the state capital. Sequencing of the positive samples revealed that both bats were infected with Leishmania (Leishmania) infantum. Considering the adaptability of some bats species to synanthropic environments, the results of the present work can contribute to a better comprehension of the leishmaniasis cycle and epidemiology.

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.

Necator americanus is a worm that parasites the small intestine of humans and is highly prevalent in regions with poor sanitary conditions. The main strategy to control this helminth is by mass benzimidazole administration, however, periodic use of these drugs can select strains of parasites resistant to treatment. Single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype 1 gene located at codons 167, 198 and 200 have been associated with benzimidazole resistance in some nematodes. Previously, our group detected the presence of some of these SNPs in populations of soil-transmitted helminths collected in different locations in Brazil. Here, we evaluated the SNP at codon 167, which has recently been shown to be associated with failure of benzimidazoles to treat N. americanus. Our ARMS-PCR analyses were performed using 524 single N. americanus eggs from 48 patients' feces collected in six Brazilian states; however, we did not detect any mutated samples at codon 167. This study builds on previous work, helping us monitor the presence of resistance-related genotypes in Brazilian helminth populations. The data presented here can assist in the implementation of future control strategies.

Marsupials of the genus Didelphis, such as black-eared opossums (Didelphis aurita), are common synanthropic animals in urban areas of Brazil. These marsupials are frequently parasitized by numerous helminth species, including ancylostomatid nematodes. This study aimed to report the occurrence of Ancylostoma caninum in black-eared opossums captured in an urban environment of Southeastern Brazil and discuss the potential impact of these findings for public health. From January to June 2019, we collected fecal samples from 49 restrained opossums and evaluated by a simple flotation method; Helminth eggs were observed at different magnifications and identified according to morphological and morphometric features. Genomic DNA was extracted from Ancylostomatidae eggs and screened by duplex PCR for Ancylostoma spp. and Necator americanus using primers that amplify a region of internal transcribed spacer 2 and the 28S ribosomal RNA (ITS2-28S rRNA). Ancylostoma spp. eggs were detected in 65.3% (32/49) of the animals. Sequence analysis revealed 100% homology with A. caninum sequences from GenBank. Our results demonstrate a new host-parasite interaction for A. caninum, suggesting that black-eared opossums may participate in the zoonotic cycle of this parasite in urban areas of Brazil.

Species of the genus Posthodiplostomum are intestinal parasites of piscivorous birds with worldwide distribution. Recent molecular surveys have focused on developmental stages from vertebrate hosts, with few sequences from larvae found in molluscs. Moreover, most published molecular data are from collections in North America, Europe and Asia, and there are no data for South American species. In the present study, cercariae found in the freshwater limpet Gundlachia ticaga from Brazil were used for morphological, experimental and molecular studies. The longifurcate cercariae, Neascus-type metacercariae obtained in experimentally infected guppies (Poecilia reticulata), and an adult parasite recovered from a mouse were morphologically identified as Posthodiplostomum nanum. Sequences of DNA from internal transcribed spacer (ITS) rDNA and cytochrome c oxidase 1 (cox1) from the cercariae and metacercariae were compared with published data, yielding no matches (ITS divergence ≥ 5.5%, cox1 ≥ 13.9%). Phylogenetic analyses of the ITS sequences suggest paraphyly within the genus Posthodiplostomum, or misidentifications of parasites in molecular studies. For example, ITS sequences of Mesoophorodiplostomum pricei and Posthodiplostomum centrarchi or the unnamed species Posthodiplostomum sp. 8 diverge by only 1.1–1.2% and 0.6%, respectively, and all three species fall within a well-supported clade, suggesting that these isolates are congeneric. The phylogenetic tree obtained for cox1 sequences, although not well resolved, reveals that the type species of the genus, Posthodiplostomum cuticola, does not group with any species previously identified as Posthodiplostomum. Overall, the analyses of molecular data here obtained for P. nanum compared with sequences of related species suggest that a review of this group is necessary. Such studies may result in a split of the genus Posthodiplostomum and the transference of some species currently assigned in this genus to other already described genera.

