Development of Two Animal Models To Study the Function of Vibrio parahaemolyticus Type III Secretion Systems

Bacterial strains.Wild-type V. parahaemolyticus NY-4 strain (serotype O3:K6) and ΔvcrD (T3SS1 knockout [KO]), ΔescV (T3SS2 KO), and ΔvcrD and ΔescV (T3SS1 and T3SS2 [T3SS1/2] KO) deletion mutants were generated previously (Table 1) (33). Bacterial strains were grown 6 to 8 h in Luria-Bertani (LB) medium supplemented with 2.5% NaCl (LB-S) and shaking (250 rpm) at 37°C. Strains were not induced to express T3SSs before use in animals. ΔexsD and ΔexsA deletion mutants (Table 1) were included in our analysis of hemolytic activity. The ΔexsD deletion mutant expresses the T3SS1 constitutively, and the ΔexsA deletion mutant serves as an alternative knockout of the T3SS1. A tdhA deletion mutant (ΔtdhA) was generated using previously described methods (26), except we employed a modified set of oligonucleotide primers (Table 1).

Comparison of growth rates.The growth rates of wild-type strain NY-4 (serotype O3:K6) and T3SS1 KO, T3SS2 KO, and T3SS1/2 KO mutant strains were compared using a Bioscreen C plate reader (Oy Growth Curves AB Ltd., Helsinki, Finland). Briefly, 20 μl of overnight culture was inoculated into 180 μl of fresh LB-S medium, and the optical density at 600 nm (OD₆₀₀) was monitored over 24 h. Growth curves were repeated three times with 10 independent wells per replicate. After 24 h, the bacterial count (CFU) was determined by the drop-plate method (9).

Hemolytic assay.The hemolytic activity of the supernatants was evaluated by first preparing overnight culture from isolated colonies grown from freezer stocks. The colonies were isolated twice on LB-S agar plates after which one colony for each strain was selected from the second plate and dispersed separately in 10-ml LB-S broth tubes. Broth cultures were grown for approximately 24 h at 37°C with shaking (250 rpm). A small aliquot (200 μl) was retrieved to quantify the OD and CFU per milliliter (final counts ranged between 2 × 10⁹ and 4.5 × 10⁹ per ml). The remaining culture was pelleted by centrifugation (4,000 × g for 10 min) at room temperature, and the supernatant was transferred to 15-ml concentrators (Amicon Ultra-15 with a PLBC Ultracel-PL membrane with a molecular size cutoff of 3 kDa). The supernatant was concentrated to ∼10% of original volume (∼1 ml). Human red blood cells or erythrocytes (HRBCs) (2% suspension in Alsever solution from a single donor; Innovative Research, Novi, MI) were pelleted, washed four times, and resuspended in an equal volume of phosphate-buffered saline (PBS) (pH 7.4) before they were added to supernatant samples (1:1). The mixtures were incubated at 37°C for 4 h after which samples were briefly centrifuged (600 × g for 3 min), and the hemolytic activity from each sample was evaluated by measuring the optical absorbance from the supernatant (541 nm) with a Multiskan MCC/340 spectrophotometer (Thermo LabSystems, Helsinki, Finland). For a positive control, PBS (150 μl) was mixed with 150 μl of HRBCs and incubated for 4 h at 37°C. After incubation, 150 μl of a 10% solution (in PBS) of Triton X-100 (Triton N-101; Sigma Chemical Co., St. Louis, MO) was added. Three biological replicates (including three technical replicates each) were used in the analysis, and hemolytic activity of each strain was then normalized against the log concentration from the original culture (CFU/ml) or using the total protein from the supernatant (quantified using a MicroBCA protein assay; Thermo Fisher Scientific, Rockford, IL).

Piglet model.The piglets were taken from the dam when they were approximately 6 h old and were raised until they were 48 h old. The piglets were fed three times a day with infant milk formula (Enfamil Lipil milk-based infant formula with iron) using individual feeders and with increasing volume from 80 ml to 120 ml until they were 5 days old. Both males and females were used in equal frequency, and the average body weight at infection was 1.4 kg (standard deviation [SD], 0.15 kg).

