JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1987, p. 2085-20890095-1137/87/112085-05$02.00/0Copyright C 1987, American Society for Microbiology

Vol. 25, No. 11

Pathogenicity Test for Listeria monocytogenes UsingImmunocompromised Mice

G. N. STELMA, JR.,t* A. L. REYES,t J. T. PEELER, D. W. FRANCIS, J. M. HUNT,§ P. L. SPAULDING,C. H. JOHNSON,t AND J. LOVETT

Division of Microbiology, Food and Drug Administration, Cincinnati, Ohio 45226

Received 20 May 1987/Accepted 27 July 1987

The lethality of Listeria isolates was determined with normal adult mice and mice that were im-munocompromised by treatment with 20 mg of carrageenan per kg. The mean 50% lethal doses (LD50s) of thepathogenic isolates were significantly lower (a = 0.05) in' the immunocompromised mice than in the untreatedmice, with an average reduction of 5.8 1glo units. Ini contrast, the mean LD50s of the nonpathogenic isolateswere lowèr in the immunocompromised mice by an average of only 0.4 loglo unit, a differehce that was notsignificant (a = 0.05). Whén immunocompromised mice were used, the LD50s of pathogenic Listeriamonocytogenes isolates were lower than those of nonpathogenic L. innocua and L. seeligeri isolates by '6 loglounits and lower than those of nonpathogenic L. ivanovii isolates by .-41glo units. Pathogenic L. monocytogenesisolates could be distinguished from nonpathogenic isolates by their ability to cause deaths in im-munocompromised mice in 3 days at a dose of -104 CFU per mouse. An alternative procedure usingiron-overloaded mice failed to effectively differentiate pathogenic Listeria isolates.

Listeria monocytogenes is a short, gram-positive, non-sporeforming rod that exhibits a characteristic tumblingmotility in a hanging drop preparation. The organism is afacultative anaerobe and is catalase positive (2). L. monocy-togenes has been cultured from a variety of natural environ-ments, including soil, vegetation, and water (12, 39). It hasalso been found in association with disease in fish, birds, anda variety of mammals, including humans (3, 8, 10-12).Most human infections fall into one of the following

categories: (i) neonatal sepsis or meningitis; (ii) sepsis ormeningitis in immunocompromised patients; or (iii) sepsis ornonspecific flulike illness in healthy women during preg-nancy, which can lead to infection of the fetus (2). Condi-tions associated with Listeria infection in nonpregnant adultsinclude diabetes, alcoholism, cancer, and treatment withcorticosteroids or other immunosuppressive drugs (2, 11).The mode of acquisition of L. monocytogenes from theenvironment is often unclear. Transplacental infection anddirect acquisition from the vaginal canal are thought to causeperinatal infections. However, the source of infection of themother is often difficult to identify (8, 30). Recently someprogress has been made, with the identification of commer-cial food products as the transmission vehicles in threeoutbreaks of listeriosis (3, 10, 30).When outbreaks of human listeriosis occur, it is important

for clinicians to be able to verify the pathogenicity ofassociated isolates, particularly isolates from the suspectedvehicle of infection. Hemolytic activity correlates with thepathogenicity of L. monocytogenes (13, 26, 33, 40). How-ever, hemnolysis is frequently weak and hemolysis tests often

* Corresponding author.t Present address: Division of Toxicology and Microbiology,

Health Effects Research Laboratory, U.S. Environmental Protec-tion Agency, Cincinnati, OH 45268.

t Present address: Environmental Monitoring and Support Labo-ratory, U.S. Environmental Protection Agency, Cincinnati, OH45268.

§ Present address: U.S. Food and Drug Administration, Cincin-nati, OH 45202.

yield ambiguous results (7). Even the CAMP test (4), whichpotentiates the hemolytic activity of L. monocytogenes, isinconclusive, and interpretations are subjective. Conse-quently, animal inoculations (28) are important in confirmingthe pathogenicity of isolates.

While screening environmental Listeria isolates for mousepathogenicity, we noted that our negative control organism,an avirulent L. innocua strain, occasionally caused deaths atdoses of approximately 109 CFU per mouse. This observa-tion suggested that (in normal mice) the lethal doses of someavirulent strains may be relatively close to those of virulentstrains and that the mouse test is not completely reliable.