The existence of cryptic species in the genus Hydatigera, cyclophillid cestodes, mainly of felids, was recently described based on molecular studies of parasites from Asia, Europe and Africa. However, the occurrence of H. taeniaeformis sensu stricto (s.s.), the species more widely distributed and with a presumed specificity for murid rodents as intermediate hosts, has not been formally described in Americas. In the present study, during necropsy of an Ingram's squirrel specimen, Guerlinguetus ingrami, found dead in the municipality of Belo Horizonte, Minas Gerais state, Brazil, strobilocerci were found in the liver. The metacestodes were subjected to morphological and molecular studies. Sequences of the COI barcode region were obtained and used for phylogenetic analyses. The morphology and measures of the rostellar hooks were compatible with the ones described for H. taeniaeformis s.s. This identification was confirmed by a molecular phylogenetic approach (96.2-99.7% similarity with isolates of the parasite from Europe and Asia). This is the first molecular confirmation of the existence of H. taeniaeformis s.s. on the American continent. Moreover, the involvement of sciurid rodents in the transmission of H. taeniaeformis s.s. is discussed here as a probable case of parasite spillover.

Considering the great efforts towards formulating a vaccine against hookworms, and the concerns about the spread of drug resistance through hookworm populations, it is justified to study the molecular diversity and population genetic structure of these nematodes. This work had the aim to develop microsatellite markers to investigate the genetic structure and the molecular diversity of Brazilian populations of Ancylostoma caninum. Seven microsatellites markers were successfully used to characterize five Brazilian populations. These findings may contribute to a better comprehension of the ecology, patterns of transmission, drug resistances and development of immunotherapeutic strategies in hookworms.

Although obesity is well established in hamsters, studies using diets with high levels of simple carbohydrate associated with lipids are necessary to assess the impact of this type of food in the body. In this study a high sugar and butter diet (HSB) and high temperature were employed towards this end. Obesity was successfully induced at a temperature of 30.3°C to 30.9°C after 38 days feeding the animals an HSB diet. It was shown that although diet is important for the induction of obesity, temperature is also essential because at a temperature slightly below the one required, obesity was not induced, even when the animals were fed for a longer period (150 days).The obese clinical condition was accompanied by biochemical and hematological changes, as increased cholesterol and triglyceride levels and increased leukocyte numbers, similar to alterations observed in obese humans. Furthermore, it was demonstrated that increasing the intake of simple carbohydrates associated with lipids provided evidence of inflammation in obese animals.

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.

Currently, a great proportion of the emerging infectious human diseases are zoonotic, with most of the pathogens originated from wildlife. In this sense, synanthropic animals such as marsupials play important role in the dissemination of pathogens due to their proximity to human dwellings. These hosts are affected by many gastrointestinal parasites, including species with zoonotic potential. The aim of this study was to assess the diversity of gastrointestinal parasites infecting the black-eared opossum D. aurita captured in urban areas of Southeastern, Brazil. In addition, the potential risk for the human population based on the One Health perspective has been discussed. Forty-nine marsupial specimens were captured with Tomahawk live traps and fecal samples were collected. The samples were evaluated by parasitological procedures. Eggs and oocysts were analyzed at different magnifications (400 × and 1000 ×), and their identification, together with adult nematodes, was established on morphological and morphometric data. Forty-three hosts (87.76%) scored positive for at least one gastrointestinal parasite, being 83.67% (41/49) for helminths, and 65.30% (32/49) for protozoa. For Cryptosporidium sp., only 13 samples were evaluated due to insufficient amount of feces obtained of some animals. A prevalence of 23.08% (3/13) was reported for this parasite. PCR analysis revealed Ancylostomatidae eggs to belong to the genus Ancylostoma. Our results demonstrated that multiparasitism is frequently found in these animals and a high percentage of potentially zoonotic parasites are observed, implying that D. aurita may be involved in zoonotic cycles in urban environments.

