Back to the future: studying cholera pathogenesis using infant rabbits

Abstract

Cholera is a severe diarrheal disease, caused by Vibrio cholerae, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation of V. cholerae into 3-day-old rabbits pretreated with cimetidine led to lethal, watery diarrhea in virtually all rabbits. The appearance and chemical composition of the rabbit diarrheal fluid were comparable to those of the "rice-water stool" produced by cholera patients. As in humans, V. cholerae mutants that do not produce cholera toxin (CT) and toxin-coregulated pilus (TCP) did not induce cholera-like disease in rabbits. CT induced extensive exocytosis of mucin from intestinal goblet cells, and wild-type V. cholerae was predominantly found in close association with mucin. Large aggregates of mucin-embedded V. cholerae were observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influences V. cholerae's association with the host intestine and its exit from the intestinal tract. Our model should facilitate identification and analyses of factors that may govern V. cholerae infection, survival, and transmission, such as mucin. In addition, our results using nontoxigenic V. cholerae suggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-V. cholerae vaccines.

FIG 1

Gross findings in infant rabbits inoculated with V. cholerae (A and C) or buffer (B and D). The boundary between wet and dry fur is shown with an arrow in panel A. The arrows in panels C and D show the fluid-filled distended cecum and the normal-appearing cecum, respectively. More than 20 V. cholerae-infected and 10 mock-infected rabbits were examined.

FIG 2

Cecal fluid accumulation ratios in infant rabbits inoculated with wild-type or mutant V. cholerae or buffer. Samples were collected 22 h after infection. The ratios were calculated as the weight of accumulated cecal fluid to the weight of drained cecal tissue. Error bars represent the standard deviations of the means. Values that were significantly lower (P < 0.001) than the ratio measured with wild-type (wt) V. cholerae infection are indicated by three asterisks. There were at least seven rabbits in each group.

FIG 3

Histological findings in the distal small intestines of rabbits inoculated with wild-type (WT) or mutant V. cholerae. (A and B) Representative H&E-stained sections taken from rabbits at 22 h postinfection showing edema (arrows) and capillary congestion (arrowheads) in V. cholerae-infected rabbits (A), which is not evident in mock-infected rabbits (B). (C) The arrows point to heterophils present in the lamina propria of V. cholerae-infected rabbits. (D to F) PAS-stained sections show that mucin (magenta stain; arrows) is absent from goblet cells in rabbits infected with wild-type V. cholerae (D) but not in mock-infected rabbits (E) or rabbits infected with the ctxAB mutant (F). Bars, 500 µm (A, B, D, E, and F) and 100 µm (C). (G to I) Histological scores for capillary congestion (G), villous edema (H), and mucin depletion from goblet cells (I) were calculated for at least 8 rabbits. Each symbol shows the score for one rabbit. Horizontal bars represent the median values. Median values that were significantly lower than those found in wild-type-vibrio infection are indicated by asterisks below the groups on the graph (*, P < 0.05; **, P < 0.01).

FIG 4

Quantitative and microscopic assessment of wild-type V. cholerae in the intestines of infant rabbits at various times postinoculation. (A) Numbers of CFU recovered from homogenates of the indicated intestinal sections or in the cecal fluid (prox SI, proximal small intestine). Bars represent the geometric mean values. Values that were significantly greater than the values at 8 h postinfection are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Each time point represented data from 5 to 15 rabbits. (B to D) Representative confocal micrographs showing the distribution of fluorescent wild-type V. cholerae at 12 h (B) and 22 h (C and D) postinoculation. Tissue sections from at least five rabbits were counterstained with phalloidin (red) to visualize filamentous actin and DAPI to stain nuclei (blue). In some sections, tissue autofluorescence (diffuse green signal within villi) is evident; this was also observed in tissues from mock-infected rabbits.

FIG 5

Recovery of wild-type or mutant V. cholerae (CFU) from homogenates of the indicated regions of the intestines or cecal fluid 22 h postinfection (SI, small intestine). Bars represent the geometric mean values from 11 to 22 rabbits. Each symbol shows the value for one rabbit.

FIG 6

Representative confocal micrographs showing fluorescent V. cholerae (green) in the rabbit distal small intestine at 22 h postinfection. Tissues were stained with wheat germ agglutinin (WGA) (red) and DAPI (blue). (A and B) Wild-type V. cholerae often colocalized with mucin-rich “clouds” of material tethered to the villi. (C) In contrast, the V. cholerae ctxAB mutant appeared less associated with mucin and was more often found in the intervillous space. The cecal fluid from rabbits infected with wild-type V. cholerae contained many large mucin-rich aggregates of V. cholerae cells compared with the fluid from rabbits infected with the ctxAB mutant (compare panels D and E). Tissues from at least 10 rabbits were examined.

FIG 7

Scanning electron micrographs of the distal small intestines of rabbits inoculated with wild-type V. cholerae (A, C, and E) or the ctxAB mutant (B, D, and F). (A and B) Low-magnification images showing macroscopic material (arrow) on the villi in rabbits inoculated with the wild type (A) but not the ctxAB mutant (B). (C to F) Higher magnification reveals that this material contains numerous V. cholerae organisms (C and E), whereas the ctxAB mutant has a patchy distribution and is in direct apposition to the brush border microvilli (D [arrows] and F). Goblet cells, identified by their shorter, darker microvilli, are seen in panel F. Tissue samples from at least five rabbits were examined.

FIG 8

V. cholerae ctxAB mutant caused mild diarrhea in infant rabbits 3 days postinoculation. (A) Diarrhea was manifest as fecal contamination of the hind legs and tail. (B) A moderate inflammatory infiltrate was observed in the colonic tissue of all infected rabbits (arrows indicate heterophils). (C and D) Representative images showing the increased numbers of TUNEL-positive cells in the colons of the three infected rabbits examined (C) compared to those in the three mock-infected control animals examined (D). Bars, 50 µm.