A new genus and a new species of Clausiidae (Crustacea, Copepoda) parasitic on Dipolydora armata (Polychaeta, Spionidae) in Brazil

A new genus and species of parasitic copepod (Clausiidae), Spionicola mystaceus, associated with the polychaete Dipolydora armata (Spionidae) is described and figured. The new copepod has an elongate body, 5-segmented antennule, 2-segmented rami on legs 1 and 2, 2 spines representing leg 3, no leg 4, leg 5 well developed and reduced armature elements on feeding limbs. The host is a mollusk-shell borer, collected off São Sebastião Island, State of São Paulo, Brazil.

The effects of the experimental infection from a focus of transmission of Schistosoma mansoni in a population of Biomphalaria tenagophila (D'Orbigny, 1835) in the region of "Vale do Ribeira de Iguape", Brazil

To observe the effects of the parasitic infection on the biology of B. tenagophila, field and laboratory populations of this mollusk from Itariri, in Vale do Ribeira, Brazil, were experimentally infected. Each mollusk received 10 miracidia of Schistosoma mansoni (SJ lineage) and was observed throughout the parasite's development. The biological variables were compared according to the criteria "group" and "infectious phase". The main damage caused by the parasitic infection manifested itself in reproduction, longevity and lesions on the shell of the mollusks in the patent phase. An infection rate of 58.8% was observed. Microanatomical study of the mollusk's digestive gland and ovotestis revealed the presence of evolving larval forms and cercariae. It was concluded that the effects of the parasitic infection on both populations were moderate, despite the low survival rate of the infected mollusks, the damage did not prevent either reproduction or the elimination of cercariae, which continued for a long time.

Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania) amazonensis in the state of Mato Grosso do Sul, mid-western Brazil

INTRODUCTION: The work was conducted to study phlebotomine fauna (Diptera: Psychodidae) and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania) amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. METHODS: The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus) as bait, from May 2004 to January 2006. RESULTS: Ten species of phlebotomine sandflies were captured: Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Psathyromyia campograndensis, Psathyromyia punctigeniculata, Psathyromyia shannoni and Sciopemyia sordellii. The two predominant species were Ev bourrouli (57.3%) and Bi flaviscutellata (41.4%), present at all sampling sites. Two of the 36 hamsters used as bait presented natural infection with Leishmania. The parasite was identified as Leishmania (Leishmania) amazonensis. CONCLUSIONS: Analysis of the results revealed the efficiency of Disney traps for capturing Bichromomyia flaviscutellata and the simultaneous presence of both vector and the Leishmania species transmitted by the same can be considered a predictive factor of the occurrence of leishmaniasis outbreaks for the human population that occupies the location.

Epidemiological and ecological aspects related to malaria in the area of influence of the lake at Porto Primavera dam, in western São Paulo State, Brazil

A study was carried out in the area of influence of the Porto Primavera Hydroelectric Power Station, in western São Paulo State, to investigate ecological and epidemiological aspects of malaria in the area and monitor the profile of the anopheline populations following the environmental changes brought about by the construction of the lake. Mosquitoes captured were analyzed by standardized indicator species analysis (ISA) before and during different flooding phases (253 m and 257 m elevations). The local human population was studied by means of parasitological (thin/thick blood smears), molecular (PCR) and serological tests. Serological tests consisted of Enzyme Linked Immunosorbent Assay (ELISA) with synthetic peptides of the circumsporozoite protein (CSP) from classic Plasmodium vivax, P. vivax variants (VK247 and "vivax-like"), P. malariae and P. falciparum and Indirect Immunofluorescence Assay (IFA) with asexual forms of P. vivax, P. malariae and P. falciparum. The results of the entomological survey indicated that, although the Anopheles darlingi population increased after the flooding, the population density remained very low. No malaria, parasite infection or DNA was detected in the inhabitants of the study area. However, there was a low frequency of antibodies against asexual forms and a significant prevalence of antibodies against P. vivax, P. vivax variants, P. falciparum and P. malariae; the presence of these antibodies may result from recent or less recent contact with human or simian Plasmodium (a parallel study in the same area revealed the existence of a sylvatic cycle). Nevertheless, these results suggest that, as in other places where malaria is present and potential vectors circulate, the local epidemiological conditions observed could potentially support the transmission of malaria in Porto Primavera Lake if infected individuals are introduced in sufficient numbers. Further studies are required to elucidate the phenomena described in this paper.

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA) and indirect ELISA

The Indirect Fluorescence Assay (IFA) and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI) with the IFA (IgG and IgM) and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI). Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2%) seropositive animals for this parasite and 149 (70.8%) negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.