Differences between virulent and avirulent isolates ofother opportunistic pathogens have been more readily dis-cernible when lethality tests were performed in compro-mised animals (15) (A. L. Reyes, J. T. Peeler, C. H.Johnson, P. L. Spaulding, and G. N. Stelma, Jr., Abstr.Annu. Meet. Am. Soc. Microbiol. 1986 P2, p. 275). There-fore, a bioassay in compromised mice appeared to be apotential niethod to differentiate virulent and avirulentListeria isolates without the uncertainties of the CAMP andmouse lethality tests. In this report, we describe the devel-opment of a pathogenicity test for L. monocytogenes whichuses mice treated with carrageenan (CG) to block macro-phage function (35, 36). In this test the 50% lethal doses(LD50s) of the virulent L. monocytogenes were reduced fromthose observed with normal mice by 25 loglo units, whereasthe LD50s of avirulent isolates were reduced only by <1.2log1o units. Preliminary results suggest that the method alsoidentifies virulent isolates of L. ivanovii.

MATERIALS AND METHODSListeria isolates. The sources of the strains used in this

study are given in Table 1. Strains that originated in ourlaboratory were isolated from raw milk or cheese by themethod described by Lovett et al. (16). The Listeria isolateswere confirmed by morphology, tumbling motility, Gramstain, catalase production, hemolytic activity on horse bloodagar, and API 20S (Analytab Products) profile. Species wereidentified by testing for nitrate reduction, urease production,

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TABLE 1. Results of CAMP test, mouse pathogenicity test, and LD50 determinations in normal andimmunocompromised mice for 11 Listeria isolates

Result of test LD50Strain Origin Source' CAMP Mouse Untreated Immunocompromised

pathogenicity" mice micee

L. monocytogenes Scott A Clinical CDC S+ + 3.5 x 107 57fL. monocytogenes V7 Raw milk CDC S + + 6.3 x 106 l6fL. monocytogenes Brie 1 Brie cheese FDA-DM S+ + 1.9 x 107 25fL. monocytogenes Murray B Clinical CDC S+ + 2.0 x 107 lofL. monocxytogenes V37 CE Raw milk CDC S+ + 1.1 x 106 6.5fL. innocua 2478KD Raw milk FDA-DM - - 4.7 x 109 3.0 x 108L. innoc-ua 2498A Raw milk FDA-DM - - 2.2 x 109 1.5 x 109L. innocua LA-1 Cheese FDA-LA - - 6.1 x 109 4.4 x 109L. ivanovii KC1714 Unknown CDC R+ + 3.8 x 108 7.3 x 106fL. ivanovii LA-30 Brie cheese FDA-LA R+ + 1.1 x 108 7.1 x 106L. seeligeri LA-15 Cheese FDA-LA S+ - 3.8 x 109 1.4 x 109

'CDC, Centers for Disease Control, Atlanta, Ga.; FDA-DM, Division of Microbiology, Food and Drug Administration, Cincinnati, Ohio; FDA-LA, Food andDrug Administration District Laboratory, Los Angeles, Calif.

h Geometric mean (CFU per 20-g mouse) of three separate determinations.S +, Double zone of hemolysis adjacent to S. aureus; R +, double zone of hemolysis adjacent to R. eqai; -, no hemolysis.

"Strains were considered lethal if at least three of five mice injected i.p. with 109 CFU died within 1 week.'Inoculated i.p. with 200 mg of CG per kg at -24 h.f'Significantly different (oa = 0.05).

H2S production (triple sugar iron), methyl red-Voges-Proskauer reaction, and ability to ferment, with acid produc-tion, dextrose, esculin, maltose, mannitol, rhamnose, andxylose (2). The CAMP test (4, 13) was used to distinguish L.innocua and L. ivanovii from L. monocytogenes.Mouse pathogenicity test. Initially, pathogenicity was de-

termined by a modification of the method of Ralovich (28).Isolates were grown for 24 h at 35°C in Trypticase soy broth(BBL Microbiology Systems) plus 0.6% yeast extract. Cul-tures were concentrated 10-fold by centrifugation and sus-

pended in 0.1% peptone. Five 16- to 18-g Swiss White micewere each given 0.1 ml of the suspension, containing 109cells, intraperitoneally (i.p.). The mice were observed for 1week, and deaths were recorded. Strains that killed three ormore mice were considered to be pathogenic. In eachexperiment, control mice were injected with known patho-genic and nonpathogenic strains; experiments in whicheither set of controls gave inappropriate results were disre-garded. This occurred in approximately 10% of the experi-ments.

