Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4% and 78.7%, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.     

Angiostrongylus costaricensis egg antigen for the immunodiagnosis of abdominal angiostrongyliasis

Angiostrongylus costaricensis is the aetiological agent of human abdominal angiostrongyliasis, a parasitic disease reported from the United States to Argentina, with a widespread occurrence of the nematode throughout Central and South America. This study assesses the performance of A. costaricensis eggs as antigen in an enzyme-linked immunosorbent assay (ELISA), for the determination of parasite-specific IgG1 antibodies. The specificity and the sensitivity of the method were 87% and 90.5%, respectively. Through this test it was possible to demonstrate a sharp and early decline in IgG1 antibody in serum samples taken from patients with histopathological diagnosis of abdominal angiostrongyliasis at different time points after surgical treatment. The present work demonstrated the usefulness of the egg antigen in the development of a specific diagnostic test for abdominal angiostrongylosis.     

Comprehensive proteomic profiling of adult Angiostrongylus costaricensis, a human parasitic nematode

Angiostrongylus costaricensis is a nematode helminth that causes an intestinal acute inflammatory process known as abdominal angiostrongyliasis, which is a poorly understood human disease occurring in Latin America. Our aim was to study the proteomic profiles of adult parasites focusing on immunogenic proteins. Total cellular extracts from both genders showed similar 2-DE profiles, with 60% of all protein spots focused between pH 5-7 and presenting molecular masses from 20.1 to 66 kDa. A total of 53 different dominant proteins were identified in our dataset and were mainly associated with the following over-represented Gene Ontology Biological Process terms: "macromolecule metabolic process", "developmental process", "response to stress", and "biological regulation". Female and male immunoblots showed similar patterns of reactive proteins. Immunoreactive spots identified by MALDI-PSD were found to represent heat shock proteins, a putative abnormal DAuer Formation family member, and galectins. To date, very few biochemical analyses have focused on the nematode Angiostrongylus costaricensis. As such, our results contribute to a better understanding of its biology and the mechanisms underlying the host-parasite relationship associated with this species. Moreover, our findings represent a first step in the search for candidate proteins for diagnostic assays and the treatment of this parasitic infection.     

An indirect immunofluorescence antibody test employing whole eggs as the antigen for the diagnosis of abdominal angiostrongyliasis

Abdominal angiostrongyliasis is a potentially fatal zoonotic disease with a broad geographical distribution throughout Central and South America. This study assessed the performance of Angiostrongylus costaricensis eggs as the antigen in an indirect immunofluorescence assay for the determination of parasite-specific IgG and IgG1 antibodies. For prevalence studies, an IgG antibody titre ≥ 16 was identified as the diagnostic threshold with the best performance, providing 93.7% sensitivity and 84.6% specificity. Cross reactivity was evaluated with 65 additional samples from patients with other known parasitic infections. Cross reactivity was observed only in samples from individuals infected with Strongyloides stercoralis. For clinical diagnosis, we recommend the determination of IgG only as a screening test. IgG1 determination may be used to increase the specificity of the results for patients with a positive screening test.     

Abdominal angiostrongyliasis in rodent experimental infection: evidence for systemic circulation of first stage larvae

Eggs of Angiostrongylus costaricensis embrionate and hatch in the course of their migration in the intestinal wall, and first stage larvae (L1) are released in feces. Aiming to investigate the possible systemic circulation of L1, we inoculated mice and, four weeks later, examined their peritoneal cavities and several organs for the presence of L1. A total of 65 larvae were found in extra-intestinal organs (kidney, heart, spleen, liver, lungs). No larvae were found in blood or in the peritoneal cavity. 320 and 578 L1 were found in intestinal wall and intestinal contents, respectively. The experiment was repeated and it confirmed that the metastrongylid larvae found in several organs were larval stages of A. costaricensis. Ten Oligoryzomis sp. rodents, a natural host for A. costaricensis, were also infected and in three animals L1 could be recovered from several organs as well as from bronchoalveolar lavage (BAL) in one of them. These data indicate that systemic circulation and bronchial elimination of L1 may represent an alternative route for release of L1 of A. costaricensis into the environment for transmission to the intermediate host.     

Chromosomal mapping of host susceptibility loci to Angiostrongylus costaricensis nematode infection in mice

Angiostrongylus costaricensis is a nematode found mainly as a rodent parasite. Laboratory mice were experimentally infected with this parasite. It is known that there is great variability in mortality among inbred mouse strains after infection with this nematode. The survival rate at 5 weeks after infection of A/J mice was 90.5%, whereas that of SM/J mice was only 33.3%, with severe anemia and decreased body weight about 3 weeks after infection. To identify host susceptibility genes for infection with this nematode, we undertook chromosomal mapping by a whole-genome scanning approach in (A/JxSM/J)F2 mice. We mapped a host susceptibility locus (here designated Acsns, for Angiostrongylus costaricensis nematode susceptibility locus) to the telomeric portion of Chromosome 19 (peak LOD=4.35). We also identified two loci on Chr 13 and Chr 17 that have epistatic effects on host survival. This is the first report on host susceptibility loci for helminth infection mapped by whole-genome scanning.     

