Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility

Abstract

Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.

Figure 1

Electron microscopy of female Litomosoides sigmodontis from BALB/c mice with or without tetracycline therapy. (a–c) Worms from untreated mouse (day 28 after infection). Bacteria (arrows) are prominent in the lateral chords of the hypodermis (a), in the rachis of the ovary (b), and in embryonic stages within the uterus (c). (d) Worm from tetracycline-treated mouse (day 28 after infection). Remnants of damaged bacteria (arrow) are seen in the hypodermis. ×14,500.

Figure 2

Immunohistology of adult L. sigmodontis from BALB/c mice with or without tetracycline therapy. Staining was performed using rabbit antiserum against Onchocerca volvulus endobacterial hsp-60, APAAP. (a) Cross-section of an untreated male worm with numerous endobacteria (red) in the lateral chord of the hypodermis (arrow); day 28, ×800. (b) Cross-section of a treated female worm with no endobacteria stained in the lateral chords (arrows); day 28, ×800. (c) Longitudinal section of an untreated female with numerous endobacteria in the hypodermis (arrow); day 28, ×800. (d) Longitudinal section of a treated female with no endobacteria stained; day 28, ×670. (e) Oblique section of a female with endobacteria in the hypodermis (black arrow), in the rachis (white arrow) of the ovary (ov), and in oocytes (arrowhead); day 49, ×800. (f) Cross-section of a treated female with an empty uterus (u) with no endobacteria stained in the hypodermis (arrow); day 42, ×500. (g) Longitudinal section of a treated female showing degenerated oocytes in the ovary (ov) with no endobacteria stained in the hypodermis; day 42, ×800. AAPAAP, alkaline phosphatase antialkaline phosphatase.

Figure 3

Phylogenetic analysis of filarial endobacterial 16S rRNA gene sequences. Phylogram derived from a maximum parsimony analysis of aligned 16S rRNA genes from Wolbachia species, filarial endobacteria, and representative members of the Rickettsiales. Branch lengths (inferred numbers of nucleotide changes) are given above each branch. Bootstrap support (100 replicates, heurisitic search) are given in italic below each supported branch.

Figure 4

Effect of tetracycline treatment on the microfilaremia of L. sigmodontis–infected cotton rats. In contrast to infected but untreated rats, there is a quick decline in microfilaremia in rats treated with tetracycline (added at 0.3% final concentration to the drinking water).