RESUMEN
Standard diagnostic methods currently in use for the identification of helminth infections in ruminants are based on the morphological analysis of immature and adult stages of parasites. This paper describes a method for the semiquantitative identification of nematodes, mainly Trichostrongyloidea, at species-level resolution. The method is based on amplification and fragment analysis followed by minisequencing of the ITS-2 region (internal transcribed spacer 2) of the ribosomal DNA of parasite eggs or larvae. This method allows for the identification of seven genera (Chabertia, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, Teladorsagia, and Trichostrongylus) and 12 species (Chabertia ovina, Cooperia curticei, Cooperia punctata, Cooperia oncophora/Cooperia surnabada, Haemonchus contortus, Haemonchus placei, Haemonchus longistipes, Oesophagostomum asperum, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei, and Trichostrongylus colubriformis) of infectious nematodes of domestic ruminants. The concordance between the morphological and molecular analyses in the detection of genera ranged from 0.84 to 0.99, suggesting the proposed detection method is specific, semiquantitative, less laborious, and highly cost-efficient.
Asunto(s)
Infecciones por Nematodos/veterinaria , Rumiantes/parasitología , Trichostrongyloidea/aislamiento & purificación , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , ADN de Helmintos , ADN Ribosómico , Cabras , Haemonchus/genética , Haemonchus/aislamiento & purificación , Infecciones por Nematodos/parasitología , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Oesophagostomum/genética , Oesophagostomum/aislamiento & purificación , Ostertagia/genética , Ostertagia/aislamiento & purificación , Ovinos , Strongyloidea/genética , Trichostrongyloidea/genética , Trichostrongylus/genéticaRESUMEN
Sulfated polysaccharides from the egg jelly of sea urchins act as species-specific inducers of the sperm acrosome reaction, which is a rare molecular mechanism of carbohydrate-induced signal-transduction event in animal cells. The sea urchin polysaccharides differ in monosaccharide composition (l-fucose or l-galactose), glycosylation, and sulfation sites, but they are always in the alpha-anomeric configuration. Herein, structural analysis of the polysaccharide from the sea urchin Glyptocidaris crenularis surprisingly revealed a unique sulfated beta-d-galactan composed by (3-beta-d-Galp-2(OSO(3))-1-->3-beta-d-Galp-1)(n) repeating units. Subsequently, we used the G. crenularis galactan to compare different 2-sulfated polysaccharides as inducers of the acrosome reaction using homologous and heterologous sperm. We also tested the effect of chemically over-sulfated galactans. Intriguingly, the anomeric configuration of the glycosidic linkage rather than the monosaccharide composition (galactose or fucose) is the preferential structural requirement for the effect of these polysaccharides on sea urchin fertilization. Nuclear magnetic resonance and molecular dynamics indicate that sulfated alpha-galactan or alpha-fucan have less dynamic structural behavior, exhibiting fewer conformational populations, with an almost exclusive conformational state with glycosidic dihedral angles Phi/Psi = -102 degrees /131 degrees . The preponderant conformer observed in the sulfated alpha-galactan or alpha-fucan is not observed among populations in the beta-form despite its more flexible structure in solution. Possibly, a proper spatial arrangement is required for interaction of the sea urchin-sulfated polysaccharides with the specific sperm receptor.
Asunto(s)
Reacción Acrosómica , Galactanos/química , Espermatozoides/metabolismo , Animales , Fertilización , Galactosa/química , Glicósidos/química , Espectroscopía de Resonancia Magnética , Masculino , Conformación Molecular , Monosacáridos/química , Óvulo/metabolismo , Polisacáridos/química , Conformación Proteica , Erizos de MarRESUMEN
The egg jellies of sea urchins contain sulfated polysaccharides with unusual structures, composed of linear chains of l-fucose or l-galactose with well-defined repetitive units. The specific pattern of sulfation and the position of the glycosidic bond vary among sulfated polysaccharides from different species. These polysaccharides show species specificity in inducing the acrosome reaction, which is a critical event for fertilization. Females of the sea urchin Lytechinus variegatus spawn eggs containing a sulfated fucan with the repetitive sequence [3-alpha-L-Fucp-2(OSO(3))-1 --> 3-alpha-L-Fucp-4(OSO(3))-1 --> 3-alpha-L-Fucp-2,4(OSO(3))-1 --> 3-alpha-L-Fucp-2(OSO(3))-1](n). We now observe that, close to winter, a period of decreased fertility for the sea urchin, the females synthesize a distinct sulfated fucan with a simple structure, composed of 4-sulfated, 3-linked alpha-fucose residues. This sulfated fucan is inactive when tested in vitro for the acrosome reaction using homologous sperm. The amount of egg jellies spawned by females (and their constituent sulfated polysaccharides) varied greatly throughout the year. Apparently, there is a correlation between the temperature of the sea water and the expression of the 4-sulfated, 3-linked sulfated fucan. Overall, we described the occurrence of two isotypes of sulfated fucan in the egg jelly of the sea urchin L. variegatus, which differ in their biological activity and may be involved in the periodicity of the reproductive cycle of the invertebrate.