Obesity and ancylostomiasis are considered public health problems. Recent studies have shown that infection by intestinal helminths in obese individuals can ameliorate metabolic disorder and improve glucose tolerance by decreasing both insulin resistance and low-intensity inflammation. However, few helminth species have been studied in this context, and some modulation mechanisms still require deeper investigation. Therefore, the present work aimed to investigate the role of experimental infection with Ancylostoma ceylanicum in the modulation of the immune response in an obese experimental model. Four groups of hamsters were used as follows: two groups were submitted to a hyperlipidic and hypercaloric diet capable of inducing obesity, one infected and the other uninfected; and two normonourished control groups, one infected and one uninfected by A. ceylanicum. Biochemical, haematological, parasitological and immunological parameters were evaluated. The results demonstrated that A. ceylanicum infection accentuated weight loss in obese animals compared to normonourished animals. However, obesity reduced the recovery of worms and oviposition of the females, and both infected groups showed decreased levels of haemoglobin, albumin, iron and erythrocytes. Significant relations were observed for pathogenesis in the following cases: infection interfered in lipid metabolism, which increased levels of total cholesterol and triglycerides in the obese group, and caused a decrease in HDL levels in both groups. Obesity led to an increase in glucose levels, and the infection exacerbated this parameter in both the normonourished and obese groups. Inflammation was intensified in obese animals that showed elevated macrophage and neutrophil activation in adipose tissue, enlargement of the spleen and accumulation of lipids in the liver and faeces. Despite the decrease in IFN-γ levels, the infection did not potentiated the expression of the Foxp3, IL-10 and IL-2 transcription factor for any of the infected groups, markers that could positively compensate the host from the damage caused by obesity.

Made available in DSpace on 2019-10-06T15:38:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01. Added 1 bitstream(s) on 2021-07-15T14:35:32Z : No. of bitstreams: 1 S0001-37652019000100616.pdf: 1663784 bytes, checksum: 6bc7f23d512c50a2be683eb226599c6d (MD5) 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. Molecular Helminthology Laboratory Department of Parasitology Institute of Biological Sciences Federal University of Minas Gerais/UFMG, Avenida Antônio Carlos, 6627 Urban Bats Laboratory Zoonosis Control Center Municipality Secretary of Health of Belo Horizonte, Rua Edna Quintel, 173 São Paulo State University/UNESP Institute of Bioscience, Rua Professor Doutor Antônio Celso Wagner Zanin, s/n São Paulo State University/UNESP Institute of Bioscience, Rua Professor Doutor Antônio Celso Wagner Zanin, s/n

Helminth parasites cause significant morbidity and mortality in endemic countries. Given the severity of symptoms that helminths may elicit in the host, intervention with prophylactic and therapeutic measures is imperative. Treatment with benzimidazoles is the most widely used means of combatting these parasites. However, widespread use of these drugs can select for drug-resistant parasite strains. In this review, we approach the problem of benzimidazole resistance in helminths in both humans and animals, focusing on the properties of the drug, the molecular mechanisms of drug resistance and how resistance is diagnosed.

Trematodes belonging to the superfamily Diplostomoidea have complex life cycles involving birds, mammals and reptiles as definitive hosts, and gastropods and different groups of invertebrates and vertebrates as intermediate hosts. Molecular studies of these parasites are numerous, but data from larval stages in molluscs remain scarce, particularly in South America. The present study focused mainly on five morphotypes of longifurcate cercariae found in Biomphalaria straminea (Dunker, 1848) from Belo Horizonte, Minas Gerais, Brazil, collected between 2009 and 2017. In each morphotype, nuclear internal transcribed spacer (ITS1-5.8S ITS-2) rDNA and mitochondrial cytochrome c oxidase 1 (COI) genes were sequenced. Laboratory-reared fish, Poecilia reticulata Peters, 1859 or snails, Biomphalaria glabrata (Say, 1818) were exposed to cercariae to obtain metacercariae. The morphology of cercariae, experimentally obtained metacercariae, and phylogenetic analyses led to the identification of three species of Diplostomidae [Austrodiplostomum compactum (Lutz, 1928), Crassiphialinae gen. sp. and Hysteromorpha sp.] and two species of Strigeidae (Cotylurus sp., Apharyngostrigea sp.). Previously published sequences allowed species-level identification for only A. compactum, although provisional identifications were possible in two cases. First, the COI from cercariae of Apharyngostrigea sp. in Brazil matched those of metacercariae from naturally infected Cnesterodon decemmaculatus (Jenyns, 1842) in Argentina; although a positive identification is not possible, the material presents morphological similarities with larval stages previously described for A. simplex. Secondly, Cotylurus sp. resembles C. lutzi. Our analysis of previously published COI sequences suggests that Cotylurus cornutus (Rudolphi, 1808) has a Holarctic distribution. Both the morphology of experimentally obtained metacercariae and COI sequences indicate that Hysteromorpha sp. in Brazil is distinct from congeners in North America [Hysteromorpha corti (Hughes, 1929)] and Europe [Hysteromorpha triloba (Rudolphi, 1819)].