Pathogenicity in compromised mice. CG (Sigma type II)was dissolved in distilled water and injected i.p. into 18- to20-g mice (200 mg/kg) 24 h before challenge (35). Bacterialcultures were grown as described in the previous section.The mice were challenged by i.p. injections of 10-fold serialdilutions of washed cells suspended in phosphate-bufferedsaline (pH 7.3). Control mice received CG, followed byphosphate-buffered saline after 24 h. The mice were ob-served for mortality over 5 days. For comparison, lethalitieswere also determined in untreated mice and in mice thatwere overloaded with iron by intramuscular injection of 250mg of iron dextran (Carter-Glagau Laboratories, Inc.) (15)per kg 2 h before infection. In the rapid procedure, CG-treated mice were inoculated with i104 CFU of the teststrain and held for 3 days. Controls consisted of groups offive mice treated with CG only, CG plus 104 L. innocua, or

CG plus 104 L. monocytogenes.Statistical methods. The LD5,s were estimated by the

method of Spearman and Karber (9). Groups of five miceeach were tested at five or more inoculum levels. Three trialswere run with mice treated with CG and with untreatedmice. Duplicate trials were run with iron-overloaded mice.

Since loglo LD50 values were assumed to be normallydistributed, the treatments were compared by computing ananalysis of variance (27).

RESULTS

Species identification. The critical tests used to identify thespecies of each Listeria isolate were fermentation ofrhamnose and xylose, the CAMP test, and the mousepathogenicity test. Isolates that produced acid from rham-nose but not xylose and had a positive CAMP test (enhancedzone of hemolysis) with Staphylococcus aureus and a posi-tive test for mouse pathogenicity were identified as L.monocytogenes (2). L. seeligeri isolates were distinguishedfrom L. monocytogenes by their ability to produce acid fromxylose but not rhamnose and their lack of pathogenicity formice (2). L. innocua isolates were distinguished by theirnegative reactions in the CAMP and mouse pathogenicitytests (2), and L. ivanovii isolates were identified by theirability to produce a positive CAMP reaction with Rhodo-coccus equi (31). All isolates chosen for this study producedunambiguous results in the CAMP test.

At the time of isolation, one of the isolates (2478KD) washemolytic on agar containing 5% horse blood but negativefor mouse pathogenicity. Subsequently, a negative CAMPtest was observed, and the isolate was designated as L.innocua. The species, origin, and other important character-istics of all of the isolates used in the development of thepathogenicity test are listed in Table 1.Development of pathogenicity test. The LD50 of all of the

isolates were estimated in untreated mice and in mice thatwere immunocompromised by treatment with CG (35). TheLD50s (Table 1) are the geometric means of three separatedeterminations. The average LD50s of the pathogenic L.inonocYtogenes were consistently lower than those of thenonpathogenic L. innocua and L. seeligeri. Considerablevariability was observed within the two groups, however,and the highest LD50s of pathogenic strains nearly over-lapped the lowest LD5Os of nonpathogenic strains (Table 2).When immunocompromised mice were used, the lethalitiesof the pathogenic strains were greatly enhanced (Tables 1and 2). The mean LD50s of the pathogenic Listeria strains

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TABLE 2. The range of LD50s' observed for five pathogenic strains of L. monocytogenes, four nonpathogenic strainsof L. innocua and L. seeligeri, and two strains of L. ivanovii

LD50oTreatment

L. monocytogenes L. innocua and L. seeligeri L. iv'anovii

None 1.2 x 108-2.6 x 105 1.0 x 1010-5.2 x 108 8.2 x 108-3.5 x 107CG (200 mg/kg) 1.1 x 102-3.7 x 100 5.1 x 109-1.3 x 108 1.9 x i07-i.1 X 106

a LD50s represent individual data points rather than geometric mean shown in Table 1.

were reduced by 5.2 to 6.3 log1o units, averaging 5.8 log1ounits. These reductions were statistically significant (a =

0.05). In contrast, the mean LD50s of the nonpathogenicstrains were reduced only by 0.1 to 1.2 log1o units in theimmunocompromised mice, averaging 0.4 log1o unit, a dif-ference that was not significant (ax = 0.05). In immuno-compromised mice the LD50s of pathogenic L. monocyto-genes were at least 6.0 log1o units below the lowest LD50observed for any nonpathogenic L. innocua or L. seeligeri.The two L. ivanovii strains were both positive in the

mouse pathogenicity test (Table 1). However, the results ofLD50 determinations in immunocompromised mice indicatedthat neither strain was pathogenic (Table 1). Although theLD50 of strain KC1714 was reduced significantly (at = 0.05)by the CG treatment, the mean LD50 was greater than 6.0log1o units, a much higher dose than would be expected foran opportunistic pathogen.The effect of iron overload on the pathogenicity of Listeria

