Virulence of Listeria Monocytogenes Serovars and Listeria Spp. in Experimental Infection of Mice

917 Journal of Food Protection, Vol. 54, No. 12, Pages 917-921 (December 1991) Copyright©, International Association of Milk, Food and Environmental Sanitarians Virulence of Listeria monocytogenes Serovars and Listeria spp. in Experimental Infection of Mice ALAIN MENLIDIER, CLALIDINE BOSIRAUD*, and JEAN-ALBERT NICOLAS Lahoraioire de Microbiologie, UFR de Pharmacie, 2 rue du Docieur Marcland, 87025 Limoges Cedex, France Downloaded from http://meridian.allenpress.com/jfp/article-pdf/54/12/917/1662574/0362-028x-54_12_917.pdf by guest on 29 September 2021 (Received for publication March 28, 1991) ABSTRACT competence (33), and route of inoculation (24). Differences in virulence between the various Listeria spp. have been Wild strains of Listeria monocytogenes, Listeria ivanovii, indicated by differences in LD50 (7), bacterial count in the Listeria seeligeri, Listeria innocua, and Listeria welshimeri were reticuloendothelial system (79), and various histomorph- isolated from infected animals and foodstuffs. Their virulence was ological features (77). Virulence has also been reported to tested in Swiss mice after intraperitoneal injection of a fixed number of organisms. The presence of hemolysin was determined be associated with the presence of a hemolysin (8,15,31,32). using the CAMP test. Bacteria were enumerated in peritoneal The present study was designed to evaluate the viru lavage fluid, liver, and spleen. Spleen weights were measured, lence of various wild strains of Listeria isolated from and the presence of L. monocytogenes in the brain was also infected animals and foodstuffs. We also compared four investigated. L. innocua, L. seeligeri, and L. welshimeri were not different serovars of L. monocytogenes (4b, l/2a, l/2b, 1/ found to be pathogenic for mice. L. ivanovii was detected in liver, 2c). After intraperitoneal injection into mice, we measured spleen, and peritoneal lavage fluid but at lower levels than L. spleen weight, Listeria count in peritoneal lavage fluid, monocytogenes (p<0.001). The pathogenic capabilities of four liver and spleen, and the presence of organisms in the brain. different serovars of L. monocytogenes (4b, l/2a, l/2b, l/2c) were We also assayed hemolysin. The results demonstrated the compared. Serovars l/2b and l/2c, which are frequently isolated from foodstuffs, were found to colonize the liver and spleen to a importance of serotype of L. monocytogenes in virulence, lesser extent than serovar 4b (p<0.01 and <0.001 respectively). which was in line with the various serovars associated with The behavior of serovar l/2a, the most commonly isolated from human and animal disease. foodstuffs, was strain dependent. Two out of the four strains tested were strongly hemolytic and were as virulent as strains of MATERIALS AND METHODS serovar 4b, while the other two were weakly hemolytic, and avirulent like L. innocua. These results could account for the Hemolysin relatively small number of human Listeria infections due to L. This was assayed using the CAMP test on blood agar base monocytogenes serogroup 1/2, despite the very frequent occur with 5% sheep erythrocytes according to Brzin and Seeliger (4) rence of this serovar in foodstuffs. using Staphylococcus aureus (CIP 5710) and Rhodococcus equi (CIP 5859). Bacterial strains and preparation of inoculum Listeria monocytogenes is now a serious concern in the The characteristics of the strains are listed in Table 1. The food industry, and outbreaks of listeriosis have been asso bacteria were grown on trypticase soy agar (TSA, BBL Microbi ciated with consumption of contaminated coleslaw (28), ology Systems, Cockeysville, MD) and stored at 4°C. Before the experiments, the virulence of the infecting strains was restored by Mexican-style fresh cheese (18), and soft ripened cheese in passage through mice according to the protocol of Mackaness (79) Switzerland (2). Serovar 4b appears to be most frequently and Wirsing von Kbnig et al. (35) which results in increased responsible for human (20,26) and animal (26) listeriosis, lethality. The strains were then cultured in brain heart infusion whereas serogroup 1/2 is the most common serovar isolated broth (Difco Laboratories, Detroit, MI) at 37°C for 18 h. The cells from foodstuffs in our laboratory (23). Listeria innocua, were centrifuged at 6,000 g for 15 min, suspended in sterile L. seeligeri, and L. welshimeri have been assumed to be physiological saline (0.85%), washed twice, and reconstituted in nonpathogenic (25,29), although L. seeligeri was shown to sterile physiological saline to the original volume, giving approxi 9 6 be responsible for a case of meningitis in a susceptible mately 10 CFU/ml. The inocula were adjusted to 2xl0 CFU/ml. individual (27). In contrast, L. ivanovii is generally re Inoculation of the mice garded as pathogenic (3,14,30), although to a lesser degree