Alteration in the endogenous intestinal flora of Swiss Webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.     

Parasitic helminths from Sigmodon hispidus (Rodentia: Cricetidae) from seasonal and evergreen habitats in Costa Rica

The helminthological fauna of the cotton rat, Sigmodon hispidus in a tropical environment varies according to habitat and feeding behavior. Six species of nematodes (Longistriata adunca, Trichostrongylus sigmodontis, Strongyloides sigmodontis, Litomosoides carinii, Monodontus sp. and Protospirura sp.) and two species of cestodes (Hymenolepis diminuta and Raillietina sp.) were found in rats from extensive dry lands in Guanacaste where hot temperatures and heterogeneous diet are the rule. Only two species of nematodes (Longistriata adunca and Angiostrongylus costaricensis) were found in rats collected in a humid pineapple plantation in the Central Plateau (Alajuela) where mild temperatures predominate. A. costaricensis, a metastrongylid of medical importance, was found in 42% of them.     

Endemic infections of Parastrongylus (=Angiostrongylus) costaricensis in two species of nonhuman primates, raccoons, and an opossum from Miami, Florida

Parastrongylus (=Angiostrongylus) costaricensis was first reported in the United States from cotton rats, Sigmodon hispidus, in Texas in 1979. Here, we report the findings of P. costaricensis in a siamang (Hylobates syndactylus) from the Miami MetroZoo, in 2 Ma's night monkeys (Aotus nancymaae) from the DuMond Conservancy located at Monkey Jungle in Miami, in 4 raccoons (Procyon lotor) trapped near the MetroZoo, and in an opossum (Didelphis virginiana) trapped at the MetroZoo. These records are the first records of P. costaricensis from all 4 species of hosts. All of the primates were zoo-born, and the raccoons and opossum were native, indicating that this parasite is now endemic at these 2 sites.     

Isolation of Angiostrongylus costaricensis first-stage larvae from rodent feces on a Percoll gradient

Several density gradients were tested for the isolation of parasitic nematode, Angiostrongylus costaricensis, first-stage larvae from rodent feces. With a 45/72% Percoll gradient, 83-99% (89.56+/-6.57%) of the larvae were recovered in a clean preparation.     

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.     

New species of Oswaldocruzia (Nematoda: Molineoidae), new species of Rhabdias (Nematoda: Rhabdiasidae), and other helminths in Rana cf. forreri (Anura: Ranidae) from Costa Rica

Oswaldocruzia costaricensis n. sp. (Strongylida: Molineidae) from the intestines and Rhabdias savagei n. sp. (Rhabditida: Rhabdiasidae) from the lungs of Rana cf. forreri (Anura: Ranidae) are described and illustrated. Oswaldocruzia costaricensis represents the 77th species assigned to the genus and differs from the other Neotropical species in the genus by possessing a Type II bursa and long cervical alae. Rhabdias savagei represents the 47th species assigned to the genus and differs from other Neotropical species in the genus by possession of 4 lips and a postequatorial vulva. Rana cf. forreri was also found to harbor the trematodes, Haematoloechus parcivitellarius and Megalodiscus temperatus, the nematodes, Aplectana incerta, Aplectana itzocanensis, Cosmocerca podicipinus, Foleyellides striatus, Subulascaris falcaustriformis, and a larva of the nematode Brevimulticaecum sp. Cosmocerca panamaensis is considered to be a synonym of Cosmocerca podicipinus.     

Infection of wild rats with Angiostrongylus costaricensis by intraperitoneal subcutaneous route

Three groups of cotton rats (Sigmodon hispidus) were infected with Angiostrongylus costaricensis; the first was inoculated by stomach tube; the second intraperitoneally and the third subcutaneously. In all the groups each rat received 50 L3. The highest rates of infection (51.5%) were obtained by the intraperitoneal route, followed by oral inoculation (47.1%). Poor results were observed (10.5%) subcutaneously.     

Phyllocaulis variegatus--an intermediate host of Angiostrongylus costaricensis in south Brazil

Molluscs collected in five localities in the State of Rio Grande do Sul (Brazil) were digested and examined. The infected slugs were identified as Phyllocaulis variegatus and the larvae found were inoculated per os into mice. After 50 days, worms with the caracteristics of Angiostrongylus costaricensis were recovered from the mesenteric arterial system. The results establish the role of P. variegatus as intermediate host of A. costaricensis in south Brazil, where many cases of abdominal angiostrongyliasis have been diagnosed.     

