Walter dos Santos Lima

First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from São Vicente, state of São Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.

Seasonal infection pattern of gastrointestinal nematodes of beef cattle in Minas Gerais State—Brazil

Tracer calves were used to assess the seasonality of infections of gastrointestinal parasites in beef cattle extensively raised at a farm in the State of Minas Gerais, Brazil. Tracer calves acquired infections during all months of the year, however, highest worm burdens were observed in the rainy season. The following nematode species were recovered from tracer calves: Cooperia punctata, C. spatulata, C. pectinata, Haemonchus similis, H. placei, Oesophagostomum radiatum, Trichostrongylus colubriformis, T. axei, Bunostomum phlebotomum and Trichuris discolor. Cooperia was most prevalent, representing 74.4% of the total of all nematodes recovered. This was followed by Haemonchus 19.2%, Oesophagostomum 4.5%, Trichostrongylus, Trichuris and Bunostomum, represented less than 1% of the total. Thirty Nellore cows with calves were used to evaluate the dynamics of the infections. The cows were the principal source of contamination to pasture and to infection of suckling calves during the pre-weaning period, and probably, also contributed to increased infection of weaned calves at the beginning of the rainy season. Strongyloides was the first infection observed in the calves. The peak egg count was observed in the 2nd month, however, all Strongyloides egg counts became negative in the 7th month. Strongylate nematode egg counts were low in calves from the 3rd month of age onward, and increased gradually until calves were 1 year old. A gradual reduction then occurred until the end of the trial. From the 3rd month onward Cooperia and Haemonchus infective larvae (L3) were most prevalent in fecal cultures. Fecal cultures after the 4th month also resulted in recoveries of Oesophagostomum, Trichostrongylus, and Bunostomum L3. Cooperia was the predominant genus in fecal cultures until the calves were 9 months old. After the calves were 9 and 12 months of age, L3 of Cooperia and Oesophagostomum, respectively decreased in prevalence. Trichostrongylus and Bunostomum L3 were only minimally present in the fecal cultures at this time.

Molecular Differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Hematological and coagulation profiles in dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866).

Abstract Hematological and coagulation profiles were studied in crossbred dogs experimentally infected with Angiostrongylus vasorum. Two groups of five dogs were experimentally inoculated with 50 and 100 third stage infective larvae (L3) of A. vasorum per kilogram of body weight. A third group of five uninfected animals was used as control. One sample of 10ml of blood was collected from each animal on the 10, 20, 30, and 45 days after inoculation (dai) and at 30-day intervals thereafter for the remainder of the 210-day experimental period. The blood sample was used for the complete hemogram and platelet count, as well as measurements of prothrombin time, partial thromboplastin time and factors V and VIII. Anemia was observed in infected dogs, 6 weeks after the infection. The eosinophils presented peaks in four periods after infection. Thrombocytopenia became accentuated on the 72dai. Decreased prothrombin time activity and increased partial thromboplastin time were observed at the 6 and 9 weeks after infection and decreased of factors VIII and V activities occurred from 4 to 6 weeks after infection. It may be conclude that infection by A. vasorum in dogs may cause a discrete anemia during the acute phase which is probably regenerative. In addition, important hemostatic alterations due to the infection suggest a chronic intravascular consumption coagulopathy.

Predatory activity of the fungi Duddingtonia flagrans, Monacrosporium thaumasium, Monacrosporium sinense and Arthrobotrys robusta on Angiostrongylus vasorum first-stage larvae.

Angiostrongylus vasorum is a nematode that parasitizes domestic dogs and wild canids. We compared the predatory capacity of isolates from the predatory fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34), Monacrosporium sinense (SF53) and Arthrobotrys robusta (I31) on first-stage larvae (L1) of A. vasorum under laboratory conditions. L1 A. vasorum were plated on 2% water-agar (WA) Petri dishes marked into 4 mm diameter fields with the four grown isolates and a control without fungus. Plates of treated groups contained each 1000 L1 A. vasorum and 1000 conidia of the fungal isolates AC001, NF34, SF53 and I31 on 2% WA. Plates of the control group (without fungus) contained only 1000 L1 A. vasorum on 2% WA. Ten random fields (4 mm diameter) were examined per plate of treated and control groups, every 24 h for 7 days. Nematophagous fungi were not observed in the control group during the experiment. There was no variation in the predatory capacity among the tested fungal isolates (P>0.05) during the 7 days of the experiment. There was a significant reduction (P < 0.05) of 80.3%, 74.5%, 74.2% and 71.8% in the means of A. vasorum L1 recovered from treatments with isolates AC001, NF34, SF53 and I31, respectively, compared to the control without fungi. In this study, the four isolates of predatory fungi were efficient in the in vitro capture and destruction of A. vasorum L1, confirming previous work on the efficiency of nematophagous fungi in the control of nematode parasites of dogs and as a possible alternative method of biological control.