Mass drug administration has been implicated as the major cause of drug resistance in nematodes of ruminants. Single-nucleotide polymorphisms (SNPs) at codons 167, 198, and 200 of the beta-tubulin isotype 1 gene are associated with albendazole resistance mechanisms. Although drug resistance is suspected to occur in nematodes of the same order, at present, there is no evidence of a strong correlation between these canonical SNPs and albendazole resistance in hookworms. In the absence of a hookworm strain that is naturally resistant to albendazole, we produced an albendazole-resistant Ancylostoma ceylanicum strain by selective drug pressure. Restriction fragment length polymorphism-PCR (RFLP-PCR) was employed to identify the presence of SNPs previously associated with drug resistance in other nematodes. However, none of the benzimidazole resistance–associated SNPs known in other nematodes were found. A beta-tubulin isotype 1 gene mini-cDNA library was constructed to obtain the complete cDNA gene sequence for the analysis of the entire gene to identify distinct SNPs associated with resistance. Some SNPs were found, but the resulting sequences were not reproducibly detected among the different clones, preventing their association with the resistance mechanism. The parasitological and hematological parameters of the albendazole-resistant strain were characterized and compared to those of the sensitive strain. Although the albendazole-resistant strain was less adapted to its host, with fewer worms recovered, all other parameters analyzed were similar between both strains. The results of the present study indicate that the mechanism of albendazole resistance of the resistant strain described herein must differ from those that have previously been characterized. Thus, new mechanistic studies are needed in the future.

Mutations in the β-tubulin isotype 1 gene at codons 167 (F167Y), 198 (E198A) and 200 (F200Y) have been associated with benzimidazole resistance in helminths. The F200Y polymorphism has previously been described for Ancylostom caninum; however, the F167Y polymorphism has not been investigated in members of the Ancylostomatidae family. The aim of this study was to screen for the F167Y polymorphism in A. caninum isolates recovered from naturally infected dogs in two Brazilian states. No mutation was observed at codon 167 in the 230 analyzed samples from the two populations; however, it is possible that this change may be present at a low frequency in other populations of the same species. These results highlight the importance of monitoring the genetic basis involved in the drug resistance process.

Single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene in codons 167, 198 and 200 have been associated with benzimidazole resistance in some nematodes, although no polymorphisms in this gene have been described in the hookworm Ancylostoma caninum to date. This study aimed to screen for SNPs in the β-tubulin isotype 1 gene at codons 198 and 200 associated with benzimidazole resistance in A. caninum worms recovered from naturally infected dogs from two Brazilian states (Minas Gerais and Piauí). To perform this analysis, a molecular technique based on the amplification refractory mutation system (ARMS-PCR) was standardized. A total of 110 individual worms recovered from 9 dogs in Piauí and 124 individual worms recovered from 10 dogs in Minas Gerais were analyzed using this methodology. The presence of a SNP at position 200 was observed at a low frequency (0.8%), which was only detected in the worm population from Minas Gerais State. A total of 75 randomly selected samples were sequenced to validate this technique, and the presence of the SNP was confirmed. Furthermore, SNP at position 198 were evaluated by sequencing, and none were detected in this region. This is the first report of the presence of a SNP in the β-tubulin isotype 1 gene associated with benzimidazole resistance in an A. caninum population.

The tetraprimer ARMS-PCR technique is efficient for SNP detection and can be used to search for polymorphisms associated with drug resistance. However, the establishment of this methodology is not always straightforward because of the constraints on primer design due to the restrictions of the polymorphic regions. Here, we describe the standardization of the tetraprimer ARMS-PCR methodology for the detection of a SNP at codon 198 of the Ancylostoma caninum β-tubulin gene. This SNP is associated with resistance to albendazole in various nematodes. The methodology was used to screen 327 individuals from 6 different locations. No mutation was found in any of the samples. This methodology will be useful for screening for the E198A SNP in the β-tubulin gene of canine hookworms in a broader population to determine whether this SNP is associated with benzimidazole resistance in this species. The method could also be adapted for the analysis of other SNPs in other nematode species.

