INTRODUCTION

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Listeria monocytogenes is a food-borne gram-positive pathogen that is able to translocate across the gut epithelium, resulting in listeriosis, i.e., systemic infection. Most healthy adults experience a limited infection, with at most mild influenza-like symptoms or, in some cases, gastroenteritis (2, 13, 30). However, listeriosis is extremely dangerous for pregnant women, causing abortions and stillbirths, and for newborn, elderly, and immunocompromised individuals, causing meningitis, meningoencephalitis, or sepsis (13, 14, 18, 21, 30). In Europe, the incidence of listeriosis is increasing (2, 14). The rate of mortality caused by listeriosis, excluding abortions, is one of the highest among bacterial infections. Overall fatality rates of 24 to 44% have been reported (2, 14, 18, 21). Because of the property of pathogens to acquire resistance to antibiotics, new approaches to prevent listeriosis deserve attention. Luminal factors such as gastric acidity, antimicrobial bile salts and fatty acids, and pancreatic enzymes contribute to the intestinal nonspecific defenses by killing pathogens. Therefore, changing the composition of the diet and thus changing the composition of gastrointestinal contents may affect colonization and translocation of L. monocytogenes. To study the efficacy of food components, an animal model of food-derived listeriosis with a quantitative, reliable, and accurate biomarker for translocation is required. Translocation of L. monocytogenes is observed in rats (24, 28) and mice (26), making them suitable models. Classically, translocation of listeria in animal models is determined by microbiological culturing of lymphoid organs (24, 26, 28). This approach has some disadvantages. First, culturing of organs is invasive, demanding killing of animals. Consequently, many animals are needed when more than one time point is necessary, e.g., when kinetics of both translocation and colonization have to be determined. Second, microbiological culturing only measures viable bacteria and not those already killed by immune cells and is therefore not representative of the total amount of translocated pathogens. Our laboratory has previously shown that urinary nitrate and nitrite (summed as NO_x), which reflects the production of nitric oxide (NO) by phagocytic cells (15), is a quantitative biomarker of salmonella translocation and is useful in studying the efficacy of functional food components on salmonella infection (5, 6, 27). Upon translocation, L. monocytogenes infects mesenteric lymph nodes (MLNs), spleen, and liver (24, 26, 28), resulting in infiltration of neutrophils and monocytes (7). These leukocytes, together with hepatocytes, Kupffer cells, and splenic macrophages, are able to induce NO synthase (12, 22, 23, 25, 33). It has been shown that L. monocytogenes induces NO synthase in murine spleen cells (33) and macrophages (25) and increases the concentration of NO_x in serum and urine when systemically administered in mice (4, 16). Thus, urinary NO_x may also be a quantitative biomarker of listeria translocation. Therefore, a strictly controlled experiment was performed with rats that were intragastrically infected with increasing doses of L. monocytogenes. Urinary NO_x and viable listeria in lymphoid organs and feces were measured.

	MATERIALS AND METHODS

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Bacterial culturing. L. monocytogenes 4B (clinical isolate, B1242 from the collection of our institute) was routinely stored at 80°C in brain heart infusion broth (Difco, Detroit, Mich.) containing 20% (vol/vol) glycerol. Stock solutions were quickly thawed, plated on listeria-selective PALCAM plates (Merck, Darmstadt, Germany), and then incubated aerobically at 37°C for 18 h. Subsequently, a few colonies were inoculated in brain heart infusion broth, followed by overnight incubation at 37°C under aerobic conditions. Bacterial cells were collected by centrifugation (15 min at 3,500 × g), washed three times in sterile saline, and resuspended in saline containing 3% (wt/vol) sodium bicarbonate. The virulence of the strain used was sustained by routine oral passage in Wistar rats, followed by isolation from spleen and liver at day 3 after oral administration.