Heartworm (Dirofilaria immitis) disease in a Brazilian oncilla (Leopardus tigrinus)

Heartworm disease is caused by the intravascular nematode Dirofilaria immitis, a pathogen of public health importance usually associated to domestic dogs and cats, and to a lesser extend to other mammal species. The oncilla (Leopardus tigrinus) is a threatened neotropic felid species that naturally occurs in Brazil. Here, we report the encounter of adult and larval stages of heartworms in a female specimen of L. tigrinus, probable of free-ranging origin, from Ubatuba, São Paulo, Brazil, which died showing clinical signals compatible with heartworm disease. This was the first reported case of D. immitis infection and associated disease in L. tigrinus, also suggesting that the oncilla acted as a definitive host for this parasite. The present findings confirmed D. immitis as a pathogenic agent for this felid species, thus supporting the recommendation for the inclusion of diagnostic testing for this pathogen in routine health screening procedures for captive and free-ranging oncillas in Brazil, especially in those localities where climate conditions support the occurrence of the parasite. Potential reservoirs as oncillas are established beyond the reach of veterinary care, thus representing a continuing risk for domestic animals and humans acquiring heartworm infection. We encourage further serologic and molecular studies aiming to establish D. immitis prevalences in L. tigrinus and other wild carnivores in the region of Ubatuba, as well as ecological and veterinary studies to access the role of this pathogen for the survival of this threatened felid species.

The effects of the experimental onfection from a focus of transmissopn of Schistosoma mansoni in a population of Biomphalaria tenagophila (D'Orbigny, 1835) in the region of "Vale do Ribeira de Iguape", Brazil

Com objetivo de observar os efeitos da infecção parasitária na biologia de B. tenagophila, foram realizadas infecções experimentais em populações de campo e laboratório, ambas procedentes de Itariri, Vale do Ribeira, Brasil. Cada molusco recebeu 10 miracídios de Schistosoma mansoni (linhagem SJ), sendo observado durante o desenvolvimento dos parasitos. As variáveis biológicas foram comparadas segundo os critérios "grupo" e "fase de infecção". Os principais danos decorrentes do parasitismo se manifestaram na reprodução, na longevidade e em lesões na concha dos moluscos na fase patente. Foi encontrada uma taxa de infecção de 58,8 por cento. O estudo microanatômico da glândula digestiva e do ovoteste do molusco revelou a presença de formas larvárias em evolução e cercárias. Concluiu-se que os efeitos da infecção parasitária, sobre as duas populações, foram moderados, uma vez que os danos não impediram a reprodução e a eliminação de cercárias que se manteve por um longo período, apesar da baixa sobrevivência dos moluscos parasitados

The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense

Background: Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks. Results: cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology. Conclusions: We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

Background: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion. Methods: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like. Results: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V-repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP. Conclusions: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.

Plasmodium vivax: Induction of CD4(+)CD25(+)FoxP3(+) Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites

Circulation CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-beta) as well as pro-inflammatory (IFN-gamma, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4(+)CD25(+)FoxP3(+)GITR(+) or CD4(+)CD25(+)FoxP3(+)CTLA-4(+) and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.

A Novel Organotellurium Compound (RT-01) as a New Antileishmanial Agent

Leishmaniasis is a neglected disease and endemic in developing countries. A lack of adequate and definitive chemotherapeutic agents to fight against this infection has led to the investigation of numerous compounds. The aim of this study was to investigate the effect of RT-01, an organotellurane compound presenting biological activities, in 2 experimental systems against Leishmania amazonensis. The in vitro system consisted of promastigotes and amastigotes forms of the parasite, and the in vivo system consisted of L. amazonensis infected BALB/c mice, an extremely susceptible mouse strain. The compound proved to be toxic against promastigotes and amastigotes. The study also showed that treatment with RT-01 produces an effect similar to that treatment with the reference antimonial drug, Glucantime, in L. amazonensis infected mice. The best results were obtained following RT-01 intralesional administration (720 mu g/kg/day); mice showed significant delay in the development of cutaneous lesions and decreased numbers of parasites obtained from the lesions. Significant differences in tissue pathology consisted mainly of no expressive accumulation of inflammatory cells and well-preserved structures in the skin tissue of RT-01-treated mice compared with expressive infiltration of infected cells replacing the skin tissue in lesions of untreated mice. These findings highlight the fact that the apparent potency of organotellurane compounds, together with their relatively simple structure, may represent a new avenue for the development of novel drugs to combat parasitic diseases.

Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background: Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector. Results: The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs). Conclusion: Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

Mucosal Leishmaniasis Caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

Background: Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis. Methodology: Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. Results: This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Para, Acre, and Rondonia and cases of ML caused by L. (V.) braziliensis in the state of Rondonia. Conclusions/Significance: L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88(-/-)) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88(-/-) mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88(-/-) mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-gamma responses by NKT, CD4(+) and CD8(+) T lymphocytes, and production of high levels of IL-10. MyD88(-/-) mice replenished with IL-12 and IFN-gamma abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-gamma-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.