This study compared the course of Ancylostoma ceylanicum infection in hamsters infected with different inocula and the consequences for the host and helminth populations. The average of adult worms recovered, according to the number of third stage larva used, were 28.0, 24.8, 24.6, and 24.8 % to inocula size of 25 L3, 75 L3, 125 L3, and 250 L3, respectively. The size of the inoculum did not affect the establishment, survival, or fecundity of adult helminths. Reductions in the red blood cell and hemoglobin levels in the infected group were inversely proportional to the number of white blood cells. Moreover, differential cell counting revealed a positive correlation between the worm load and leucocyte numbers. The humoral response against excretion-secretion antigens was more robust and sensitive compared with the response against crude extract, with no direct linear correlation with the number of worms. The effect of the population density was more evident in females.

Tick-borne infections can result in serious health problems for wild ruminants, and some of these infectious agents can be considered zoonosis. The aim of the present study was the post-mortem detection of hemoparasites in free-living Mazama gouazoubira from Minas Gerais state, Brazil. The deer samples consisted of free-living M. gouazoubira (n = 9) individuals that died after capture. Necropsy examinations of the carcasses were performed to search for macroscopic alterations. Organ samples were collected for subsequent imprint slides, and nested PCR assays were performed to detect hemoparasite species. Imprint slide assays from four deer showed erythrocytes infected with Piroplasmida small trophozoites, and A. marginale corpuscles were observed in erythrocytes from two animals. A. marginale and trophozoite co-infections occurred in two deer. A nested PCR analysis of the organs showed that six of the nine samples were positive for Theileria sp., five were positive for A. phagocytophilum and three were positive for A. marginale, with co-infection occurring in four deer. The results of the present study demonstrate that post-mortemdiagnostics using imprint slides and molecular assays are an effective method for detecting hemoparasites in organs. Patógenos transmitidos por carrapatos podem resultar em sérios problemas de saúde para os ruminantes selvagens, e alguns podem ser zoonoses. O objetivo do presente estudo foi a detecção post mortem de hemoparasitos, em Mazama gouazoubira de vida livre, oriundos de Minas Gerais, através da análise de lâminas de impressão e nested PCR. Foram amostrados nove M. gouazoubira de vida livre, que morreram após a captura. Exames de necropsia foram realizados, e as carcaças foram examinadas para detectar alterações macroscópicas. Amostras dos órgãos foram coletadas para a realização de imprint em lâminas e para nested PCR à procura de hemoparasitos. A análise das lâminas mostrou pequenos trofozoítos de Piriplasmida nos eritrócitos de quatro dos oito animais examinados, e corpúsculos de Anaplasma marginale foram observados nos eritrócitos de dois dos cervídeos. A coinfecção com A. marginale e trofozoítos de piroplasmas ocorreu em dois animais. As análises de nPCR dos órgãos mostraram que seis dos nove animais estavam positivos para Theileria sp., cinco para A. phagocytophilum e três para A. marginale, sendo que a coinfecção ocorreu em quatro cervídeos. Os resultados do presente estudo demonstram que os diagnósticos post-mortem, pelas imprints em lâminas e ensaios moleculares, são métodos eficazes de detecção de hemoparasitos nos principais órgãos parasitados.

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, 1 19,151 1 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.

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.

This study compared the course of infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs infected with L3 larvae. Dogs infected with A. caninum eliminated more eggs than did those infected with A. braziliense. A total of 38 % of A. caninum and 44 % of A. braziliense larvae were recovered as adult worms. There were no marked clinical abnormalities in dogs with either infection. A. caninum was associated with anemia and an increased number of circulating neutrophils, whereas infection with A. braziliense led to a decrease in the number of leukocytes. The humoral response against excreted and secreted antigens from adult worms was more sensitive and specific than the response induced with the crude antigen. No immune response was observed for either crude or excreted-secreted (ES) antigens from larvae of either species. A nonspecific response against the crude antigen of A. braziliense was found at 0 and 7 days postinfection and maintained throughout the infection period. However, antibody titers against ES antigens were elevated in A. caninum infection at patency and death, showing that this antigen has a higher specificity. The immune response elicited by infection with A. braziliense in dogs has not been described previously. No significant differences were observed in the infection processes of the two Ancylostoma species, except for the higher number of eggs eliminated from dogs infected with A. caninum, which may indicate a better evolutionary adaptation of the parasite to its host in comparison with A. braziliense.