strains was tested in an effort to simplify and possiblyshorten the bioassay procedure. The results observed withfive pathogenic L. monocytogenes strains (Table 3) indicatedthat iron overload significantly (a = 0.05) enhanced thelethality of only three of the strains and that the LD50s of thethree strains were lowered only by s2.0 log1o units.Rapid screening of Listeria isolates. Although the LD50

determinations required 5 days from the time of inoculationof the organisms until the completion of the experiment, itwas observed that immunocompromised mice inoculatedwith 103 to 104 CFU of pathogenic Listeria isolates died onday 3. Because nonpathogenic isolates were never observedto cause deaths at these low doses, a pathogenicity test inwhich immunocompromised mice were inoculated with ap-proximately 103 to 104 CFU and held for 3 days appeared tobe feasible. Seven additional isolates were tested by thisprocedure. The isolates chosen were (i) a strain of L.monocytogenes that was CAMP positive but negative forpathogenicity in normal mice, (ii) two isolates from cheesethat were negative in the CAMP and mouse pathogenicitytests, (iii) two isolates from cheese that were positive in theCAMP and mouse pathogenicity tests, and (iv) two L.ivanovii isolates that were pathogenic to normal mice. The

TABLE 3. Effect of iron overload on virulence ofL. monocytogenes isolates

LDOaStrain

Normal mice Iron-overloaded micel

Murray B 2.0 x 107 4.7 x 105<Brie 1 1.9 x 107 2.2 x 105'V37CE 1.1 x 106 8.4 x 105<V7 6.3 x 106 1.2 x 106Scott A 3.5 x 107 6.0 x 106

a Expressed as CFU per 20-g mouse.b Injected i.p. with 250 mg of iron dextran per kg of body weight.c Significantly different (et = 0.05).

LD50s were also determined to verify the results of the 3-daypathogenicity test. The results (Table 4) showed that onlythe two L. monocytogenes isolates that were CAMP positiveand pathogenic to normal mice caused deaths in 3 days at adose of 104 CFU per mouse. The other five strains werenonpathogenic by this test. The LD50 determinations (Table4) confirmed the results of the 3-day test.

DISCUSSION

An unambiguous and rapid procedure for the identificationof potentially pathogenic Listeria strains is needed becauseof difficulties in interpreting the results of the CAMP andmouse pathogenicity tests, along with the requirement ofwaiting a full week for mouse pathogenicity test results. Thefirst step in the development of such a procedure was tochoose an animal model in which illness mimics that inhumans.The type of illness caused by pathogenic L. monocyto-

genes varies. In some species it attacks the finest andstrongest of the herd or flock; in humans, however, it usuallyattacks the unborn, the very young, or those weakened byother disorders (1, 12). The mouse was chosen as the animalmodel because of its similarities to humans in diseasemanifestations and the susceptible population. Neonate (25)and immunocompromised (24, 36) mice are highly suscepti-ble to infection by L. monocytogenes, and with one excep-tion (37), studies performed with pregnant mice have asso-ciated pregnancy with an increased risk of infection (14, 17,22, 29) and subsequent fetal loss (17, 38). In contrast, the ratprobably is not an appropriate model since pregnant ratsinfected with L. monocytogenes have been observed todeliver large litters of healthy pups (6). This suggests that therat pups were highly resistant to L. monocytogenes, eventhough they were not immunocompetent.The protection of mice against L. monocytogenes is de-

pendent upon macrophages and T cells (18, 19, 21), withoutthe involvement of antibody (20) or polymorphonuclear cells(36). Therefore, an effective immunocompromised mousemodel requires the impairment of either macrophage orT-cell function. Since infection of nude mice, which lackT-cell function, has been shown to result in prolongedinfections without death (23), impairment of macrophagefunction was deemed more appropriate for a rapid pathoge-nicity test with death as the endpoint. CG was selected as themacrophage-suppressing agent because of its ability to causean eightfold decrease in the number of macrophages inmouse peripheral blood within 2 days and because of itslong-lasting effect, with full recovery requiring 15 days (36).Cortisone acetate has also been shown to suppress cellularimmunity and to increase the susceptibility of mice toListeria infection (24). However, its effect waned rapidlyafter 24 h (24), indicating that it would probably not beeffective for the duration of the infection.The advantages of using immunocompromised mice for

determining the lethality of Listeria isolates can be seen by

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TABLE 4. Ability to cause death in 3 days at a dose of 104 CFU per immunocompromised mouse as a measureof pathogenicity of Listeria isolates

Activity in immunocompromiseda miceResult of test:

Cumulative deathsStrain SourceMouse LD5,e Pathogenicity

CAMPh pathogenicity( Day Day 2 Day 3

L. monocytogenes ATCC 15313 Rabbit + - 0 0 0 1.9 x 109L. innocua D15 Cheese - - 0 0 0 7.6 x 109L. innocua D18 Cheese - - 0 0 0 5.4 x 109L. monocytogenes MC147 Cheese S+ + 0 0 3 45 +L. monocytogenes MC148 Cheese S+ + 0 0 4 14 +L. ivanovii 4827KA Raw miik R+ + 0 0 0 1.1 x 109L. ivanovii 4827KB Raw milk R + + 0 0 0 2.0 x 108

a Treated with 200 mg of CG 24 h before challenge.b S +, Double zone of hemolysis adjacent to S. aureus; R +, double zone of hemolysis adjacent to R. equi; -, no hemolysis.'Strains were considered pathogenic if at least three of five mice injected i.p. with 109 CFU died within 1 week.d Cumulative deaths in groups of five mice challenged with approximately 104 CFU.e CFU per 20-g mouse.

examining the data shown in Tables 1 and 2. When untreatedmice were used, the geometric means and all of the individ-ual LD50s of the pathogenic L. monocytogenes strains Werelower than those of the nonpathogenic L. innocua and L.seeligeri strains. The differences were sometimes small, andthe LD50 of one L. innocua isolate was as low as 5.2 x 108CFU (Table 2). If the standard mouse pathogenicity test hadbeen used on that occasion, the result would have beenfalse-positive. When immunocompromised mice were used,however, all of thé LD50s of the pathogenic L. monocyto-genes strains were -1.1 x 102 CFU, whereas those of thenonpathogenic L. innocua and L. seeligeri strains were .1.3x 108 CFU (Table 2), a difference of 6 log1o units. Thisdifference between the LD50s of pathogenic and nonpatho-genic strains is so great that false-positive or false-negativetests are unlikely.

In addition, the use of imtnunocompromised mice im-proved the reliability of the in vivo pathogenicity test. Thevariance for the LD50 determinations was 0.18311 when theimmunocompromised mice were used, compared with0.33291 when untreated mice were used.Because L. ivanôvii has occasionally been associated with

human infection (32), two isolates were included in the LD50determinations. Both appeared to be pathogenic by thestandard mouse test (Table 1). However, in CG-treated miceneither strain was lethal at low doses, suggesting that patho-genicity tests in normal mice may frequently yield false-positive results.On the basis of reports that administering iron compounds

to mice significantly reduced the LD50 of a pathogenic strainof L. monocytogenes (34) and that virulent strains of L.monocytogenes exhibited faster growth rates as a function ofiron concentration than did avirulent strains (5), we investi-gatéd the effects of iron overload on our Listeria isolates.Pretreatment of the mice with iron dextran caused a signif-icant lowering of the LD50 of three of five pathogenicisolates; however, the differences were small (-2 log10units). Therefore, we concluded that the iron-overloadmouse model would not be useful for determining thepathogenicity of Listeria isolates. Our data differ from thoseof Sword (34), who reported that iron effectively enhancedthe virulence of L. monocytogenes. It should be noted thatSword tested only one pathogenic isolate, with an exception-ally low LD50 (2.5 x 103), in untreated mice (34).The hypothesis that pathogenic Listeria isolates can be

identified 3 days after injection of immunocompromised

mice with approximately 104 CFU is supported by the datashown in Table 4. The two L. monocytogenes isolates thatwere CAMP positive and pathogenic to normal mice causeddeaths within 3 days; the two isolates that were CAMPnegative and not pathogenic to normal mice did not causedeaths. The data obtained with the other three isolatessuggest that the in vivo test using immunocompromised micecan differentiate isolates for which ambiguous results areobtained by the other two methods (Table 4). L. monocy-togenes ATCC 15313, which would have been deemedpathogenic if tested only by the CAMP procedure butnonpathogenic if tested only for mouse pathogenicity, wasnonpathogenic by thé 3-day test. The absence of pathoge-nicity of that organism was confirmed by the observationthat its LD50 was 1.9 x 109 in immunocompromised mice.The two L. ivanovii isolates were both pathogenic to normalmice but were not pathogenic in the 3-day test in im-munocompromised mice. The LD50s of these isolates were1.1 x 109 and 2.0 x 108 CFU in immunocompromised mice,confirming their lack of pathogenicity. The results obtainedwith the four L. ivanovii strains we have tésted suggest thatL. ivanovii is not a pathogenic species even though it killsnormal mice at -109 CFU. However, additional isolates,including strains implicated in human illness, will be studiedto substantiate these observations.

ACKNOWLEDGMENTS

We thank Robert M. Twedt for reviewing the manuscript andDiana Redmond for typing the manuscript.

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