RESUMO
Fractionated excretory/secretory products (ES) of adult Haemonchus contortus were evaluated as protective antigens. The proteins were successively eluted from a Thiol Sepharose column using 25 mM cysteine followed by 25 mM Dl-dithiothreitol (DTT). Sheep were vaccinated three times and challenged with 5000 third stage infective larvae (L3) of H. contortus. Highest level of protection was found in sheep vaccinated with the DTT-eluted fraction in which egg output and worm burden were reduced by 52 and 50%, respectively, compared to the adjuvant control group. There was a positive correlation between fecundity (number of eggs per female) and the cumulative EPG or worm burden. Serum and mucus antibody levels of ES-specific immunoglobulins increased after immunizations and after challenge for IgG, IgA and IgE. The harvesting of H. contortus from animals clustered per group revealed the presence of cysteine protease activity in the ES of all groups but in addition to that, metalloprotease activity was also detected in the groups vaccinated with the DTT-eluted fraction, total ES and adjuvant only, in contrast to previous batches of ES (completely inhibited by E64) obtained from non vaccinated animals.
Assuntos
Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Hemoncose/prevenção & controle , Hemoncose/veterinária , Haemonchus/química , Haemonchus/imunologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/prevenção & controle , Abomaso/parasitologia , Animais , Anticorpos Anti-Helmínticos/análise , Anticorpos Anti-Helmínticos/biossíntese , Cromatografia em Agarose , Cisteína Endopeptidases/química , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Hemoncose/imunologia , Imunização , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Contagem de Linfócitos , Muco/imunologia , Contagem de Ovos de Parasitas , Ovinos , Doenças dos Ovinos/parasitologia , Compostos de Sulfidrila/metabolismoRESUMO
Activation-associated secreted proteins (ASP) of nematodes have been studied as potential vaccine components. In this study we report the cloning and analysis of cDNA and genomic sequences of Cooperia punctata and establish the presence of two 75% identical ASP-1 genes in C. punctata. Additional C. punctata ASP paralogues were shown to be present. Analysis of PCR products amplified from genomic DNA from a pool of worms revealed extensive sequence diversity within this family of proteins, reflecting the presence of different ASP paralogues in a single worm as well as extensive polymorphisms between different worms. ASP proteins contain a conserved region called the sperm-coating protein (SCP) domain of unknown function, which is present as a single copy in proteins from yeast and a wide range of multi-cellular organisms. Only in three nematodes has a protein composed of duplicated SCP-domains been identified. C. punctata is the first organism in which at least two such genes are found. Database searches identified similarity of the C-terminal cysteine-rich domain of ASP proteins to a nematode metallothionein motif. Cp-asp-1b was expressed in Escherichia coli and both the N-terminal and C-terminal domain were shown to be recognized by sera of C. punctata infected bovines. The description of the asp gene family of C. punctata provides the basis for more detailed studies into the extent of variation and immunological recognition of this family that may assist in rational vaccine design.
Assuntos
Doenças dos Bovinos/parasitologia , Proteínas de Helminto/genética , Trichostrongyloidea/genética , Tricostrongiloidíase/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Bovinos , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Proteínas de Helminto/metabolismo , Proteínas de Helminto/fisiologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , Técnica de Amplificação ao Acaso de DNA Polimórfico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Trichostrongyloidea/metabolismo , Trichostrongyloidea/fisiologia , Tricostrongiloidíase/parasitologiaRESUMO
In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species