Oral Inoculation of A/J Mice for Detection of Invasiveness Differences between Listeria monocytogenes Epidemic and Environmental Strains

Abstract

Four-week-old Harlan A/J mice were orally infected with six epidemic and six environmental strains of Listeria monocytogenes. Epidemic strains were significantly more invasive as a group than were environmental strains (P < 0.05), and the intestines of some mice infected with epidemic strains had extensive hemorrhage. Mice inoculated with epidemic strains were significantly more likely to become systemically infected than mice inoculated with environmental strains (P < 0.01).

Listeria monocytogenes is a food-borne bacterial pathogen that can cause severe disease and high mortality in humans and animals (27). Although most epidemics are caused by L. monocytogenes serotype 4b, serotype 1/2a is the most common serotype isolated from food (4). This implies different virulence potential according to serotype. Other factors that may affect infectivity are the infective dose, survival of the bacteria in the gastrointestinal tract, and host immunity (15, 27). Penetration of the intestinal barrier is the primary step for the infection (16), and subsequent invasion of organs such as the liver and spleen are also critical for establishing a systemic infection (27). Therefore, it is important to determine the ability of L. monocytogenes strains to penetrate the intestinal mucosa and invade organs by using an in vivo model to assess invasiveness.

L. monocytogenes infection elicits a comparable innate and acquired cellular immune response in humans and rodents; therefore, the murine model of L. monocytogenes infection has been widely used (1). Most previous mouse models used to assess strain invasiveness used intraperitoneal or intravenous inoculation of immunocompetent or immunocompromised mice (9, 23, 24, 26). However, most human infections are acquired orally, and these models do not address survival and translocation in the gut. Furthermore, studies describing intragastric infection of mice or guinea pigs required relatively high numbers of bacteria (10⁹ CFU) (2, 3, 16, 18, 26).

Recently, an A/J mouse model in which intragastric inoculation was used was described by Czuprynski et al. (6). It was found that 6- to 8-week-old A/J mice develop systemic infection following intragastric inoculation by using numbers of organisms similar to what is detected in L. monocytogenes-contaminated food products. However, only two strains (Scott A and EGD) were tested (6), and sodium pentobarbital was used for anesthesia, which was later shown to increase susceptibility to infection (7). Therefore, it is important to test the efficacy of this model by using more strains and an alternative means of anesthesia.

Mice.

A previous study (22) tested NCR mice of various ages to assess the effect of mouse weight on the L. monocytogenes infection and demonstrated that smaller mice were more sensitive to oral infection and further suggested the use of 14- to 15-g mice (approximately 4 weeks old) for L. monocytogenes infection. Therefore, 4-week-old A/J mice (Harlan Sprague-Dawley, Indianapolis, Ind.) were used in this study (average weight, 14.1 ± 1.6 g). Mice were divided randomly (six mice per group), housed under sterile microisolator caps, and acclimated for 5 days before experiments were initiated. Mice were given sterile food and water ad libitum except during the 5 h before inoculation, when food was removed.

Oral inoculation.

Prior to infection, mice were anesthetized by isoflurane gas (1 liter/min) and an inoculum (10⁶ CFU/ml, log-phase growth) was introduced intragastrically in a total volume of 0.1 ml by using a stainless steel feeding needle (22-gauge by 1.5 in.; Fisher Scientific, Pittsburgh, Pa.) attached to a 1-ml syringe. Six epidemic strains and six environmental strains were tested (Table 1). For controls, mice were inoculated with sterile phosphate-buffered saline. Seventy-two hours after infection, the mice were euthanized with ketamine (0.06 mg) and xylaxine (0.03 mg), followed by cervical dislocation. The liver and spleen were removed aseptically, weighed, and homogenized in 1 ml of cold, sterile phosphate-buffered saline. Homogenates were diluted in sterile phosphate-buffered saline, plated on blood agar, and incubated at 37°C for 48 h. Colonies phenotypically characteristic of L. monocytogenes were spotted onto ALOA plates (Microbiology International, Frederick, Md.) and incubated at 37°C for 24 h. Phospholipase-positive colonies were counted, and the number of CFU per gram was calculated for each organ. Data were expressed as log₁₀ CFU of L. monocytogenes per gram of tissue, and both general linear model analysis of variance and unpaired t tests (NCSS Statistical Software) were performed to compare the differences between epidemic and environmental strains.

