Hookworm infection as a model for deepen knowledge of iron metabolism and erythropoiesis in anemia.

Over the years, there has been progress in understanding the molecular aspects of iron metabolism and erythropoiesis. However, despite research conducted both in laboratories and living organisms, there are still unanswered questions due to the complex nature of these fields. In this study we investigated the effects of hookworm infection on iron metabolism and how the hosts response to anemia is affected using hamsters infected with Ancylostoma ceylanicum as a model. Our data revealed interesting relationships between infection-induced anemia, erythropoiesis, iron metabolism, and immune modulation, such that the elevated production of erythropoietin (EPO) in renal tissue indicated intensified erythropoiesis in response to anemia. Additionally, the increased expression of the erythroferrone (ERFE) gene in the spleen suggested its involvement in iron regulation and erythropoiesis. Gene expression patterns of genes related to iron metabolism varied in different tissues, indicating tissue-specific adaptations to hypoxia. The modulation of pro-inflammatory and anti-inflammatory cytokines highlighted the delicate balance between immune response and erythropoiesis. Data derived from the investigation of changes induced in iron metabolism and stress erythropoiesis following anemia aid in our understanding of mechanisms related to blood spoliation and anemia, which could potentially be extrapolated or compared to other types or causes of anemia. These findings also contribute to our understanding of the pathophysiology of erythropoiesis in the context of blood loss.

Molecular variability of the Ancylostoma secreted Protein-2 (Aca-asp-2) gene from Ancylostoma caninum contributes to expand information on population genetic studies of hookworms.

Hookworm infection is a major public health problem in many regions of the world. Given the high levels of host morbidity and even mortality of the host caused by these infections, it is crucial to understand the genetic structure of hookworm populations. This understanding can provide insights into the ecology, transmission patterns, mechanisms of drug resistance, and the development of vaccines and immunotherapeutic strategies. Previously, we examined presumably neutral molecular markers, such as microsatellites and COI (Cytochrome C oxidase subunit 1) in Brazilian populations of Ancylostoma caninum. Here we analyze the molecular variability of a genomic fragment of the Aca-asp-2 (Ancylostoma secreted protein-2) gene from Ancylostoma caninum. This gene is a highly expressed and activated following the infection of the L3 larvae in the host. We obtained individuals of A. caninum from five different geographic locations in Brazil, sequenced and analyzed parts of the gene. The results revealed extensive polymorphism at this fragment, especially in the intronic region, indicating low selective pressure acting on these sequences. However, we also observed irregular distributions of nucleotides and polymorphisms in the coding region of this gene, resulting in the identification of 27 alleles. The data presented here contribute to expanding the understanding of population genetic studies of hookworms.

Role of the IL-33/ST2 Activation Pathway in the Development of the Hepatic Fibrosis Induced by Schistosoma mansoni Granulomas in Mice.

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.

From field to laboratory: isolation, genetic assessment, and parasitological behavior of Schistosoma mansoni obtained from naturally infected wild rodent Holochilus sciureus (Rodentia, Cricetidae), collected in Northeastern Brazil.

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 tRNACys-12S (referred to as 16S herein) 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 the 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.

Phenotypic characterization and multiple resistance analysis in an experimentally selected albendazole-resistant hookworm isolate.

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.

Detection of Benzimidazole Resistance-Associated Single-Nucleotide Polymorphisms in the Beta-Tubulin Gene in Trichuris trichiura from Brazilian Populations.

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.

Bats as hosts of Leishmania (Leishmania) infantum in Minas Gerais, an endemic area for visceral leishmaniasis.

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.

Development of allele-specific PCR methodology (AS-PCR) to screening A. lumbricoides and A. suum.

Ascaris lumbricoides and Ascaris suum are described as helminths that infect humans and pigs, respectively. It is estimated that infection by A. lumbricoides affects about 447 million individuals living in tropical regions of developing countries. However, there is an increasing number of cases of human ascariasis in countries with no recent history of autochthonous infection by A. lumbricoides. In these places, pigs have been incriminated as the main source of human infection. Conventional parasitological diagnosis does not allow species-specific identification, and the real epidemiological scenario of human and swine ascariasis is still uncertain. Therefore, this work presents the application of a species-specific molecular diagnosis, based on the allele-specific PCR methodology (AS-PCR), using the Internal Transcript Space 1 (ITS-1) of the ribosomal DNA, as a target for differentiating between the two species, using DNA obtained from eggs. To validate the methodology, stool samples positive for Ascaris spp, were obtained from 68 humans from seven Brazilian states and from six pigs from the state of Minas Gerais. All samples obtained from humans were genotyped as A. lumbricoides and all samples obtained from swine were genotyped as A. suum. These results are in agreement with the literature, which demonstrates that in most endemic regions, transmission cycles are separate. Therefore, the execution of this work allowed the availability of a useful methodology for the differential diagnosis of the species, which may contribute to the characterization of the real epidemiological profile of human and swine ascariasis, and to the implementation of future control strategies.