(i) Dose determination.To determine the dose, 20-ml samples of 6-h-old cultures of bacteria with an OD₆₀₀ of 2.5 to 2.7 were centrifuged at 4,000 × g for 10 min at room temperature and were resuspended in 10 ml of sterile PBS (pH 7.4). Serial 10-fold dilutions were generated corresponding to 10⁶ to 10¹² CFU/ml. The inoculum was mixed with 10 ml of infant milk formula and placed in clean and disinfected feeders, allowing each pig to drink the complete dose. The feeders were refilled several times with a small amount of infant formula (5 ml) to ensure that the bulk of the inoculum was ingested.

(ii) Experimental design.After dose determination trials, we performed three independent experiments consisting of two piglets per bacterial strain (treatment), for a total of 6 piglets per treatment. Treatments consisted of a dose of 10¹¹ CFU of either the wild-type strain NY-4 (serotype O3:K6) or T3SS1 KO or T3SS2 KO mutant strain or PBS (pH 7.4) as a vehicle control.

(iii) Clinical observations.The body temperature of each piglet was evaluated rectally, and individual rectal swabs were taken and placed immediately in 10 ml sterile PBS prior to infection. All piglets were observed every 2 h postinoculation, and clinical signs were evaluated (diarrhea, vomiting, feed consumption, and alertness). In addition, body temperature and rectal swabs were taken to assess pyrexia and bacterial shedding, respectively. The occurrence of diarrhea was evaluated by a subjective score as follows: presence of soft to liquid feces on swab (+), presence of soft to liquid feces on the swab and stained perineum (++), and presence of soft to liquid feces on the swab, stained perineum, and presence of a large amount of loose fecal discharge in the cage (+++). Vomiting was recorded as present or absent. At 8 h and 24 h after inoculation, one piglet from each group was euthanized with an intravenous overdose of 2 ml of pentobarbital. A full necropsy was performed immediately after death, and gross changes were recorded. Samples for bacteriology (lung, liver, spleen, kidney, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) were immediately placed in 10 ml sterile PBS. Samples for histological analysis were placed in 10% formaldehyde for fixation. All tissues were processed and stained with hematoxylin and eosin for histological evaluation.

(iv) Bacteriology.Fecal samples obtained immediately before inoculation, follow-up rectal swabs, and necropsy tissues were cultured on thiosulfate-citrate-bile salts-sucrose agar (TCBS; Difco Laboratories, Detroit, MI) containing 100 μg/ml ampicillin (NY-4 and derivative strains are resistant to ampicillin). The plates were incubated at 37°C overnight and were then examined for the presence of smooth translucent green-blue colonies. Isolation of V. parahaemolyticus from rectal swabs was recorded as present or absent. The tissue samples were processed with a stomacher 80 lab blender (Seward Medical London, United Kingdom), and when possible, the number of CFU per gram of tissue was determined.

Intrapulmonary mouse model.Mice were housed in microisolator cages at the Animal Science Experimental Animal Laboratory Building (Pullman, WA) and allowed ad libitum access to food and water. The age and body weight of infected mice ranged from 4 to 5 months old and 30 to 40 g, respectively, and males and females were used at similar frequencies. Mice were injected intraperitoneally with a combination of 60 to 70 mg of ketamine per kg of body weight and 12 to 14 mg of xylazine per kg of body weight (24). Upon deep sedation, each mouse was placed on a board with its mouth kept open by holding the upper and lower incisors with plastic forceps. The tongue was then extended with forceps to one side, and a microsyringe was used to deposit 50 μl of inoculum onto the back of the tongue and proximal to the larynx. The inoculum was inhaled into the lungs by normal breathing. The mouse was then held in an upright position for 10 min and allowed to recover (32). The presence of clinical signs in mice was observed every hour during the first 12 h and every 3 h thereafter. Those animals that survived up to 24 h postinoculation were euthanized in a CO₂ chamber followed by cervical dislocation; mice judged to be moribund were euthanized immediately. A full necropsy was performed immediately upon death, and gross changes were recorded. Samples for bacteriology (lung, liver, spleen, and gastrointestinal tract) were immediately placed in sterile PBS. The histological samples were obtained from the same tissues as described above with lung and several sections of the small intestine insufflated and placed in 10% formaldehyde until they were processed.