TABLE 1.

L. monocytogenes strains used in this study and oral infection results

Type of strain	Strain	Previous strain designation(s)	Serotype	Source informationc	Donorsa	Reference(s)	Avg log CFU/g for:
							Liver	Spleen
Epidemic	LMB469	Scott A/ILSI-1	4b	Mass., 1983	ILSI	5	5.15 ± 0.9	6.22 ± 0.7
	LMB472	F2365/TS29/ILSI-4	4b	L.A., 1985	ILSI	17	3.11 ± 1.1	3.47 ± 1.6
	LMB496	TS27/ILSI-28	4b	Halifax, Canada, 1981	ILSI	25	1.74 ± 0.8	1.53 ± 0.9
	LMB498	TS38/ILSI-30	4b	UK, 1988-1990	ILSI	11, 19	2.14 ± 0.8	3.06 ± 1.2
	LMB503	J0161/ILSI-35	1/2a	U.S., 2000	ILSI	13	5.21 ± 0.7	6.34 ± 0.6
	LMB504	H7738/ILSI-36	4b	U.S., 1998-1999	ILSI	12, 21	4.92 ± 1.5	5.52 ± 1.4
	LMB508b	G6054/ILSI-40	1/2b	Illinois, 1994	ILSI	8	0	0
Environmental	LMB429	3276	4b	Cheese	FDA		1.09 ± 1.0	1.12 ± 1.1
	LMB431	15a90	3a	Turkey	FDA		0.96 ± 0.6	1.74 ± 1.1
	LMB437	15c03	1/2a	Avocado	FDA		1.30 ± 1.3	1.52 ± 1.5
	LMB446	3515	4b	Shrimp	FDA		3.20 ± 0.9	4.05 ± 1.1
	LMB582	H7788	1/2a	Hot dog, 1998	CDC		0.71 ± 0.7	1.65 ± 1.0
	LMB622	RM3000	1/2c	Soil	USDA		1.09 ± 1.0	1.03 ± 1.0

^aStrain sources are as follows: Centers for Disease Control and Prevention, Atlanta, Ga. (CDC); International Life Sciences Institute, L. monocytogenes strains collection (http://www.foodscience.cornell.edu/wiedmann/listeriadbase.htm) (ILSI); U.S. Food and Drug Administration, Bothel, Wash. (FDA); and U.S. Department of Agriculture, Albany, Calif. (USDA).

^bStrain LMB508 was associated with an outbreak of noninvasive disease and was not included in the statistical analysis.

^cL.A., Los Angeles, Calif; UK, United Kingdom.

Strain invasiveness.

A greater percentage of mice in groups inoculated with epidemic strains were infected (average, 77%) than were mice inoculated with environmental strains (average, 32%; P < 0.01) (Table 2). The L. monocytogenes counts in the livers and spleens of mice infected with epidemic strains were significantly higher than those of mice inoculated with environmental strains (P < 0.05) (Fig. 1A). Most mice infected with environmental strains had weight gain, while the majority of mice infected with epidemic strains had weight loss (P < 0.05). In addition, the intestines of five mice infected with epidemic strains (two mice with LMB469, one mouse with LMB472, and two mice with LMB504) had extensive hemorrhage. The average bacterial numbers in the livers and spleens of mice with extensive intestinal hemorrhage were significantly higher than those of mice with no hemorrhage (P < 0.05).

TABLE 2.