Female Swiss mice weighing 18 to 20 g were purchased from than L. monocytogenes (13). In the mouse, the pathogenic Elevage Depre (St. Doulchard, France). They were given pelleted ity and virulence of Listeria spp. have been established on chow (Extralabo, Longueville, France) with ad libitum access to the basis of their genetic origin (21), age (70), immuno- water. After a 48 h acclimitization period, the animals were JOURNAL OF FOOD PROTECTION, VOL. 54, DECEMBER 1991 918 MENUDIER, BOSGIRAUD AND NICOLAS TABLE 1. Strains of Listeria spp. tested. Two strains were weakly hemolytic (wh), whereas the other two were strongly hemolytic (sh), like the other strains of L. monocy Strains Species Serovars" Origins togenes, L. ivanovii, and L. seeligeri. 31386 Listeria monocytogenes 4b Trout meat Mortality 362 L. monocytogenes 4b Sheep fetus Good colonization of organs was obtained with a dose of 106 24631 L. monocytogenes l/2b Rib of lamb CFU per mouse even with the most pathogenic strains. In two of 23185 L. monocytogenes l/2b Cheese the experiments, several animals died within 72 h after inocula 3670 L. monocytogenes l/2a Frozen minced meat tion. Inoculation of strain 23185 (serovar 1 /2b) killed one out of 28607 L. monocytogenes- l/2a Cows milk 15 mice, strain 362 (serovar 4b) killed one out of 20 mice in the 2143 L. monocytogenes l/2a Frozen minced meat first set of experiments, and five out of the 15 in the second set. 27795 L. monocytogenes l/2a Frozen minced meat 2141 L. monocytogenes l/2c Frozen minced meat Listeria count in peritoneal lavage fluid 72 h after inoculation 4133 L. monocytogenes l/2c Frozen minced meat Mean number of CFU expressed as log10 Listeria per 0.1 ml PI L. monocytogenes l/2c Frozen minced meat of peritoneal lavage fluid was calculated for each species (Fig.l), 28423 L. monocytogenes l/2c Frozen minced meat each L. monocytogenes serovar (Fig.l), and for each separate Downloaded from http://meridian.allenpress.com/jfp/article-pdf/54/12/917/1662574/0362-028x-54_12_917.pdf by guest on 29 September 2021 94415 L. ivanovii 5 Lambs brain strain (Table 2). L. innocua and L. seeligeri were not found in the 94669 L. ivanovii 5 Sheep fetus peritoneal cavity. L. ivanovii and L. welshimeri were only isolated 55009 L. seeligeri 3b Water in low levels from a few mice. Significant numbers of all strains 26033 L. seeligeri l/2b Milk products of L. monocytogenes were isolated from peritoneal lavage fluid, 20878 L. welshimeri 6a Pork spare rib except for the two serovar l/2a wh strains (2143; 27795) and a 21070 L. welshimeri 6b Pork back rib serovar l/2b strain (24631). For these latter three strains, bacteria 2138 L. innocua 6a Frozen minced meat appeared to be rapidly eliminated from the peritoneal cavity, as was observed with L. innocua, L. seeligeri, L. welshimeri, and L. a Serovars of the wild strains were determined by the Listeria ivanovii. Levels of L. monocytogenes (serovar l/2b) strain 23185 National Reference Center (CHR de Nantes, France). in lavage peritoneal fluid were higher than those of strain 24631, although it belongs to the same serovar (P<0.001; cf. Table. 2). inoculated i.p. with 0.5 ml of inoculum using a 1 ml tuberculin The same result was observed for strain 28423, L. monocytogenes syringe fitted with a 12 gauge (4.5-mm) needle. Control mice (serovar l/2c) and the three other strains belonging to the same were injected i.p. with 0.5 ml of sterile physiological saline. Two serovar. Strain 28423, L. monocytogenes serovar l/2c, was also sets of experiments were carried out with different groups of found in peritoneal lavage fluid at much higher levels than the animals. In the first, nine groups of 20 mice were infected with three other strains belonging to the same serovar (P<0.001; cf. nine different strains: 31386, 362, 3670, 28607, 2143, 27795, Table. 2). The mean numbers of CFUs for L monocytogenes 2141, 4133, and 2138. In the second, 11 groups of 15 mice were serovars l/2c and l/2a wh, L. ivanovii, L. seeligeri, L. welshimeri, infected with 11 different strains: 362, 24631, 23185, PI, 28423, and L. innocua were significantly different from that of serovars 94415, 94669, 55009, 26033 , 20878, and 21070. 4b and l/2a sh (P<0.001; cf. Fig. 1 ) and serovar l/2b (P< 0.05). Bacterial count in organs The surviving mice were killed by cervical dislocation 72 h after inoculation. After injection of 2.0 ml of RPMI 1640 (Labysystems France, Les Ulis, France) into the peritoneal cavity, peritoneal lavage fluid was removed and serially diluted in sterile physiological saline. Appropriate dilutions were plated on TSA. Liver and spleen were removed and weighed under aseptic condi tions. Organs were homogenized in sterile physiological saline in separate sterile glass tissue grinders. The homogenates were plated in 10-fold dilutions on TSA. The plates were incubated at 37°C for 24 to 48 h before counting the colonies.

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