(i) Dose determination.To establish a suitable dose for the wild-type strain (NY-4), 10-fold serial dilutions (10⁴ to 10¹⁰) of bacteria were inoculated into 28 adult C57BL/6 mice. The number of CFU per milliliter was calculated using the OD₆₀₀ measurement of overnight cultures and then verified through bacterial enumeration using a drop-plate technique (9).

(ii) Mortality studies.The same methods for preparing inoculum were used for comparative studies. To achieve a mortality rate between 80 and 100%, we used a dose of 5 × 10⁵ CFU (see Results). Four groups of 10 adult mice (C57BL/6) were infected with wild-type strain NY-4 or T3SS1 KO, T3SS2 KO, or T3SS1/2 KO mutant strain. Two replicate studies were completed. Vehicle controls were used in one trial (n = 5 mice) to confirm that our procedures did not confound the experimental outcome.

(iii) Bacteriology.All tissue samples were first weighed and then placed either in 2 ml PBS (spleen and lung) or in 10 ml PBS (liver and gastrointestinal tract). All samples were plated on TCBS and incubated at 37°C overnight. When possible, the number of bacterial CFU per gram of tissue was estimated.

The experimental protocols described herein were approved by the Washington State University Institutional Animal Use and Care Committee (ASAF 3841 and 3638).

Statistical analysis.Analysis of variance (ANOVA) was used to compare culture growth and hemolytic activity, and a P value of <0.05 was considered statistically significant. For the hemolytic assay results, we employed a Dunnett's upper one-sided multiple-comparison test with either the PBS-only or ΔtdhA results used as the “control” for comparison purposes. The clinical score was analyzed using repeated-measures ANOVA, and pair-wise comparisons were performed using a Tukey-Kramer test. These analyses were conducted using NCSS 2004 (Number Cruncher Statistical System, Kaysville, UT). Analysis of the mouse survival ratio was performed with the Kaplan-Meier and log-rank test using Graph Pad Prism 5.01 (GraphPad Software Inc., San Diego, CA).

Growth rates and hemolytic activities are equivalent for the strains employed in challenge experiments.The growth rates and hemolytic activities of the wild-type and mutant strains used in this study were evaluated to determine whether mutant strains with deletion mutations were compromised for these traits. There was no difference in the shape of the growth curves or the final OD₆₀₀ at 24 h (P > 0.05) (Fig. 1 A). All strains except the ΔtdhA strain retained hemolytic activity against HRBCs relative to negative control (P > 0.05) (Fig. 1B). We included ΔexsA and ΔexsD strains in this analysis, which serve as positive and negative regulators of the T3SS1 expression, respectively (34, 35). Hemolytic activity was not different for either strain (Fig. 1B). The inclusion of ΔtdhA deletion mutant confirmed that hemolysis in this assay was responsive to the absence of TDH (P < 0.0001) (Fig. 1B), and this result was similar when raw optical density data were used or when these data were normalized by CFU/ml (data not shown). We have shown in a separate experiment that normalizing by total protein concentration does not change the conclusion that only the ΔtdhA strain is attenuated for hemolytic activity (data not shown). From these data, we concluded that the results from our challenge experiments are not likely to be confounded by inadvertent differences in growth rates or by differences in TDH activity.

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FIG. 1.

(A) The growth rates of V. parahaemolyticus strains (wild-type strain NY-4 [serotype O3:K6] and T3SS1 knockout KO, T3SS2 KO, and T3SS1/2 KO mutant strains) were compared over 24 h in broth culture. The value at each hour is an average of three biological replicates (variance not shown). The final OD₆₀₀ was not different for the different strains (P > 0.05). Ctrl, control. (B) The hemolytic activity of the supernatants was evaluated by measuring the optical absorbance at 541 nm 4 h after mixing culture supernatant with human red blood cells. The values (bars) are the means plus standard errors of the means (error bars) for three biological replicates. For simplicity, the values shown here were normalized by dividing the values by the value for the lysis positive control. The ΔtdhA strain and the negative control (Neg Ctrl) had significantly lower hemolytic activity (P < 0.05) than the other strains (indicated by an asterisk). The outcome of the analysis did not change when the values were normalized by CFU/ml (except the negative control was not included in the latter analysis).

