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.

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.

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.

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.