João Carlos Minozzo

Experimental bovine infection with Taenia saginata eggs: recovery rates and cysticerci location

Studies were carried out on the recovery rate and cysticerci location in bovines experimentally infected with Taenia saginata eggs. Three calves of 6.5 months and one with 19 months of age were infected orally with 2 x 104 eggs of Taenia saginata. A fifth calf served as control. After 90 days of infection, the animals were slaughtered and organs and skeletal muscles were inspected using a slicing technique every 5 mm. From the four infected calves, 702 cysticerci were recovered, of which 570 (81.2%) were alive and 132 (18.8%) were degenerated. The recovery rate ranged from 0.01 to 1.43% with an average of 0.88%. The cysticerci presented the following anatomical distribution: hioideos muscles 02 (0.28%), kidneys 03 (0.43%), tongue 07 (1.00%), liver 12 (1.71%), lungs 15 (2.14%), diaphragm 18 (2.56%), mastication muscles 25 (3.56%), heart 49 (6.98%), anterior muscle 323 (46.00%) and posterior muscle 248 (35.33%).

Teste imunoenzimático (enzyme-linked immunosorbent assay) para diagnóstico da cisitcercose bovina e estudo da cinética de produção de anticorpos contra-Cysticercus bovis

An indirect ELISA (ENZYME-LINKED IMMUNOSORBENT ASSAY) was developed for searching of antibodies against-Cysticercus bovis in bovine. Three antigens were studied: partial antigen of C. cellulosae, total antigen of C. bovis, and total antigen of C. longicollis. In the standardization of the ELISA the following combinations were analyzed: antigen 250 and 500 ng of protein/well, dilution of the sera 50, 100, 200, and 400 times, dilution of the conjugated (anti bovine -IgG conjugated IgG of goat with peroxidase) 400 and 800 times. The crossing of the conditions above resulted in the following standardization: antigen 250 ng/well, sera and conjugated diluted 100 and 400 times respectively. The reaction cut-off between reagents and non-reagents animals was determined by the average of the optic densities of 54 negative sera plus three standard deviation resulting in the value of 0,303. The reactivity of the three antigens used in the ELISA test was compared using sera from experimentally infected calves, using sera dilutions and conjugated standardized previously. Using the antigen of C. bovis was verified high correlation with the test standardized with C. cellulosae. However, the absorbance values were significantly smaller. With C. longicollis was observed low reactivity, but increasing the amount of antigen, up to 3000ng/well, there was a proportional increase of the response. The kinetics of antibodies anti-C. bovis production was studied in ten calves experimentally infected with 2 x 104 T. saginata eggs. Six non-infected calves were used as control. After 90 days from the infection date, the animals were killed. Thirteen samples of sera of each animal were analyzed. The first was picked in the day of the infection and the remaining at each 15 days. The maximum production of antibody was observed between 30 and 60 days post infection. With the standardized test it was detected antibodies against-C. bovis, in 2 from 20 cattle considerated as non-holder of cyst by the inspection service. These animals could be considered possible cyst holders.

Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Evaluation of the protective potential of a Taenia solium cysticercus mimotope on murine cysticercosis

An NC-1 mimotope from Taenia solium cysticerci can help identify patients with neurocysticercosis through immunoassay. After chemical synthesis, an NC-1 peptide was coupled to bovine serum albumin (NC-1/BSA) for used as an immunogen in murine Taenia crassiceps cysticercosis, which is an experimental model of cysticercosis caused by T. solium. NC-1/BSA immunisation decreased parasitaemia by inducing 74% protection compared to the 77% protection obtained with T. crassiceps crude antigen. The influence of immunisation was also observed on the size and stage of development of the parasite. Antibodies from NC-1/BSA-immunised mice recognised proteins from the tegument and from the buddings, and intense immunostaining was observed in the final stage of the metacestode. The capacity of NC-1/BSA to induce protective antibodies which are reactive to proteins from the tegument of the metacestode suggests that this mimotope is a potential candidate for a vaccine against human and animal cysticercosis.

Determination of sphingomyelinase-D activity of Loxosceles venoms in sphingomyelin/cholesterol liposomes containing horseradish peroxidase

Based on degradation of sphingomyelin/cholesterol liposomes containing entrapped horseradish peroxidase, we evaluated the Sphingomyelinase-D (SMase-D) activity of scorpion, spider and snake venoms by monitoring spectrophotometrically the product of oxidation of HRP released. The results indicate that Loxosceles crude venoms (Loxosceles intermedia, Loxosceles laeta, Loxosceles gaucho and Loxosceles similis) displayed SMase-D activity in a concentration-dependent manner. Furthermore, this activity was blocked by the anti-loxoscelic antivenom. However, Tityus serrulatus scorpion venom, Phoneutria nigriventer spider venom and Bothrops jararaca, Crotalus durissus, Lachesis muta and Micrurus frontalis snake venoms did not show measurable SMase-D activity.