Moving beyond the state of the art of understanding resistance mechanisms in hookworms: confirming old and suggesting new associated SNPs.

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.

Genotypic profile of benzimidazole resistance associated with SNP F167Y in the beta-tubulin gene of Necator americanus helminths obtained from Brazilian populations.

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.

Gastrointestinal parasites in the opossum Didelphis aurita: Are they a potential threat to human health?

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.

High prevalence of Ancylostoma caninum infection in black-eared opossums (Didelphis aurita) in an urban environment.

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.

Egg genotyping reveals the possibility of patent Ancylostoma caninum infection in human intestine.

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.

Use of polyclonal IgY antibodies to detect serum immune complexes in patients with active hookworm infection.

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 with Ancylostoma ceylanicum saline 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.

First identification of the benzimidazole resistance-associated F200Y SNP in the beta-tubulin gene in Ascaris lumbricoides.

The main control strategy for Ascaris lumbricoides is mass drug administration (especially with benzimidazoles), which can select strains of parasites resistant to treatment. Mutations in the beta-tubulin isotype-1 gene at codons 167, 198 and 200 have been linked to benzimidazole resistance in several nematodes. The mutation in codon 200 is the most frequent in different species of parasites, as previously observed in Necator americanus and Trichuris trichiura; however, this mutation has never been found in populations of A. lumbricoides. This study aimed to screen for single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codon 200 in A. lumbricoides. We developed a technique based on an amplification refractory mutation system (ARMS-PCR) for the analysis of 854 single A. lumbricoides eggs collected from 68 human stool samples from seven Brazilian states. We detected the mutation in codon 200 at a frequency of 0.5% (4/854). This is the first report of this mutation in A. lumbricoides. Although the observed frequency is low, its presence indicates that these parasite populations have the potential to develop high levels of resistance in the future. The methodology proposed here provides a powerful tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations.

Hookworm infection aggravates metabolic disorder in obesity.

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.

Molecular, morphological and experimental-infection studies of cercariae of five species in the superfamily Diplostomoidea (Trematoda: Digenea) infecting Biomphalaria straminea (Mollusca: Planorbidae) in Brazil.

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)].

Albendazole resistance induced in Ancylostoma ceylanicum is not due to single-nucleotide polymorphisms (SNPs) at codons 167, 198, or 200 of the beta-tubulin gene, indicating another resistance mechanism.

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.

Standardization and application of a modified RFLP-PCR methodology for analysis of polymorphisms linked to treatment resistance in Ancylostoma braziliense.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the beta-tubulin isotype 1 gene are associated with benzimidazoles resistance in many helminths. Codon 167 mutation has never been described in hookworms; however, polymorphisms in codons 198 and 200 have been described for Ancylostoma caninum and Necator americanus. These mutations have never been investigated in Ancylostoma braziliense; therefore, it is not known if they are present in this species and whether they are correlated with treatment resistance. The RFLP-PCR technique has been used to analyze these polymorphisms in some nematodes, but depending on the species, these alterations do not create or eliminate any restriction enzyme cleavage site, making it impossible to use this technique. Here, we describe the standardization and application of a modified RFLP-PCR technique for detecting polymorphisms in individual A. braziliense worms recovered from naturally infected dogs in two Brazilian states. RESULTS: The molecular techniques used were sensitive, specific, and easy to apply. To our knowledge, we report for the first time the presence of a polymorphism at codon 198 of the beta-tubulin gene of A. braziliense (1/81; 95% CI: 0-3.69%). CONCLUSIONS: It is not known whether the presence of the mutation in codon 198 of the beta-tubulin gene of A. braziliense has importance for this parasite. However, based on studies of other helminths, it is possible that this polymorphism is directly related to the resistance to benzimidazoles. This may be a major concern, since this nematode has considerable relevance as a parasite of canids and felids and as one of the agents of cutaneous larva migrans in humans. Standardized methodologies will be useful for screening for polymorphisms in the beta-tubulin gene of canine hookworms in a broader population. The method could also be adapted for the analysis of other SNPs in other nematode species.

Molecular, morphological and experimental assessment of the life cycle of Posthodiplostomum nanum Dubois, 1937 (Trematoda: Diplostomidae) from Brazil, with phylogenetic evidence of the paraphyly of the genus Posthodiplostomum Dubois, 1936.

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.