Oral transfer of adult Ancylostoma ceylanicum hookworms into permissive and nonpermissive host species

Abstract

Syrian hamsters become anemic and exhibit delayed growth following oral infection with third-stage Ancylostoma ceylanicum hookworm larvae. Here we describe experiments designed to determine the feasibility of adult worm transfer (AWT) between hosts, a technique that would facilitate the specific study of bloodfeeding hookworms in vivo without prior exposure of the host to larva-specific antigens, permit the ex vivo manipulation of adult parasites prior to reimplantation, and also allow for cross-species transfer of worms. Weanling hamsters given an oral AWT of 40 or 60 mixed-sex A. ceylanicum worms rapidly developed anemia; in the higher-dose group, hemoglobin levels declined from prechallenge levels by 44% within 4 days following AWT. Long-term survival of transferred worms was demonstrated by recovery of parasites from the intestines 42 days after AWT. AWT hamsters acquired humoral immune responses against soluble adult hookworm extracts and excretory-secretory products that were comparable in magnitude to those of animals given a typical infection with larvae. In AWT experiments employing the nonpermissive murine model, C57BL/6 mice given adult worms rapidly became anemic and lost weight in a manner similar to AWT hamsters. Infection of additional mouse strains demonstrated that while C57BL/10 and CD-1 mice also developed anemia following AWT, BALB/c mice were resistant. The technique of AWT to mice may further our understanding of hookworm pathogenesis by allowing the study of adult hookworm infections in a species with well-characterized genetics and an abundance of available reagents.

FIG. 1.

Blood hemoglobin levels (left panel) and weight gain (right panel) in the hamster AWT experiment. Twenty-four-day-old male hamsters (initial weight range, 19 to 33 g) were either infected with 40 (n = 3) or 60 (n = 3) adult worms each or left uninfected (n = 2). All values are means ± SEM. Asterisks indicate statistical significance versus the 0-worm group.

FIG. 2.

Kinetics of hookworm-specific antibody responses in hamsters following oral infection with adult A. ceylanicum. ELISAs were performed using adult worm ES products (left panel) and soluble HEX (right panel). All values are means ± SEM. For reference, the titer of the positive-control standard used to normalize experimental values is indicated by the vertical bar in each panel.

FIG. 3.

Analysis of hookworm-specific humoral immune responses in hamsters by immunoblotting. (Left) Coomassie-stained SDS-PAGE gel of LEX, HEX, and ES products, each at 2 μg of protein per well. (Right) Immunoblot of LEX, HEX, and ES products (2 μg per well) probed with pooled sera from AWT animals (infected with 0 or 60 worms) collected 42 days postinfection (dpi) or with sera, collected 56 dpi, from animals infected with 50 L₃. All sera were diluted 1:1,000. Molecular size markers are indicated on the right of each panel.

FIG. 4.

Blood hemoglobin levels (left) and weight change (right) in mice after AWT. Data are representative of two experiments. Female C57BL/6 mice (initial weight range, 15.7 to 19.0 g) were either infected with 20 adult worms (n = 5) or left uninfected (n = 3). All values are means ± SEM. Asterisks indicate statistical significance versus the 0-worm group.

FIG. 5.

Comparison of blood hemoglobin levels in various mouse strains 3 days after AWT. Female mice of each strain (n = 3 per group) were either infected with 20 adult worms or left uninfected. All values are means ± SEM. Asterisks indicate statistical significance (P < 0.02) versus the 0-worm group for each strain.

FIG. 6.

Analysis of hookworm-specific humoral immune responses in mice by immunoblotting. HEX and ES products (1 μg per well) were probed with pooled sera collected on day 56 from AWT animals. Twice-infected (2×) mice were infected on days 0 and 42 with 20 adult worms. Singly infected (1×) mice were infected on day 42. Mouse sera were diluted 1:200, and the blot was exposed to film for 45 min; immune hamster serum was diluted 1:1,000, and the blot was exposed for 2 min. Molecular size markers, in kilodaltons, are indicated on the right.