Rhabdias paraensis sp. nov.: a parasite of the lungs of Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

The nematode parasites of Rhinella marina include species of the genus Rhabdias (Rhabdiasidae: Rhabditoidea). The present study describes Rhabdias paraensis sp. nov., which parasitizes the lungs of R. marina in Brazilian Amazonia. Of the more than 70 known species of this genus, 18 are parasites of bufonids, of which, eight are Neotropical. The new species described here is similar to Rhabdias alabialis in the absence of lips is different by the presence of conspicuous cephalic papillae. We describe details of the four rows of pores, which are distributed equally along the whole of the length of the body and connected with hypodermal cells, using histology and scanning electron microscopy. Other histological aspects of the internal structure of this nematode are also described.

Myxidium volitans sp. nov., a parasite of the gallbladder of the fish, Dactylopterus volitans (Teleostei: Triglidae) from the Brazilian Atlantic coast: morphology and pathology

Myxidium volitans sp. nov. (Myxozoa: Myxidiidae) parasitizing the hypertrophied green-brownish gallbladder of the teleost Dactylopterus volitans, collected in the Atlantic coast near Niterói, Brazil was described based on ultrastructural studies. The spores were fusiform, sometimes slightly crescent-shaped on average 21.7 ± 0.3 µm (mean ± standard deviation) (n = 50) long and 5.6 ± 0.4 µm (n = 30) wide. The spore wall was thin and smooth, comprising two equally-sized valves joined by a hardly visible sutural ridge. Spores containing two pyriform polar capsules (PC) (5.0 ± 0.4 × 2.3 ± 0.3 µm) (n = 30) are situated in each extremity of the spore. The PC wall was composed of hyaline layer (0.20-0.29 µm thick) and by a thin external granular layer. Each PC contains a polar filament (PF) with irregular arrangements that was projected from its apical region to the bases of PC and coiled laterally from bases to the tip of PC. Some regular striations and S-like structures in the periphery of the PFs with four-five irregular sections were observed. Based on the spore morphology, ultrastructural differences and the specificity of the host we describe this parasite as a new myxosporidian, named M. volitans sp. nov.

Lanfrediella amphicirrus gen. nov. sp. nov. Nematotaeniidae (Cestoda: Cyclophyllidea), a tapeworm parasite of Rhinella marina (Linnaeus, 1758) (Amphibia: Bufonidae)

The family Nematotaeniidae, tapeworms commonly found in the small intestines of amphibians and reptiles, includes 27 recognised species distributed among four genera: Bitegmen Jones, Cylindrotaenia Jewell, Distoichometra Dickey and Nematotaenia Lühe. The taxonomy of these cestodes is poorly defined, due in part to the difficulties of observing many anatomical traits. This study presents and describes a new genus and species of nematotaeniid parasite found in cane toads (Rhinella marina) from eastern Brazilian Amazonia. The cestodes were collected during the necropsy of 20 hosts captured in the urban area of Belém, Pará. The specimens were fixed and processed for light microscopy, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. Samples were also collected for molecular analyses. The specimens presented a cylindrical body, two testes and paruterine organs. However, they could not be allocated to any of the four existing nematotaeniid genera due to the presence of two each of dorsal compact medullary testes, cirri, cirrus pouches, genital pores, ovaries and vitelline glands per mature segment. Lanfrediella amphicirrus gen. nov. sp. nov. is the first nematotaeniid studied using Historesin analysis, SEM and 3D reconstruction, and it is the second taxon for which molecular data have been deposited in GenBank.

Plasmodium falciparum gametocyte carriage, sex ratios and asexual parasite rates in Nigerian children before and after a treatment protocol policy change instituting the use of artemisinin-based combination therapies