Animals and infection. The experimental protocol was approved by the animal welfare committee of Wageningen University, Wageningen, The Netherlands. Male Wistar rats (specific pathogen free, WU; Harlan, Horst, The Netherlands), 9 weeks old with a body weight of approximately 325 g, were individually housed in metabolic cages. The environmental temperature (22 to 24°C), relative humidity (50 to 60%), and dark-light cycle (light, 0600 to 1800 h) were kept constant. The rats were fed purified diets consisting of 20% casein, 63% glucose, 5% cellulose, 4% corn oil, and vitamins and minerals according to the AIN-93 recommendation (25), except for choline, which was added as choline chloride instead of choline tartrate, and calcium, which was added as calcium phosphate (CaHPO₄ · 2H₂O; 180-mmol/kg diet) instead of calcium carbonate (125-mmol/kg diet). Diets were supplied as a porridge with 68% dry weight (dry diets mixed with double-distilled water) to minimize food spilling and subsequent contamination of urine and feces. Rats were given free access to food and demineralized drinking water. Food intake and body weight were recorded every 2 to 4 days preinfection and daily postinfection.

Statistics. Results are expressed as means ± standard errors (n = 8). Data were checked for normality and homogeneity of variances by using Shapiro-Wilks's test and Levene's test, respectively. When normally distributed, differences between groups were tested by analysis of variance (ANOVA), followed by Student's t test (one sided) with Bonferroni correction for multiple comparisons. Otherwise, differences between groups were tested with the nonparametric Kruskal-Wallis test. Correlation was determined by using Pearson's product moment. Differences were regarded significant if P < 0.05. Statistical measurements were performed with a commercially available statistical package (STATISTICA edition 1999; StatSoft, Inc., Tulsa, Okla.).

	RESULTS

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Rat growth and food intake. Body weight gain was not affected by the dose of L. monocytogenes administered. Mean body weight was 324 g at the start of the experiment, whereas the mean final body weight was 375 g. Food intake was not significantly affected by the dose of L. monocytogenes given. Before infection, average food intake was 23.1 g/day. After infection, mean food intake was 21.1 g/day.

Intestinal colonization and translocation of Listeria. Intestinal colonization was determined by enumerating viable L. monocytogenes cells in feces. Figure 1 shows the kinetics of fecal excretion of viable L. monocytogenes. On day 1 after inoculation, high levels of listeria were excreted. L. monocytogenes excretion declined gradually to the detection limit within 1 week after inoculation. Fecal excretion of viable L. monocytogenes was dose dependent. As expected, no viable L. monocytogenes could be detected in feces of rats that received 8 × 10⁹ heat-killed bacteria. No clinical signs of diarrhea were observed during the course of infection.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Fecal excretion of L. monocytogenes before and during infection with different doses of this pathogen. Viable or heat-killed cells (8 × 109) were orally administered on day 0. Data are expressed as means ± standard errors of eight rats per group. DL, detection limit. Values on the same day not sharing the same letter are significantly different (P < 0.05), as determined by ANOVA followed by Student's t test with Bonferroni correction.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Viable counts of L. monocytogenes in MLNs after oral challenge with different doses of this pathogen. Heat-killed or viable cells were orally administered on day 0. Listeria cells were enumerated in MLNs by standard plating techniques 1 day after inoculation. Data are expressed as means ± standard errors of eight rats per group. DL, detection limit. Values not sharing the same letter are significantly different (P < 0.05), as determined by ANOVA followed by Student's t test with Bonferroni correction.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Kinetics of urinary NOx excretion (A) and total infection-induced urinary NOx (B) after oral challenge with different doses of L. monocytogenes. Viable or heat-killed cells (8 × 109) were orally administered on day 0. Data are expressed as means ± standard errors of eight rats per group. Values not sharing the same letter are significantly different (P < 0.05), as determined by ANOVA followed by Student's t test with Bonferroni correction.

	DISCUSSION

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This study was performed to investigate the efficacy of urinary NO_x as a quantitative biomarker of translocation of L. monocytogenes. This food-borne pathogen can colonize the intestinal tract, invade the intestinal mucosa, and spread to systemic organs, causing sepsis and meningitis in individuals with hampered immune function. Colonization of listeria depended on the oral dose (Fig. 1). Our study also showed that the number of viable listeria cells in MLNs increased with higher doses of orally administered L. monocytogenes (Fig. 2). We were, however, unable to detect L. monocytogenes in spleen and liver. This may be explained by the time point at which organs were sampled, (i.e., day 1 after infection). Although Hirose et al. (17) described that dissimination of L. monocytogenes to spleen and liver can be detected on day 1 after intragastric inoculation of 3 × 10⁹ CFU in male F344/Slc rats, other oral models using mice showed that this pathogen can be detected in the spleen only from the second day and beyond (24, 26). Species and strain differences (F344/Slc versus Wistar rats in our study) and, probably, the distinct diets (rodent chow versus purified diets in our study) may account for this discrepancy. Literature data on the kinetics of translocation to MLNs are consistent; all studies showed translocation to MLNs on day 1 after inoculation (17, 24, 26). Although the time of spreading to spleen and liver may depend on the animal model used, these studies as well as our experiment clearly show that extraintestinal dissemination of L. monocytogenes occurs in a dose-dependent manner in both rats and mice (26, 30).

