A histochemical study of the Nras/let-60 activity in filarial nematodes

Abstract

Background: Control and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides.

Methods: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy.

Results: Nras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi.

Conclusion: Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae.

Fig. 1

a Anti-Nras staining of host cellular reaction associated with adult O. volvulus demonstrating the intensity of Nras staining in the plasma cells and the general absence of staining in other host cells. b 8-bit grayscale converted image of host plasma cell zone, showing the selection of positive cells (arrow) as control values for quantification purposes

Fig. 2

a Anti-Nras/let-60 stained section of an untreated adult female O. volvulus demonstrating the staining pattern most commonly observed with staining in the hypodermis and uterine epithelium. b 8-bit grayscale converted image of Fig. 4a demonstrating the selection of areas for analysis. c An anti-Nras/let-60 stained section of an ivermectin-treated adult female O. volvulus demonstrating the decrease in staining pattern most commonly seen in ivermectin-treated nodules. d 8-bit grayscale converted image of Fig. 4c demonstrating the selection of the area for analysis

Fig. 3

A healthy adult untreated female O. volvulus demonstrating multiple developing forms in the uterus (H&E stain). Key: Ct: cuticle, Hy: hypodermis, Lm: longitudinal muscle, In: intestine, Ut: uterus

Fig. 4

Positive anti-Nras/let-60 staining: a Oocytes. b Morulae. c Coiled microfilariae. d Limited staining is seen in stretched microfilariae

Fig. 5

a Female O.volvulus stained with anti-WSP demonstrating the presence of Wolbachia. b A serial section stained the anti-Nras stain showing an intense punctate pattern associated with Wolbachia

Fig. 6

The primary growth zone of a female O. volvulus. a Differential interference contrast (DIC) image of the area shown in 6B. b Laser scanning miscroscope (LSM) image of mature primary oocytes showing virtually no co-localization as demonstrated by the lack of yellow color in the image

Fig. 7

a LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow). b DIC image of the site shown in 7C. c LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow)

Fig. 8

Adult female Brugia malayi. a Anti-Nras stained section showing a punctate staining present in the hypodermis. b Anti-WSP stain of a serial section showing punctate staining associated with the presence of Wolbachia

Fig. 9

Adult female Dirofilaria immitis. a Anti-Nras stained section showing punctate staining associated with the Wolbachia. b An anti-WSP stained serial section

Fig. 10

Adult female L. sigmodontis. a An anti-WSP stained section showing the presence of Wolbachia. b An anti-Nras stained serial section of 10A. c A higher magnification of 10B showing the punctate staining associated with the presence of Wolbachia

Fig. 11

Adult Onchocerca ochengi stained with anti-Nras. a The hypodermis (H) showing punctate staining consistent with the location of Wolbachia. Nuclei in the uterine (U) developing microfilariae are also positive. b The nuclei in the developing morulae in the uterus (U), and the intestinal epithelium (I) are also positive. The body wall (W) cuticle is negative