The effects of artemisinin-based combination therapies (ACTs) on transmission of Plasmodium falciparum were evaluated after a policy change instituting the use of ACTs in an endemic area. P. falciparum gametocyte carriage, sex ratios and inbreeding rates were examined in 2,585 children at presentation with acute falciparum malaria during a 10-year period from 2001-2010. Asexual parasite rates were also evaluated from 2003-2010 in 10,615 children before and after the policy change. Gametocyte carriage declined significantly from 12.4% in 2001 to 3.6% in 2010 (@@χ² for trend = 44.3, p < 0.0001), but sex ratios and inbreeding rates remained unchanged. Additionally, overall parasite rates remained unchanged before and after the policy change (47.2% vs. 45.4%), but these rates declined significantly from 2003-2010 (@@χ² for trend 35.4, p < 0.0001). Chloroquine (CQ) and artemether-lumefantrine (AL) were used as prototype drugs before and after the policy change, respectively. AL significantly shortened the duration of male gametocyte carriage in individual patients after treatment began compared with CQ (log rank statistic = 7.92, p = 0.005). ACTs reduced the rate of gametocyte carriage in children with acute falciparum infections at presentation and shortened the duration of male gametocyte carriage after treatment. However, parasite population sex ratios, inbreeding rates and overall parasite rate were unaffected.

Interaction of Trichomonas vaginalis and Tritrichomonas foetus with keratin: an important role in parasite infection

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such infections could cause serious risks to the infected host.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy

Leprosy is a spectral disease exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. We studied the influence of costimulatory molecules on the immune responses of subjects along the leprosy spectrum. The expression of the costimulatory molecules was evaluated in in vitro-stimulated peripheral blood mononuclear cells of lepromatous and tuberculoid patients and healthy exposed individuals (contacts). We show that LL patients have defective monocyte CD86 expression, which likely contributes to the impairment of the antigen presentation process and to patients anergy. Accordingly, CD86 but not CD80 blockade inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the LL anergy, there was reduced expression of the positive signalling costimulatory molecules CD28 and CD86 on the T-cells in these patients. In contrast, tuberculoid leprosy patients displayed increased expression of the negative signalling molecules CD152 and programmed death-1 (PD-1), which represents a probable means of modulating an exacerbated immune response and avoiding immunopathology. Notably, the contacts exhibited proper CD86 and CD28 expression but not exacerbated CD152 or PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signalling.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.

Ultrastructural description of Ceratomyxa microlepis sp. nov. (Phylum Myxozoa): a parasite infecting the gall bladder of Hemiodus microlepis, a freshwater teleost from the Amazon River

A new ceratomyxid parasite was examined for taxonomic identification, upon being found infecting the gall bladder of Hemiodus microlepis (Teleostei: Hemiodontidae), a freshwater teleost collected from the Amazon River, Brazil. Light and transmission electron microscopy revealed elongated crescent-shaped spores constituted by two asymmetrical shell valves united along a straight sutural line, each possessing a lateral projection. The spores body measured 5.2 ± 0.4 µm (n = 25) in length and 35.5 ± 0.9 µm (n = 25) in total thickness. The lateral projections were asymmetric, one measuring 18.1 ± 0.5 µm (n = 25) in thickness and the other measuring 17.5 ± 0.5 µm (n = 25) in thickness. Two equal-sized subspherical polar capsules measuring 2.2 ± 0.3 µm in diameter were located at the same level, each possessing a polar filament with 5-6 coils. The sporoplasm was binucleate. Considering the morphometric data analyzed from the microscopic observations, as well as the host species and its geographical location, this paper describes a new myxosporean species, herein named Ceratomyxa microlepis sp. nov.; therefore representing the first description of a freshwater ceratomyxid from the South American region.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region

Mansonella ozzardi infections are common in the riverside communities along the Solimões, Negro and Purus Rivers in the state of Amazonas (AM). However, little is known about the presence of this parasite in communities located in regions bordering AM and the state of Acre. The prevalence rate of M. ozzardi infections was determined in blood samples from volunteers according to the Knott method. A total of 355 volunteers from six riverine communities were enrolled in the study and 65 (18.3%) were found to be infected with M. ozzardi. As expected, most of the infections (25%) occurred in individuals involved in agriculture, cattle rearing and fishing and an age/sex group analysis revealed that the prevalence increased beginning in the 40-50-years-of-age group and reached 33% in both sexes in individuals over 50 years of age. Based on the described symptomatology, articular pain and headache were found to be significantly higher among infected individuals (56 and 65% prevalence, respectively, p < 0.05). Sera from volunteers were subjected to ELISA using a cocktail of recombinant proteins from Onchocerca volvulus to evaluate the specificity of the test in an endemic M. ozzardi region. No cross-reactions between M. ozzardi-infected individuals and recombinant O. volvulusproteins were detected, thus providing information on the secure use of this particular cocktail in areas where these parasites are sympatric.