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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. Results were expressed as the mean + SEM log10 viable Listeria per 0.1 ml of peritoneal lavage fluid and per gram of organ (10 to 20 mice per strain, and 30 to 70 mice per serovar of L. monocytogenes or per 4b 1/2b 1/2a 1/2a 1/2c L.ivan L.seel L.wels Linn Listeria species). In the second set of experiments, brains were Listeria monocvtoeenes removed under aseptic conditions and homogenized in 2.0 ml of tryptose broth (BBL Microbiology Systems); 0.1-ml aliquots of Figure 1. Presence of L. ivanovii (L. ivan), L. seeligeri (L. seel), the homogenates were plated on TSA and incubated for 24 to 48 L. welshimeri (L. wels), L. innocua (L. inn) and the various h at 37°C. One ml was then enriched in 9.0 ml of tryptose broth serovars of L. monocytogenes in peritoneal lavage fluid 72 h for 7 d at 30°C. The percentage of homogenates positive for 6 Listeria was then determined. after an i.p. injection of 10 CFU per mouse. L. monocytogenes serovar 112a is split into two groups: strongly hemolytic (sh), weakly hemolytic (wh). Results are expressed as the mean ±_ SEM Statistical analysis CFU of Listeria per 0.1 ml of peritoneal lavage fluid (30 to 70 Differences between the means were compared using Student's mice per group) for each serovar and species. *** indicates a t-test after analysis of variance. Differences with a P value of less than 0.05 were regarded as significant. significant difference with respect to L. monocytogenes serovar 4b (P<0.001). RESULTS Spleen weight Hemolysin Infection with most of the strains of L. monocytogenes led to It can be seen from the results in Table 2, that the four wild a significant increase in spleen weight 72 h after injection (Table strains of L. monocytogenes serovar l/2a gave different results. 2). In contrast, spleen weights of animals infected with L. innocua

JOURNAL OF FOOD PROTECTION, VOL. 54, DECEMBER 1991 VIRULENCE OF LISTERIA ON MICE 919 TABLE 2. Effects of intraperitoneal injection of 106 CFU of the various strains of Listeria.

Peritoneal lavage fluid Spleen Liver Strains Species Serovar Hemolysin Positive Bacterial count3 Weight" Positive Bacterial counf Positive Bacterial counf % % %

31386" Listeria monocytogenes 4b + 80 1.91 +0.31 0.194 ± 0.012k 100 6.80 + 0.19 100 5.91 ± 0.22 362" L. monocytogenes 4b + 79 2.34 ± 0.34 0.176 ± 0.008' 95 6.89 ± 0.27 95 6.56 + 0.24 362" L. monocytogenes 4b + 90 3.50 ± 0.74 0.157 ± 0.024 100 7.70 ± 0.49 100 6.66 + 0.51 24631" L. monocytogenes l/2b + 20 0.64 ± 0.38" 0.209 ± 0.018J 100 5.12 ± .023" 100 3.65 + 0.42" 23185" L. monocytogenes l/2b + 93 3.19 ± 0.49 0.193 + 0.017' 100 7.09 ± 0.29 100 6.31 ± 0.29 3670" L. monocytogenes l/2a + 100 2.50 ± 0.25 0.191 + Q.011' 100 6.93 ± 0.21 100 5.91 ± 0.23 28607" L. monocytogenes l/2a + 95 2.23 + 0.20 0.184 ±0.011' 100 7.00 + 0.29 100 6.01 ± 0.31 2143" L. monocytogenes l/2a + (weakly) 0 0" 0.133 ± 0.007 0 0" 0 0" 27795" L. monocytogenes l/2a + (weakly) 0 0" 0.116 ±0.006k 10 0.27 ±0.19" 5 0.11 +0.11" 2141" L. monocytogenes l/2c + 95 1.12 + 0.18" 0.190 ± 0.014k 95 5.24 ± 0.32E 95 3.93 ± 0.308 4133" L. monocytogenes l/2c + 70 0.99 ± 0.20" 0.183 ±0.011k 100 5.61 ± 0.16" 100 4.33 ± 0.22s PI" L. monocytogenes l/2c + 67 1.02 ± 0.25" 0.191 ± 0.016k 100 5.99 ± 0.20f 100 4.27 + 0.19s 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 28423" L. monocytogenes l/2c + 93 2.61 + 0.28 0.172 ± 0.019 100 6.68 ± 0.24 100 5.28 ± 0.24 94415" L. ivanovii 5 + 7 0.07 ± 0.07 0.162 ± 0.011 27 0.72 ± 0.32" 47 1.43 ± 0.45 94669" L. ivanovii 5 + 33 0.63 ± 0.29 0.151 ± 0.009 87 3.58 ± 0.46 67 1.79 ± 0.35 55009" L. seeligeri 3b + 0 0 0.161 ± 0.009 0 0 0 0 26033" L. seeligeri l/2b + 0 0 0.157 ± 0.008 0 0 0 0 20878" L. welshimeri 6a - 27 0.58 + 0.28 0.135 + 0.011 0 0 0 0 21070" L. welshimeri 6b - 20 0.29 ±0.17 0.150 ± 0.006 0 0 7 0.15 + 0.15 2138" L. innocua 6a - 0 0 0.118 ±0.007' 5 0.13 ±0.131 10 0.24 ±0.16 Control 0 0 0.142 + 0.007 0 0 0 0