Angiostrongylus vasorum (Baillet, 1866) Nematoda: Prostostrongylidae, em cães de Minas Gerais, Brasil

For the first time Angiostrongylus vasorum in Canis familiaris in Minas Gerais State, Brazil, is described. The description and measurements of three males and ten females are presented together with a diagram of the parasite.

Cytological and parasitological analysis of bronchoalveolar lavage fluid for the diagnosis of Angiostrongylus vasorum infection in dogs

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of many pulmonary diseases. The aims of this work were to perform this procedure in dogs in the acute and chronic phases of an Angiostrongylus vasorum infection for cytological analysis and to evaluate the potential of this technique as a diagnostic method for this lung-heart worm. The BAL procedure was performed through the use of an endotracheal tube on seven A. vasorum infected dogs and on five non-infected dogs lined as a control group. Sixty days post-infection (dpi) active and live larvae were retrieved from the bronchoalveolar fluid (BALF) of all infected dogs. Furthermore, in one animal it was possible to retrieve larvae in its BALF before the pre-patent period. This work reports that the A. vasorum infection resulted in an increase of relative neutrophils and eosinophils counts. In contrast, there was a significant decrease in the alveolar macrophage relative count in infected animals from 60 to 330 dpi. This study shows that the BAL is an accurate technique for the diagnosis of canine angiostrongylosis. Moreover, the technique allows us to retrieve cells and other elements that line the lung surface for cytological evaluation, which provides information about inflammatory diseases, and the diagnosis and prognosis of pulmonary parasites such as A. vasorum.

Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866)

Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20-22 and 10kDa.

Use of a candidate gene array to delineate gene expression patterns in cattle selected for resistance or susceptibility to intestinal nematodes.

In the present study, we use microarray technology to investigate the expression patterns of 381 genes with known association to host immune responses. Hybridization targets were derived from previously characterized bovine cDNAs. A total of 576 reporters (473 sequence-validated cDNAs and 77 controls) were spotted onto glass slides in two sets of four replicates. Two color, comparative hybridizations across both mesenteric lymph node (MLN) and small intestine mucosa (SIM) RNA samples were done between animals with previously demonstrated phenotypic differences based on natural exposure to gastrointestinal (GI) nematodes over a 6-month exposure period. A total of 138 significant hybridization differences were detected by mixed model analysis of variance. A subset of these significant differences was validated by quantitative, real-time RT-PCR to assay transcript levels for 18 genes. These results confirmed that in the SIM, susceptible animals showed significantly higher levels in the genes encoding IGHG1, CD3E, ACTB, IRF1, CCL5 and C3, while in the MLN of resistant animals, higher levels of expression were confirmed for PTPRC, CD1D and ITGA4. Combined, the results indicate that immune responses against GI nematode infections involve multiple response pathways. Higher levels of expression for IgE receptor, integrins, complement, monocyte/macrophage and tissue factors are related to resistance. In contrast, higher levels of expression for immunoglobulin chains and TCRs are related to susceptibility. Identification of these genes provides a framework to better understand the genetic variation underlying parasite resistance.

Biochemical serum profiles in dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866).

Abstract The biochemical profiles of crossbred dogs experimentally infected with the parasite Angiostrongylus vasorum were studied. Two groups of five dogs were experimentally inoculated with 50 and 100 third stage infective larvae (L3) of A. vasorum per kilogram of body weight. A third group of five uninfected animals were used as control. Serum from these animals were used for biochemical tests to measure total and fractioned proteins, urea, creatinine and to determine the activities of aspartate (AST), alanine (ALT) aminotransferase, gamma-glutamyl transferase (GGT), alkaline phosphatase (PAL) and creatine kinase isoenzyme MB (CK-MB). The α-1, α-2 and β-globulins fractions showed alterations during acute phase of the infection. No modifications were observed in the biochemical profiles of ALT, AST, GGT, PAL, urea and creatinine. CK-MB was shown to be a good early indicator of cardiac injury in dogs experimentally infected with A. vasorum.