NO synthesis is a primary reaction of phagocytic cells to microbes (19, 23) or bacterial components such as lipopolysaccharide (19, 31) and is known to respond in a dose-dependent manner (3, 19). NO is rapidly oxidized to NO₂ and NO₃ (20), which are excreted in urine (15). Our laboratory has previously shown that urinary NO_x is a more discriminative biomarker for salmonella translocation than enumeration of systemic bacteria from organs (27). Analysis of urinary NO_x is also noninvasive, not requiring killing of the animals. L. monocytogenes is also known to stimulate NO production in murine peritoneal macrophages (25) and spleen cells in vitro (33). In vivo studies with mice have shown that systemically administered L. monocytogenes increases NO_x in blood and urine in a dose-dependent manner (4, 16). Using the NO synthase inhibitor N^G-monomethyl-L-arginine, Boockvar et al. (4) proved that listeria-mediated urinary NO_x was derived from induced NO synthase activity. Our study showed that orally administered L. monocytogenes provoked urinary NO_x excretion in rats. Values peaked at days 3 and 4 of the infection and gradually declined thereafter, with baseline levels reached at day 6. These kinetics are remarkably consistent with the kinetics reported for bacterial counts in lymphoid organs (24, 26) after oral listeria infection and with the kinetics of inducible NO synthase mRNA in murine peritoneal macrophages (4) after intravenous administration of L. monocytogenes. Urinary NO_x was proportional to the amount of orally administered L. monocytogenes (Fig. 3) and correlated with the number of viable counts of listeria in MLNs (r = 0.99). Using enteroinvasive and noninvasive pathogens, Witthöft et al. (32) showed that invasion is a prerequisite for activation of the inducible NO synthase. In addition, oral inoculation of a pathogenic E. coli strain that colonizes the intestinal tract, but is incapable of translocation, does not result in increased urinary NO_x (I. M. J. Bovee-Oudenhoven, unpublished results). Therefore, translocation is a prerequisite for induction of urinary NO_x excretion. The observation that heat-killed L. monocytogenes is ineffective in stimulating urinary NO_x excretion also indicates that translocation is indeed obligatory for induction of the NO response by this pathogen. Thus, urinary NO_x is a suitable biomarker for L. monocytogenes translocation and can therefore be used to study the efficacy of functional food components in animal models of listeriosis.

The kinetics of the urinary NO_x response after listeria infection is different from that observed after salmonella infection (6, 27), which increases from day 3 and reaches peak values on days 6 and 7, returning to baseline levels on day 12 after inoculation. This may be explained by the time course of infection. Compared with listeria, salmonella infection is slowly cleared in rats. Whereas viable listeria counts in systemic organs increase to peak values at day 3 of the infection and are completely cleared 7 days after inoculation (24, 28), viable salmonella counts in lymphoid tissue start to increase at day 3 and beyond (10), with considerable amounts still detectable in MLNs at day 7 of infection (27).

It has been shown that NO_x is increased in plasma and urine of patients with acute infective gastroenteritis induced by pathogens such as salmonella, shigella, and campylobacter (1, 8, 9, 11). Plasma nitrate correlated with the severity of infection (8). Thus, besides application to the quantitative determination of orally acquired listeriosis in animal models, urinary NO_x might also be useful to monitor the efficacy of treatment of listeriosis in humans.

In conclusion, a dose-dependent relationship between urinary NO_x and translocation of listeria exists in a rat model of orally acquired L. monocytogenes infection. Therefore, excretion of NO_x in urine can be used as a noninvasive biomarker for quantifying translocation of this pathogen in animal models and may provide a tool to study the efficacy of functional food components. Besides this application, urinary NO_x may also be used to monitor the severity of listeriosis in humans.