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Listeria monocytogenes: comparative interpretation of mouse virulence assay
FEMS Microbiology Letters 233 (2004) 159–164 www.fems-microbiology.org
Listeria monocytogenes: comparative interpretation of mouse virulence assay Dongyou Liu
*
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, P.O. Box 6100, Mississippi State, MS 39762, USA Received 9 January 2004; received in revised form 4 February 2004; accepted 6 February 2004 First published online 21 February 2004
Abstract Being an opportunistic bacterial pathogen, Listeria monocytogenes demonstrates significant strain variations in virulence and pathogenicity. The availability of laboratory procedures to ascertain the pathogenic potential of L. monocytogenes bacteria would greatly enhance the control and prevention of listerial infections. As a method that measures all virulent determinants, mouse virulence assay has been frequently used for assessing L. monocytogenes virulence. The pathogenic potential of a given L. monocytogenes strain as determined by mouse virulence assay is often calculated from mouse mortality data in combination with colony forming units (CFUs) derived from plate counts, and expressed by medium lethal dose (LD50 ). In this report, we describe an alternative method [i.e., relative virulence (%)] that does not involve CFU estimation, and is comparable to LD50 for interpretation of mouse virulence assay for L. monocytogenes. The relative virulence (%) is obtained by dividing the number of dead mice with the total number of mice tested for a particular strain using a known virulent strain (e.g., L. monocytogenes EGD) as reference. Besides providing a more direct interpretation in comparison with LD50 values for mouse virulence assay, this method requires fewer dosage groups per L. monocytogenes strain, and eliminates CFU estimation that is step subject to variations between runs and also between laboratories. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Listeria monocytogenes; Mouse virulence assay; Medium lethal dose; Colony forming units; Relative virulence
1. Introduction Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that is capable of causing severe illness in man and animals. With its ubiquitous distribution and its tolerance to many manufacturing processes such as high concentrations of salt, extreme pH and temperature, L. monocytogenes has accounted for an increasing proportion of human foodborne diseases worldwide [1]. Indeed, with mortality rates on average approaching 30% [2,3], this bacterium far exceeds the mortality caused by other common foodborne pathogens such as Salmonella enteritidis (with a mortality of 0.38%), Campylobacter species (0.02–0.1%) and Vibrio species (0.005–0.01%) [4]. *
Tel.: +1-662-325-1286; fax: +1-662-325-1031. E-mail address: [email protected] (D. Liu).
Despite being pathogenic at the species level, L. monocytogenes encompasses a diversity of strains or genotypes with varying pathogenic potential. While many L. monocytogenes strains are highly pathogenic and sometimes deadly, others are relatively avirulent and produce little harm in the host [5–8]. The availability of methods to accurately assess the pathogenic potential of L. monocytogenes strains would therefore contribute to the improved control and prevention strategies for listeriosis. Over the years, a number of methods have been developed for gauging the virulence of L. monocytogenes. These include mouse virulence assay, in vitro culture technique using established cell lines or chicken embryos, detection of virulence associated genes or proteins, and genetic typing [1,5–8]. Among these procedures, only mouse virulence assay provides an in vivo measurement of all virulent determinants and is
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.02.005
160
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
often used as a reference standard for any newly developed tests for L. monocytogenes virulence. Since its first application to L. monocytogenes over 40 years ago [9], the mouse virulence assay has played an essential role in the determination of virulence potential and pathological effects as well as elucidation of invasion mechanisms of L. monocytogenes bacteria. In general, mouse virulence assay is carried out by inoculating groups of mice with various doses of L. monocytogenes bacteria via oral, nostril, intraperitoneal, intravenous or subcutaneous route. The virulence of a given L. monocytogenes strain is then determined by the mouse mortality resulting from the infection of this bacterium after estimation of corresponding colony forming unit (CFU) through plate counts, and is commonly expressed as medium lethal dose (LD50 ) [10,11]. Being a vital step in LD50 calculation, estimation of CFU through plate counts can be a delicate task with a narrow margin of error. Not surprisingly, CFUs for a given L. monocytogenes strain may vary from run to run and from laboratory to laboratory, resulting in somewhat different LD50 values for an identical test strain. In this report, we describe the use of relative virulence (%) as an alternative to LD50 for interpretation of mouse virulence assay for L. monocytogenes. The relative virulence (%) is calculated by dividing the number of dead mice with the total number of mice tested per L. monocytogenes strain using the mortality data from a control strain [L. monocytogenes EGD (NCTC 7973) in this study] as reference. As the relative virulence (%) is independent of CFU, it represents a more direct measurement of L. monocytogenes virulence, and eliminates a potential source of variability in the interpretation of mouse virulence assay. Furthermore, with two instead of three to four dosage groups per strain commonly used in the LD50 calculation, the relative virulence (%) offers a practical and economical approach to the assessment of L. monocytogenes virulence based on mouse virulence assay.