T3SS2 is required for gastrointestinal disease in 2-day-old piglets.Two-day-old piglets were inoculated with 10⁶ to 10¹² CFU of wild-type strain NY-4 (n = 2 animals per dose), and the 50% infective dose (ID₅₀) from this experiment was estimated to be 10¹¹ CFU when the primary response variable was the presence or absence of diarrhea. We used 2-day-old piglets in these experiments, because they were readily available, but other ages may work as well, although ID₅₀ experiments may need to be repeated. Upon infection with the wild-type NY-4 strain, piglets developed vomiting and yellow watery diarrhea within 2 h postinoculation. The clinical scores among the different infected groups and controls during the time course of the experiment were clearly different. Wild-type- and T3SS1 KO-inoculated piglets produced obvious clinical symptoms, and knocking out T3SS2 abrogated this effect on the piglets (Fig. 2 A). Vomiting, ranging from mild to severe, was observed for all piglets inoculated with wild-type strain NY-4 and in 50% of the piglets inoculated with the T3SS1 KO strain, but vomiting was not observed in the piglets inoculated with the T3SS2 KO strain or PBS. None of the inoculated piglets displayed signs of dehydration or anorexia. We observed no significant differences in body temperature among the three infected groups and controls during the time course of the infection. Fecal shedding of V. parahaemolyticus was observed only in the piglets infected with NY-4 and T3SS1 KO strains and only during the first 12 h postinoculation (Fig. 2B).

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FIG. 2.

(A) The presence or absence of diarrhea was evaluated every 2 h during the first 12 h postinoculation and every 3 h thereafter. The average diarrhea severity score (see Materials and Methods) was estimated for six piglets in each treatment group. Piglets infected with wild-type strain NY-4 or T3SS1 KO mutant strain had significantly higher clinical scores (P < 0.003) than piglets infected with T3SS2 KO strain or piglets in the PBS control group at 2 and 4 h postinoculation (p.i) (indicated by three asterisks). N.S, not significant. (B) Rectal swabs were cultured every 2 h during the first 12 h postinoculation and every 3 h thereafter. Bars show the percentages of piglets shedding V. parahaemolyticus. Numeric values below bars indicate hours p.i.

We observed no gross lesions related with V. parahaemolyticus infection in any of the piglets evaluated. At 8 h postinfection, piglets treated with wild-type strain NY-4 or T3SS1 KO strain had a mildly distended jejunum and ileum with a moderate amount of yellow liquid digesta and gas. In addition, the cecum and spiral colon were filled with moderate to abundant amounts of soft pasty to liquid green digest. Histological examination showed that the cecal submucosa and serosa were moderately expanded by edema (Fig. 3). No other histological changes were observed in all the tissues evaluated. Neither gross lesions nor histological changes were observed in piglets inoculated with the T3SS2 KO strain or PBS.

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FIG. 3.

(A) Piglets infected with wild-type strain NY-4 and T3SS1 KO mutant strain showed a mild to severe colonic submucosal and serosal edema (arrows). (B) No histological lesions were observed in the piglets infected with the T3SS2 KO strain. The sections were stained with hematoxylin and eosin. Magnification, ×100.

Bacteria were consistently recovered at 8 h and 24 h postinfection from the gastrointestinal tracts of piglets infected with wild-type strain NY-4 or T3SS1 KO strain. Only one piglet infected with T3SS2 KO strain had recoverable bacteria from the gastrointestinal samples at 8 h postinfection (Table 2). No V. parahaemolyticus was recovered in control animals or from the other tissue samples, indicating that cross-contamination was unlikely to be a confounding factor in these experiments. Collectively, these data indicate that T3SS2 is required to cause gastrointestinal disease (acute diarrhea and vomiting) in neonatal pigs, and deletion of the functional T3SS2 results in an absence of clinical symptoms characteristic of V. parahaemolyticus infection.