Wind creates crown shyness,asymmetry, and orientation in a tropical montane oak forest

In a cloud forest dominated by Quercus costaricensis, the gap size between the crowns was consistent and crowns were asymmetrical, being elongated perpendicular to the prevailing wind direction. A wind storm of 20 m/s removed 1.5% of the leaves, consistent with the idea that wind abrasion shapes tree crowns.     

Two new ascomycetes from rainforest litter in Costa Rica

Two new ascomycetes, Boerlagiomyces costaricensis (Pleosporales) and Scopinella musciformis (Sordariales sensu lato), from litter samples collected in rainforests of Costa Rica, are described and illustrated. Boerlagiomyces costaricensis has globose, ostiolate ascomata covered by numerous setae-like hairs; cylindrical, fissitunicate asci without apical structures; and large, fusiform, muriform, hyaline to pale brown ascospores. Scopinella musciformis is characterized by ostiolate ascomata with a few compact clusters of hypha-like hairs distributed on the peridial surface and a long neck; ovate to ellipsoidal unitunicate asci; and small quadrangular ascospores with diagonal germ slits.     

A simple mathematical method revealing the early stage of acute leukemia in patients with cytopenias

On the basis of findings in examinations made in 50 patients (25 with hypoplastic anaemia and 15 in the hypoplastic stage of acute leukemia) a simple mathematical method was developed for the differential diagnosis of these two diseases. The following laboratory findings were used for calculating a formula: cellularity of the bone-marrow, percentage of blast cells in the myelogram, percentage of blast cells in leukocyte concentrate, morphology of erythrocytes, presence of normoblastosis, percentage of monocytes and absolute number of neutrophils in the peripheral blood. This method allows the correct diagnosis to be made in 91% of cases with hypoplastic anaemia and in 100% of cases with acute leukemia. By using 7 laboratory findings to be obtained easily this method will enable a correct diagnosis to be made even in those situations where hitherto it was very difficult to make a differential diagnosis or where it was impossible at all.     

Low susceptibility of Achatina fulica from Brazil to infection with AngioIylus costaricensis and A. cantonensis

Introduction of Achatina fulica in Brazil has led to serious concerns about its role as vector for metaIylid worms: AngioIylus costaricensis and A. cantonensis. Experimental infection with both parasites was performed to evaluate the potential risk for their transmission by the giant African snail. Groups of 5 animals, both wild and bred at captivity were exposed at different inocula: 1, 5, and 10 x 10(3) L1 of A. costaricensis and A. cantonensis. In all groups, few snails got infected and parasitic burden was low. Two different ways of infection were tested: ingestion produced higher numbers of L3 than the inoculation through an artificial hole in the shell. We also report the parasitological examination of 6 batches of wild A. fulica from Florianópolis, state of Santa Catarina, Brazil: only 1 out of 244 animals were infected with metaIylid larvae. Taken together these data indicate that the giant African snail occurring in Southern Brazil is not a permissive host for both AngioIylus species and does not represent a significant risk for transmission of these parasites.     

Individual serological follow-up of patients with suspected or confirmed abdominal angiostrongyliasis

Abdominal angiostrongyliasis (AA) is a zoonotic nematode infection caused by Angiostrongylus costaricensis, with widespread occurrence in the Americas. Although the human infection may be highly prevalent, morbidity is low in Southern Brazil. Confirmed diagnosis is based on finding parasitic structures in pathological examination of biopsies or surgical resections. Serology stands as an important diagnostic tool in the less severe courses of the infection. Our objective is to describe the follow up of humoral reactivity every 2-4 weeks up to one year, in six individuals with confirmed (C) and ten suspected (S) AA. Antibody (IgG) detection was performed by ELISA and resulted in gradually declining curves of reactivity in nine subjects (56%) (4C + 5S), that were consistently negative in only three of them (2C + 1S) after 221, 121 and 298 days. Three individuals (2C + 1S) presented with low persistent reacitivity, other two (1C + 1S) were serologically negative from the beginning, but also presenting a declining tendency. The study shows indications that abdominal angiostrongyliasis is usually not a persistent infection: although serological negativation may take many months, IgG reactivity is usually declining along time and serum samples pairing may add valuable information to the diagnostic workout.     

Ophiorosellinia costaricensis gen. et sp. nov., a xylariaceous fungus with scolecosporous ascospores

A pyrenomycete featuring uniperitheciate stromata embedded in a subiculum and asci with iodine-positive apical ascal rings that bear scolecosporous ascospores is described as new. The fungus, Ophiorosellinia costaricensis, is known only from the type location in Costa Rica. It has been cultured, but no anamorph was discovered.