Percentage of systemically infected mice following oral inoculation with L. monocytogenes strains

Infected organ	% Infected mice inoculated with:
	Epidemic strains						Environmental strains
	LMB469	LMB472	LMB496	LMB498	LMB503	LMB504	LMB429	LMB431	LMB437	LMB446	LMB582	LMB622
Liver	100	67	50	60	100	83	25	33	20	80	20	17
Spleen	100	50	33	60	100	83	25	33	20	80	40	17

FIG. 1.

(A) Epidemic strains were significantly more invasive than environmental strains in both the liver and spleen of 4-week-old Harlan A/J mice (P < 0.05). (B) Serotype 4b strains (n = 7) were generally more invasive than non-serotype 4b strains (n = 5). (C) Taconic mice were significantly more sensitive than Harlan mice to L. monocytogenes infection (P < 0.05). The data are presented as the mean (log₁₀ CFU per gram) ± the standard error.

Mice were not systemically infected following oral inoculation with LMB508, a noninvasive strain that caused an outbreak of human gastroenteritis in 1994 (Table 1) (8). These results suggest that A/J mice can be used to differentiate invasive strains from noninvasive strains.

The relationship between serotype 4b and epidemics led us to hypothesize that serotype 4b strains are more invasive than non-serotype 4b strains. Although the average CFU per gram of liver and spleen was higher for mice infected with 4b strains than those infected with non-4b strains (120 and 12.5 times higher, respectively), the difference was not statistically significant (P = 0.28) (Fig. 1B). Additionally, one environmental 4b strain (LMB446) had a high infection rate, while a different environmental 4b strain (LMB429) had a low infection rate (Table 2). These data suggest that although serotype is related to epidemic outbreaks, it is not the only factor involved in outbreaks.

Susceptibility of mice from different vendors to L. monocytogenes infection.

During the course of the study, it became necessary to order A/J mice from a different vendor (Taconic, Germantown, N.Y.), and a difference in susceptibility of mice from the new vendor was observed. In order to determine if the observed susceptibility difference between mice from different vendors was significant, mice were purchased from both Harlan and Taconic and inoculated orally with 10⁵ CFU (0.1 50% lethal dose) of four epidemic strains (LMB469, LMB472, LMB498, and LMB503) and four environmental strains (LMB437, LMB446, LMB582, and LMB622). Taconic A/J mice were significantly more susceptible than Harlan A/J mice to L. monocytogenes infection (P < 0.05) (Fig. 1C). Additionally, there were no significant differences in invasiveness between epidemic and environmental strains and between 4b and non-4b serotypes in Taconic A/J mice.

Histopathology.

To test the accuracy of the culture system, a subset of Harlan (n = 30) and Taconic (n = 36) A/J mice were examined by histopathology. Livers and spleens with one to three areas of necrosis, four to seven areas of necrosis, or more than eight areas of multifocal necrosis were categorized as mild, moderate, or severe, respectively. The average bacterial numbers in the livers and spleens of A/J mice correlated with the degree of observed necrosis (r = 1.0; P < 0.001). Necrosis in the livers and spleens of Taconic A/J mice was greater than that of Harlan A/J mice (data not shown).

Conclusion.

In summary, 4-week-old Harlan A/J mice were systemically infected after oral inoculation with L. monocytogenes strains with a relatively low dose. Epidemic strains were significantly more invasive as a group than environmental strains and caused extensive hemorrhage in the intestines of some mice. A/J mice are relatively inefficient at mobilizing inflammatory cells, initiate a slower and less exuberant production of inflammatory cytokines, and have diminished anti-Listeria phagocytic activity; however, they are not considered an immunodeficient strain (6, 10, 14). Additionally, the macrophage-mediated immune response of younger mice against L. monocytogenes infection is not fully developed (7, 20). Therefore, it is possible that the 4-week-old A/J mice used in this study were more sensitive to L. monocytogenes infection than the mice used in other studies due age- and strain-related differences. The results of this study suggest that 4-week-old Harlan A/J mice are an appropriate in vivo model for investigating the oral infectivity and invasiveness of L. monocytogenes strains.