Surface characterization of the cuticle of dirofilaria immitis

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Abstract
The surface reactivity of the dog heartworm (D. immitis) was evaluated by comprehensive contact angle measurements and a platelet retention test. Contact angle data yielded calculated surface energy terms very similar to those previously reported for intact vascular endothelium. The platelet test revealed the native worm surface to be nonreactive, retaining fewer platelets than glass or worms whose surfaces had been modified by extraction with acid and high salt solutions. The cuticular morphology of the heartworm was studied with both light and electron microscopy, the latter coupled with ferritin-conjugated double-layer immunolabeling to reveal adsorbed host protein on the cuticle surfaces. Multiple attenuated internal reflection (MAIR) IR spectroscopy confirmed the general composition of this surface layer to be glycoproteinaceous. Morphological and histochemical studies confirmed and extended previous descriptions of nematode cuticle, adding ultrastructural detail on cortical, medial, and basal layers. A trilaminar membrane, apparently corresponding to a mammalian cell membrane (plasmalemma), constituted the external cortical layer as observed in high magnifications. The existence of a glycocalyx of varying thickness was demonstrated in ruthenium red-stained sections. MAIR IR spectra showed this glycoproteinaceous film to appear, in fully hydrated samples, as a loose biological gel. Ferritin-antibody conjugate labeling confirmed the presence of adsorbed dog albumin, dog immunoglobulin class G (IgG) and dog complement fraction 3 (C3) in the cuticular surface layer. It is likely, therefore, that D. immitis heartworms demonstrate long-term thromboresistance at least in part due to their passive low-surface-energy overcoating with host proteins.