Innovative immunization protocols using chimeric recombinant protein for the production of polyspecific loxoscelic antivenom in horses.

A chimeric protein (rCpLi) was constructed expressing three epitopes of rLiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. We have analyzed the neutralization potential of sera obtained by immunization of horses with rCpLi and rCpLi combined with initial doses of venoms and compared these with antivenom traditionally produced in horses using crude Loxosceles gaucho, Loxosceles laeta and L. intermedia venoms as antigens. We have demonstrated by ELISA that horses immunized with three initial doses of crude venom containing mixtures of L. intermedia, L. gaucho and L. laeta followed by nine doses of rCpLi generate antibodies with the same reactivity as those produced following immunization with traditional antivenom, towards the venoms of the three Loxosceles sp. species. Results from in vivo and in vitro neutralization assays showed that the new horse sera are able to neutralize the dermonecrotic activity of Loxosceles venoms, which are of medical importance in Brazil and some of these sera are capable of meeting the necessary potency requirements that could allow for their therapeutic use in humans. This immunization strategy combining both antigens used approximately 67% less crude Loxosceles venoms compared to traditional immunization protocol and can mean the development of Loxosceles antivenoms with the consequent reduction of devastation of arachnid fauna.

ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes

Human growth hormone (hGH) signal transduction initiates with a receptor dimerization in which one molecule binds to the receptor through sites 1 and 2. A sandwich enzyme-linked immunosorbent assay was developed for quantifying hGH molecules that present helix 4 from binding site 1. For this, horse anti-rhGH antibodies were eluted by an immunoaffinity column constituted by sepharose-rhGH. These antibodies were purified through a second column with synthetic peptide correspondent to hGH helix 4, immobilized to sepharose, and used as capture antibodies. Those that did not recognize synthetic peptide were used as a marker antibody. The working range was of 1.95 to 31.25 ng/mL of hGH. The intra-assay coefficient of variation (CV) was between 4.53% and 6.33%, while the interassay CV was between 6.00% and 8.27%. The recovery range was between 96.0% to 103.8%. There was no cross-reactivity with human prolactin. These features show that our assay is an efficient method for the determination of hGH.

Specific immunoassays for placental alkaline phosphatase as a tumor marker.

Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57–71) of hPLAP (ICA-PEP assay); the working range was 0.1–11 U/L and cutoff value was 0.2 U/L EA for nonsmokers. The intra- and interassay coefficients of variation were 3.7%–6.5% (ICA-PLAP assay) and 9.0%–9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes.

Resposta imune-humoral e celular em bovinos da raça Nelore imunizados com extrato de larvas (L2 e L3) de Dermatobia hominis (Linnaeus Jr., 1781)

Dermatobia hominis larvae cause ulcerative lesions and damage to subcutaneous tissue and skin of the host. Leather is the subproduct which undergoes major depreciation, making it difficult to industrial use. Nowadays, the chemical control is utilized against dermatobiosis, therefore it leads to rising toxic chemicals in the animals and environment. The immunological challenge with D. hominis larval extract may represent an important altervative for this parasitosis control. Humoral and cellular immune responses were tested in bovine using an antigenic extract prepared with D. hominis larvae. Three groups of 10 months old Nelore females were used. The first group (A) received immunogenic larval extract of D. hominis with fifteen-days interval between injections; the group (B) was the control and has not received any sort of treatment; and the group (C) received an ectoparasitecide treatment based on Dichlorvos associated to Cypermetrina. Aditionally, leucogram and levels of IgG against D. hominis by immunoassay technique were evaluated. As for the humoral immunity, animals from group A presented higher IgG production against D. hominis with maximum levels of circulating antibodies at the 45th day after the first injection. These animals also showed higher production of neutrophils, eosinophils and monocytes than those from groups B and C. The number of D. hominis larvae nodules observed in animals from the group C was 148.3% larger than those from group A and B. The evidence concerning both cellular and humoral immune responses as well as the reduction on nodules number are an indication that the immunization against D. hominis was partially protective for the immunized bovines.

Crude antigen from Taenia crassiceps cysticercus used as heterologous antigen in ELISA and in EITB for neurocysticercosis diagnosis of patients from Paraná-Brazil

Neurocysticercosis (NCC), the cerebral presence of Taenia solium metacestode (Cysticercus cellulosae), is responsible for neurological disorders worldwide. In order to validate an immunodiagnosis for public-health patients in the State of Parana-Brazil, crude antigen of Taenia crassicepsmetacestode (Cysticercus longicollis) was used as an alternative heterologous antigen to be used in ELISA and in electroimmunotransfer blotting (EITB) for active and inactive NCC diagnosis. Indirect ELISA was able to discriminate between active and inactive samples and presented high specificity and sensitivity. Any immunodominant band was able to distinguish the NCC stages, although the EITB showed 100% specificity. The immunological results proved to be an important auxiliary toll for NCC diagnosis, mainly for public-health systems in developing countries, where either the neuroimage techniques are not accessible or the resources are scarce.