" Mean ± SEM log10 CFU Listeria/Q.l ml of peritoneal lavage fluid. b Mean + SEM per gram spleen. c Mean + SEM log10 CFU Listeria/g organ (liver or spleen). " Strains used in first set of experiments. " Strains used in second set of experiments. ' P<0.05 with respect to highest mean obtained with strain belonging to same serovar. B P<0.01 with respect to highest mean obtained with strain belonging to same serovar. " P<0.001 with respect to highest mean obtained with strain belonging to same serovar. ' P< 0.05 with respect to controls. ' P<0.01 with respect to controls. k P<0.001 with respect to controls. and L. monocytogenes serovar l/2a wh were less than that of Listeria count in liver and spleen controls (Table 2). In Fig. 2, it can be seen that inoculation of the First, we studied the percentage of organs colonized in each two pathogenic species of Listeria, L. ivanovii and L. monocyto­ group of mice. L. monocytogenes were found in at least 95% of genes, except serovar l/2a wh, led to a significant increase in organs, except for the two serovar l/2a wh strains. L. ivanovii spleen weight. was found to varying extents depending on the strain and organ. For the other species of Listeria considered to be nonpathogenic 0,25 (31), the percentage was low or undetectable (Table 2). Second, because the bacterial count in the reticuloendothelial system 72 h after inoculation provides an indication of the virulence of the particular strain, we compared the extent of colonization of liver and spleen between all strains. Apart from strain 94415 of L. ivanovii and the strains of L. welshimeri, spleens were consis­ tently better colonized than livers (Table 2). Third, we demon­ strated differences in virulence between strains belonging to the same serovar, i.e., L. monocytogenes strain 23185 (serovar l/2b) colonized more organs than did strain 24631 (P<0.001). The two IshB I wh| serovar l/2a wh strains were found in negligible amounts in the 0,10 4b 1/2b 1/2a 1/2a 1/2c L.ivan L.seel L.wels Linn control liver and spleen in contrast to the two sh strains. For L. ivanovii, strain 94669 was more frequently isolated from the liver and Listeria monocytogenes spleen than strain 94415 (Table 2). Generally, three levels of colonization could be distinguished (Fig. 3a and 3b): i) high Figure 2. Effects of L. ivanovii (L. ivan), L. seeligeri (L. seel), levels of bacteria in organs (L. monocytogenes serovars 4b, l/2b, L. welshimeri (L. wels), L. innocua (L. inn) and the various serovars of L. monocytogenes 72 h after an i.p. injection of 106 l/2a sh, l/2c); ii) moderate colonization (e.g., L. ivanovii); and CFU per mouse on weight of spleen. L. monocytogenes serovar iii) little or no colonization (e.g., L. monocytogenes l/2a wh, L. 112a is split into two groups: strongly hemolytic (sh) and weakly seeligeri, L. welshimeri, L innocua). Thus, it can be seen that 6 hemolytic (wh). Results are expressed as the mean ± SEM per g with an initial inoculation dose of 10 CFU of Listeria per mouse, of spleen (30 to 70 mice per group) for each serovar and species. some bacterial strains multiplied to an extent that was propor­ *,** and *** indicate significant differences with respect to tional to their virulence, whereas other bacteria were almost controls (P<0.05, 0.01, and 0.001, respectively). completely eliminated.