T3SS1 is necessary for mortality of infected mice.We estimated the ID₅₀ for the intrapulmonary mouse model to be 1.4 × 10⁵ CFU. Consequently, we adopted a dose of 5 × 10⁵ CFU for these experiments to ensure a mortality rate between 80% and 100% after infection with the wild-type strain. We used 30- to 40-g mice in these experiments, because they were readily available, but other sizes may work, although the 50% lethal dose (LD₅₀) may need to be recalculated and a smaller inoculum volume (<50 μl) may be needed for smaller mice. To determine whether the presence of T3SS1 or T3SS2 is necessary for mortality in the intrapulmonary model, the mortality rates of mice infected with wild-type strain NY-4 or T3SS1 KO, T3SS2 KO, or T3SS1/2 KO mutant strain were compared (Fig. 4). High mortality was observed for mice infected with NY-4 or T3SS2 KO strain (40 to 80% mortality by 8 h postinoculation and 80 to 100% by 24 h). No mortality was observed in mice during the 24-h period after challenge with T3SS1 KO or T3SS1/2 KO strain. All mice infected with wild-type and T3SS2 KO strains showed diffuse and severe pulmonary hemorrhage. No gross lesions were observed in mice infected with the T3SS1 KO and T3SS1/2 KO strains. Histological evaluation of the lung in all mice showed severe and diffuse capillary congestion with perivascular edema and large areas of alveolar hemorrhage. The alveolar lumen was severely distended by large aggregates of neutrophils intermixed with some foamy macrophages, abundant edema, and cellular debris. The interstitium was randomly infiltrated by small aggregates of neutrophils (Fig. 5). To better assess how V. parahaemolyticus strains were impacting mice at death, we repeated the challenge trial twice and collected bacterial counts when mice died or were euthanized between 8 and 12 h postinoculation (p.i.). Importantly, during the latter experiment when one mouse died or was moribund and euthanized, one mouse from each of the remaining treatments was simultaneously euthanized (regardless of condition) to obtain time-matched bacteriology data; the samples were processed immediately to limit changes in microbial population density after death. No differences were observed in the numbers of bacteria recovered from the lung, gastrointestinal tract, or liver regardless of the strain used (Fig. 6). Mice inoculated with NY-4 and T3SS2 KO strains became bacteremic (70% and 30%, respectively) with similar blood CFU levels (0.7 ± 0.6 and 0.4 ± 0.1 log₁₀ CFU ± SD, respectively). Spleen samples from bacteremic animals were also positive in both animals inoculated with wild-type strain NY-4 (45%) and animals inoculated with T3SS2 KO mutant strain (100%). Mice inoculated with T3SS1 KO strain remained culture positive for V. parahaemolyticus in the lungs, gastrointestinal tract, and liver after 24, but only one animal had a culture-positive spleen at 24 h. These data indicate that T3SS1 is necessary to produce mortality following pulmonary challenge and that T3SS1 is strongly associated with systemic V. parahaemolyticus infection.

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FIG. 4.

Four groups of C57BL/6 adult mice (10 mice in each group) were inoculated with different strains of V. parahaemolyticus. The survival rate was evaluated for two independent replicates shown in panels A and B by Kaplan-Meier and log-rank tests. Values that are significantly different (P < 0.0001) are indicated by three asterisks. Values that are not significantly different (N.S) are indicated.

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FIG. 5.

Histopathological examination of the mouse lung specimens. (A) At 8 h postinoculation for all infected groups (wild-type strain NY-4 and T3SS2 KO, T3SS1 KO, and T3SS1/2 KO mutant strains), the mice presented diffuse capillary congestion with perivascular edema and large areas of alveolar hemorrhage. The alveolar lumen is severely distended by large aggregates of neutrophils intermixed with a few foamy macrophages and cellular debris. (B) At 12 h postinfection (NY-4, T3SS1 KO, and T3SS1/2 KO strains), there was severe neutrophilic infiltration. The bronchi were completely plugged by abundant suppurative exudates and infiltrated by large peribronchiolar cuff of neutrophils and macrophages. (C) Section in control animals (PBS only) are characterized by diffuse capillary hyperemia. The sections were stained with hematoxylin and eosin. Magnification, ×200.

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FIG. 6.

Average CFU recovered from lung (A), gastrointestinal tract (B), and liver (C) for mice from which bacteria were recovered. Mice infected with wild-type strain NY-4 and T3SS2 KO mutant strain died or were euthanized between 8 and 12 h postinoculation. The mice inoculated with the T3SS1 KO mutant strain were euthanized at the same time to have matched bacterial counts over time. The number of animals with recoverable bacteria/number of inoculated mice (n/n°) is shown on the x axes.