Background Presence of tumor markers in serum might be connected to the number of secreting cells and with the stage of the neoplasm. However, there are few studies regarding these markers in veterinary clinical oncology. Objectives To determine the serum concentrations of cancer antigen 15.3 (CA 15.3), carcinoembryonic antigen (CEA), and lactate dehydrogenase (LDH) in female dogs with different stages of mammary cancer. Animals Ninety female dogs, including 30 that were healthy, 40 that had nonmetastatic cancer, 12 with regional metastasis, and 8 with distant lymph node metastasis. Methods Prospective case-controlled observational study. Serum samples were collected to measure CA15.3, CEA, and LDH from 60 female dogs with mammary cancer during mastectomy and 30 healthy female dogs during routine check-up. CA15.3 and CEA were determined by chemiluminescent immunoassay and LDH by ultraviolet kinetic method. Western blotting analysis was performed to confirm the specificity and possible cross-reactivity of human CA15.3 and CEA antibodies with canine serum. Group data were compared by ANOVA followed by Student-Newman-Keuls and Tukey's tests. Correlations were investigated by Pearson and Spearman tests. Results CEA, CA15.3, and LDH were measurable in all groups. Higher serum concentration of CA15.3 and LDH was associated with regional and distant metastases. There was a significantly higher serum CA15.3 concentration in animals with lymph node metastasis when compared with animals without metastasis. There were no significant differences in CEA among groups. Expression of CA15.3 and CEA in canine serum was confirmed by Western blotting. Conclusions and Clinical Importance Serum CA15.3 can be used to distinguish nonmetastatic from metastatic carcinomas.

Despite reports that food-borne parasitic infections have been increasing worldwide, the methodologies employed to detect food contamination by helminths are still largely based on methodologies used to detect these pathogens in feces and water. This study sought to improve the diagnosis of parasitic contaminants in lettuce by standardizing a method for detecting helminth eggs and larvae and estimating their percentage of recovery. Sanitized lettuces were artificially contaminated with different amounts of Ascaris suum and hookworm eggs and larvae. To standardize the method, we tested liquid extractors, vegetable washing steps, and spontaneous sedimentation times. Higher percentages of egg and larvae recovery were obtained using 1 M glycine as the liquid extractor, manual shaking for 3 min and 2 h of sedimentation. Five different levels of artificial contamination (ten replicates each; n = 50) were tested using these standardized conditions, yielding an average recovery of 62.6 % (±20.2), 51.9 % (±20.0), and 50.0 % (±27.3) for A. suum eggs, hookworm eggs, and larvae, respectively. Tests were performed with a different matrix to evaluate the performance of the method. Furthermore, collaborative analytical studies performed by different laboratories produced satisfactory results. The method for the identification of helminth eggs and larvae proposed in this study proved to be simpler and more efficient than previously published procedures, thereby demonstrating its potential contribution to health surveillance and epidemiological studies.

Summary Deer are important natural reservoir hosts of Anaplasmataceae. The present study used nested PCR and nucleotide sequencing to evaluate the occurrence of Anaplasmataceae species in 23 free-living and six captive specimens of the cervids Mazama gouazoubira and Blastocerus dichotomus in Minas Gerais State, Brazil. Blood samples were tested for the presence of Ehrlichia and Anaplasma spp. using nPCR assays and sequencing of the msp4, msp1 and 16S rRNA genes. The identity of each sequence was confirmed by comparison with sequences available from GenBank using BLAST software. Of the animals investigated, 93.1% (27/29) were infected with haemoparasites including Anaplasma marginale (79.3%), Ehrlichia chaffeensis (3.4%), Anaplasma bovis (3.4%) and Anaplasma spp. (assigned to A. platys and A. phagocytophilum) (17.2%). Co-infection occurred in 20% (6/29) of the deer examined. Four (13.8%) were infected with A. marginale and Anaplasma sp., one (3.4%) was infected with A. marginale and E. chaffeensis, and one (3.4%) was infected with A. marginale and A. bovis. The results of the present study suggest that cross-protection does not occur in these deer. Immunological cross-reaction occurs when sera are tested diagnostically because these bacteria are closely related taxonomically, reinforcing the importance of molecular diagnosis followed by nucleotide sequencing.

Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infec tions 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 par asite 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 fre quency 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 resis tance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs Ascaris lumbricoides e Necator americanus são parasitas transmitidos pelo solo com distribuição geográfica, e representam algumas das infecções mais comuns e negligenciadas no mundo. Tratamento periódico com administração em massa de medicamentos (MDA) em áreas endêmicas é a ação recomendada pela Organização Mundial da Saúde. No entanto, o MDA pode causar a seleção de subpopulações que possuem a capacidade genética de superar o mecanismo de ação da droga. De fato, mutações no gene da beta-tubulina (códons 167, 198 e 200) estão correlacionados com a resistência ao benzimidazol em nematoides de importância veterinária. Isto é possível que esses SNPs também tenham forte correlação com a resistência ao tratamento no geohelmintos humanos A. lumbricoides, Trichuris trichiura e ancilostomídeos. Aqui, tivemos como objetivo investigar a presença de alguns desses marcadores moleculares canônicos associados à resistência parasitária ao benzimidazol em N. americanus e A. lumbricoides coletados de seis estados brasileiros. Nested-PCR e PCR-RFLP foram usados ​​para detectar mutações nos códons 167 e 198 em 601 ovos individuais de A. lumbricoides coletados de 62 amostras de fezes humanas; no entanto, nenhuma mutação foi encontrada. Os códons 198 e 200 foram testados em 552 N. americanus ovos coletados de 48 pacientes pela mesma metodologia, que apresentaram frequência relativa de 1,4% e 1,1%, respectivamente. A presença desses SNPs em N. americanus ovos é um achado importante, indicando que com alta pressão do medicamento benzimidazol há potencial de resistência ao benzimidazol a ser selecionado neste ancilostomídeo. No entanto, nestas baixas frequências, isso não indica que exista atualmente qualquer resistência ao benzimidazol problema. Este é o primeiro estudo sistemático realizado na América do Sul, e o estudo produziu um panorama das variantes genéticas no gene da beta-tubulina e resistência anti-helmíntica a parasitas transmitidos pelo solo detectados por uma genotipagem simples, rápida e acessível ensaio de ovos individuais

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n=92; Belo Horizonte, n=50; Nanuque, n=102) and the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=28; Nanuque, n=66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.

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.

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.

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.

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.

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., Author Summary Hookworm's infection is a health problem that affects areas of poverty worldwide. Nutritional diseases, including dietary protein deficiency are also prevalent in those areas. It is well known that nutritional status can influence parasite infection, reducing host responses to infection. However, the influence of nutritional status and Hookworm's infection is less explored. Our goal was to study the consequences of coexistence of low-protein malnutrition and Hookworm's infection in hamsters that are good models of this infection. For that, we established a model of protein malnutrition by feeding hamsters on standard diet or isocaloric, low-protein diet for 50 days. Hamsters fed on those diets were infected with hookworm's larvae for assessment of biochemical and parasitological parameters compared to non-infected controls. Our results confirm that malnutrition was installed, as suggested by low antibody response and by increased fecal lipid excretion in malnourished group. Regarding Hookworm's infection, animals fed low-protein diet presented a more severe infection, with higher number of worms recovered in intestine. Nonetheless, egg production was reduced in malnourished hamsters, suggesting that protein restriction was determinant on egg formation. These results confirm the hypothesis that host nutritional status greatly influences the course of Hookworm's infection.

Entamoeba histolyticais 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 ofE. histolyticain 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 byE. histolyticagenerating new pathways for diagnosis and treatment of amoebiasis.

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 rMSP1 a from A. marginale using carbon nanotubes as a carrier molecule. The fragment of rMSP1 a 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 (rMSP1 a ), G2 (MWNT + rMSP1 a ), 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 rMSP1 a (G1 and G2) produced high levels of anti-rMSP1 a IgG, demonstrating that the functionalization to carbon nanotubes did not interfere with protein immunogenicity. Immunization with MWNT + rMSP1 a 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 rMSP1 a alone (G1 group). Therefore, additional experiments using cattle should be performed to determine the efficacy, safety, immunogenicity and protection induced by rMSP1 a associated with MWNT.

ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase δ subunit and peripheral-type benzodiazepine receptor is discussed.