Onchocerca armillata contains the endosymbiotic bacterium Wolbachia and elicits a limited inflammatory response

Abstract

Human onchocerciasis, also known as River Blindness, is a debilitating disease caused by the filarial nematode Onchocerca volvulus. Many, but not all, filarial nematodes carry within their tissues endosymbiotic, Rickettsia-like bacteria of the genus Wolbachia. Onchocerca spp. infections in cattle offer the most relevant, analogous host-parasite model system. West African cattle are commonly co-infected with four Onchocerca spp.; two of these are Wolbachia-positive (Onchocerca gutturosa and Onchocerca ochengi), and the remainder are of unknown Wolbachia status (Onchocerca dukei and Onchocerca armillata). Previous studies have suggested that worm survival is dependent on this bacterium. O. armillata, an abundant parasite of African cattle that has received little attention, is a primitive species that may lack Wolbachia. The objectives of this study were to determine if O. armillata carries Wolbachia and to provide preliminary descriptions of the host inflammatory cell environment around the adult worms. The findings may support or refute the hypothesis that a prime contribution of Wolbachia is to permit long-term survival and reproduction of certain Onchocerca spp. (including O. volvulus in humans). O. armillata adult worms were found in the aorta of 90.7% of cattle (n=54) slaughtered at an abattoir in Ngaoundéré, Adamawa Region, Cameroon. The presence of Wolbachia in O. armillata was confirmed by a specific anti-Wolbachia surface protein antibody detected using a peroxidase conjugate (immunohistochemistry) and PCR for detection of Wolbachia-specific sequences within DNA extracts from frozen worms. Tissue sections stained with haematoxylin and eosin showed the host cell response to be dominated by macrophages and fibroblasts. This is unusual compared with nodule-dwelling Wolbachia-positive Onchocerca spp., where the host response is typically characterised by granulocytes, and suggests that the mechanisms for worm survival employed by this species (which is probably motile) may differ.

Fig. 1

Staining for Wolbachia surface protein (WSP) in adult female worms. (A) O. armillata negative control (normal rabbit serum) with absence of punctate staining in the hypodermal chords (arrow). (B) O. armillata probed with anti-WSP antibody; note heavily stained groups of bacteria in the hypodermis (arrows). (C) O. armillata intrauterine microfilariae showing small clusters of stained Wolbachia (arrows). (D) O. ochengi positive control; note punctate staining of organisms in the hypodermal chords (arrows). Scale bars: A and B = 20 μm; C = 5 μm; D = 10 μm.

Fig. 2

1% agarose gels loaded with PCR reactions for Wolbachia ftsZ (A) and the Wolbachia 16S rRNA gene (B). Lanes 2–4 (from left) and 6–9 show PCR products for O. armillata adult female worm DNA extracts (≥5 individuals); lane 5 represents a DNA extract from a single male worm (arrow). Lane 10, negative control (water); lane 11, positive control (O. ochengi DNA extract); lane 1, HyperLadder™ II molecular weight marker (Bioline). The images have been black-white inverted for clarity.

Fig. 3

Gross pathology of O. armillata infection in the aorta. The intimal surface of the aortic trunk from a 4-year-old cow (A) can be compared with that of an 8-year-old female (B). An O. armillata nodule is visible in the foreground (B, arrow) extending into the aortic lumen of the older cow. In (C), the aortic arch and associated serosa of an 8-year-old cow (same subject as in B) exhibits both calcified (arrowhead) and caseous (arrow) nodules that extend from the tunica media into the tunica adventitia.

Fig. 4

An adult female O. armillata residing within a tunnel inside the tunica media of the aorta. No inflammatory cells are present. The uterus of the worm contains mature Mf (arrows). Scale bar: 20 μm.

Fig. 5

Inflammatory response to O. armillata. (A) Macrophages separate two cross-sections of adult worms. The foamy cytoplasm of the macrophages suggests that they are actively phagocytosing material. Neutrophils (arrows) and extravasated erythrocytes (arrowheads) are present in low numbers. (B) Foamy macrophages with extensive fat-filled cytoplasm surround the worm cuticle. Many of the foamy macrophages have undergone necrosis. Small numbers of eosinophils can be seen in the periphery (arrowheads). Note the area of vascular injury (lower centre). (C) Granulomatous response to an adult worm. Macrophages up to several cells thick line the cavity. This is surrounded by a cuff of fibroblasts and more peripherally, granulocytes. (D) Multinucleate giant cells, macrophages and neutrophils within the tunica media (no parasite visible). Scale bars: 10 μm.