JOURNAL OF FOOD PROTECTION, VOL. 54, DECEMBER 1991 920 MENUDIER, BOSGIRAUD AND NICOLAS DISCUSSION

> In this study, we demonstrated differences in virulence o between different species and different serovars of L. mono­ S < cytogenes after injection of a fixed dose prepared from 04 a various wild strains isolated in our laboratory. Virulence Bi W 0. was expressed by the existence of a hemolysin, an increase < in spleen weight, and a high bacterial count in peritoneal lavage fluid, liver, spleen and brain. In agreement with the results of Wirsing von Konig et al. (35), L. monocytogenes o serovar 4b was found to be the most virulent. For this o serovar, strain 31386 isolated from a foodstuff was as 4b 1/2b 1/2a 1/2a 1/2c L.ivan L.seel L.wels Linn virulent as strain 362 isolated from an infected animal. In Listeria monocytogenes contrast and in agreement with the findings of Wirsing von

Figure 3a. Bacterial count of L. ivanovii (L. ivan), L. seeligeri 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 Konig et al. (35), we found that serovar l/2b was less (L. seel), L. welshimeri (L. wels), L. innocua (L. inn) and the virulent than serovar 4b (P<0.01; cf. Fig. 3a, 3b). However, various serovars of L. monocytogenes 72 h after an i.p. injection organ colonization was more extensive for strain 23185 of 106 CFU per mouse in liver. L. monocytogenes serovar 112a (serovar l/2b) than for strain 24631 and equal to the two is split into two groups: strongly hemolytic (sh) and weakly hemolytic (wh). Results are expressed as the mean ± SEM per g strains belonging to the serovar 4b. The four strains of of liver (30 to 70 mice per group) for each serovar and species. serovar l/2a could be split into two groups. The two ** and *** indicate significant differences with respect to value strongly hemolytic strains, 3670 and 28607, were as viru­ for L. monocytogenes serovar 4b at P<0.01 and 0.001, respec­ lent as the strains of serovar 4b. Strain 3670 was isolated tively. from frozen minced meat and strain 28607 from the milk of a cow with no apparent signs of mastitis. The milk compo­ sition was normal apart from a high leucocyte count. Gitter et al. (9) has previously described a cow with a severe mastitis and producing infected milk darker in color than normal milk and flaky as well. Our study gives an indica­ tion of the possible risk of contamination in the dairy industry from healthy carriers. On the other hand, the two weakly hemolytic strains, 27795 and 2143, were not de­ tected in peritoneal lavage fluid, and were only occasion­ ally detected in the liver and spleen. Hof (12) has reported the existence of a nonhemolytic and avirulent strain of serovar l/2a. In our laboratory, 11 out of 23 wild strains of 1/2b 1/2a 1/2a 1/2c L.ivan L.seel L.wels L.inn serovar l/2a isolated from foodstuffs were weakly hemo­ Listeria monocytogenes lytic. The four wild strains of serovar l/2c were all isolated Figure 3b. Bacterial count of L. ivanovii (L. ivan), L. seeligeri from meat products. They were significantly less virulent in (L. seel), L. welshimeri (L. wels), L. innocua (L. inn) and the the mouse than serovar 4b. However, strain 28423 viru­ various serovars of L. monocytogenes 72 h after an i.p. injection lence was higher than the other three strains belonging to of 106 CFU per mouse in spleen. L. monocytogenes serovar 112a the same serovar and equal to serovar 4b strains. is split into two groups: strongly hemolytic (sh) and weakly In accordance with well accepted clinical evidence and hemolytic (wh). Results are expressed as the mean ±_ SEM per g in agreement with the findings of Hof et al. (13) who of spleen (30 to 70 mice per group) for each serovar and species. utilized i.v. injection in the mouse, our results show that ** and *** indicate significant differences with respect to value for L. monocytogenes serovar 4b at P<0.01 and 0.001, respec­ wild strains of L. ivanovii were much less virulent than tively. most strains of L. monocytogenes. Differences in virulence between these two species have also been observed in vitro by some workers. Although they are both cytotoxic (6) and Listeria count in the brain able to invade nonphagocytic cells like Caco-2 (7), only L. L. ivanovii, L. seeligeri, and L. welshimeri were not detected monocytogenes is able to penetrate mouse embryo fibro­ in the brain even after 7 d of incubation at 30°C. All five strains blast cells (cell line 3T6) (76). Del Corral et al. (5) showed of L. monocytogenes were isolated, albeit to varying extents. that L. ivanovii is a nonpathogenic species for mice and Serovar 4b was found in 60% of homogenates on agar and in 80% appears to induce microfilament projection to a lesser after enrichment. The presence of serovar 1/2b depended on the extent than L. monocytogenes in Hep-2 cells. Mounier et particular strain. Strain 23185 was isolated in 50% of cases on agar and 57% of cases after enrichment. However, strain 24631 al. (22) have previously concluded that interaction with was only isolated after enrichment in one out of 15 homogenates. host filaments may represent a major virulence factor for L. The two serovar l/2c strains were detected in 13% of brain monocytogenes. This species utilizes the host cell samples cultured on agar. After enrichment, strain PI was cytoskeleton to promote intacellular movement (34) and isolated in a third of the samples, and strain 28423 in two-thirds Kuhn et al. (17) found one mutant which does not move of the samples. These results provide a further indication of the intracellularly and is significantly less virulent than the tropism of L. monocytogenes for the brain .

JOURNAL OF FOOD PROTECTION, VOL. 54, DECEMBER 1991 VIRULENCE OF LISTERIA ON MICE 921 parental strain althought it is still invasive and hemolytic. 16. Kuhn, M., S. Kathariou, and W. Goebel. 1988. Hemolysin supports The tests for virulence that we employed confirmed the survival but not entry of the intracellular bacterium Listeria mono­ cytogenes. Infect. Immun. 56:79-82. nonvirulence of L. innocua, L. welshimeri, and L. seeligeri 17. Kuhn, M„ M. C. Prevost, J. Mounier, and P. J. Sansonetti. 1990. A despite the existence of a hemolysin in the latter species nonvirulent mutant of Listeria monocytogenes does not move intra- (31). In fact there was good agreement between the various cellularly but still induces polymerization of actin. Infect. Immun. tests we employed. The virulence of Listeria was also 58:3477-3486. 18. Linnan, M. J., L. Mascola, D. L. Xiao, V. Goulet, S. May, C. found to depend on serovar. Although serovar 4b of L. Salimen, D. W. Hird, M. L. Yonekura, P. Hayes, R. Weaver, A. monocytogenes is the most virulent in human and animal Audurier, B. D. Plikatis, S. L. Fannin, A. Kleks, and C. V. Broome. disease, it is only rarely isolated from foodstuffs, in con­ 1988. Epidemic listeriosis associated with Mexican-style cheese. N. trast to the more common serovars l/2a and l/2c (23). The Engl. J. Med. 319:823-828. 19. Mackaness, G. B. 1962. Cellular resistance to infection. J, Exp. existence of avirulent serovars l/2a wh strains may account Med. 116:381-406. for the infrequent involvement of this serovar in human 20. McLauchlin, J. 1990. Distribution of serovars of Listeria monocyto­ disease. Regarding serovar l/2c, we found it to be less genes isolated from different categories of patients with listeriosis. virulent than serovar 4b, which may help explain its rela­ Eur. J. Clin. Microbiol. Infect. Dis. 9:210-213. tively low involvement in human listeriosis (20). 21. Mainou-Fowler, T., A. P. Macgowan, and R. Postlethwaite. 1988. 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JOURNAL OF FOOD PROTECTION, VOL. 54, DECEMBER 1991