2. Materials and methods 2.1. Bacteria and culture conditions Twelve L. monocytogenes strains were examined in the study (Table 1). These strains were either acquired from the American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC), or isolated from catfish or food samples [5,7]. The virulence of these strains was previously determined by PCR using several novel virulence specific gene markers of L. monocytogenes including a putative internalin gene lmo2821 (Table 1) [7]. The benefit of using the putative internalin gene lmo2821 for L. monocytogenes virulence assessment was further demonstrated in a recent DNA array study, as this gene was only found in L. monocytogenes strains/serotypes that are responsible for listerial outbreaks in human populations [12]. L. monocytogenes bacteria were initially grown on 5% sheep blood agar plates (TSA II, Becton–Dickinson Microbiology Systems, Cockeysville, MD). Several colonies from each strain were transferred to flasks containing 25 ml of brain heart infusion (BHI) broth (Difco Laboratories, Detroit, MI) and incubated at 37°C for 18 h with aeration prior to use in mouse virulence assay. 2.2. Mouse virulence assay Six- to eight-week-old female A/J mice (Jackson Laboratory, Bar Harbor, ME) were housed five per cage and allowed to acclimate for 1 week. On the day of inoculation, the optical densities (at OD540 nm ) of L. monocytogenes BHI broth cultures were adjusted to 1.35, and about 1 ml from each strain was pelleted by centrifugation, washed twice, and resuspended in 1 ml of sterile saline (0.9% NaCl). Serial dilutions (100 , 10 1 , 10 2 and 10 3 ) of L. monocytogenes suspensions were prepared in saline. For each L. monocytogenes strain, four groups of mice were inoculated intraperitoneally
Table 1 Listeria monocytogenes strains examined in this study No.
Strain
1 2 3 4 5 6 7 8 9 10 11 12
L. L. L. L. L. L. L. L. L. L. L. L.
monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes
ATCC 19112 ATCC 19114 ATCC 19115 ATCC 19116 ATCC 19117 ATCC 19118 ATCC 15313 EGD (NCTC7973) HCC8 HCC25 874 1002
Serovar
Source
Lmo2821PCRa
2 4a 4b 4c 4d 4e 1 1/2a 1 4
Human Human Human Chicken Sheep Chicken Rabbit Guinea pig Catfish brain Catfish kidney Cow brain Pork sausage
+ ) + + + + + + + ) + +
a The virulence of these strains was evaluated in PCR using virulence specific primers derived from a L. monocytogenes putative internalin gene lmo2821 [7].
Table 2 Summary of the mouse virulence assay results Strain
19112
19114
19115
19117
19118
15313
EGD
HCC8
HCC25
874
Dilution 0
10 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3
Mortality
CFUa
Day 0
Day 1
10
3 10 3 109 3 108 3 107 4.4 1010 4.4 109 4.4 108 4.4 107 11 1010 11 109 11 108 11 107 6 1010 6 109 6 108 6 107 1.6 1010 1.6 109 1.6 108 1.6 107 18 1010 18 109 18 108 18 107 2 1010 2 109 2 108 2 107 18 1010 18 109 18 108 18 107 21 1010 21 109 21 108 21 107 6.4 1010 6.4 109 6.4 108 6.4 107 12 1010 12 109 12 108 12 107
Day 2
Day 3
Day 6
1
1
(a) 10/20 (b) 5/15 (c) 3/10
(a) 50% (b) 33% (c)30%
1.6 109
3
(a) 6/20 (b) 1/15 (c) 0/10
(a) 30% (b) 7% (c) 0%
1.9 1010
1
(a) 14/20 (b) 9/15 (c) 7/10
(a) 70% (b) 60% (c) 70%
6.0 108
(a) 16/20 (b) 10/15 (c) 10/10
(a) 80% (b) 73% (c) 100%
2.6 108
(a) 10/20 (b) 5/15 (c) 4/10
(a) 50% (b) 33% (c) 40%
8.8 108
(a) 10/20 (b) 6/15 (c) 5/10
(a) 50% (b) 40% (c) 50%
7.8 109
(a) 0/20 (b) 0/15 (c) 0/10
(a) 0% (b) 0% (c) 0%
>1.2 1011
(c) 20/20 (b) 15/15 (c) 10/10
(a) 100% (b) 100% (c) 100%
<1.1 107
(a) 18/20 (b) 13/15 (c) 7/10
(a) 90% (b) 87% (c) 70%
<7 108
(a) 5/20 (b) 1/15 (c) 0/10
(a) 40% (b) 7% (c) 0%
3.5 1010
(a) 18/20 (b) 14/15 (c) 10/10
(a) 90% (b) 93% (c) 100%
<8.0 107
1 3
2 2
1
1
5 4
1 2
LD50
Day 5
4
4
Relative virulence
Day 4
5
1
Dead/testedb
3 1
5 3
1
1
4 5 1
5 5 3 5 3
2 4
1
2 2
1 4
1
4 1
2
1 4
2 1 3 1
2 3
161
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
Dose per mouse
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
19116
Mice per group
Saline Not treated
b
0/5 0/3
21 1010 21 109 21 108 21 107 0 0 5 5 5 5 5 3 1002
a
1 1 1 1 1 2
(a) 80% (b) 77% (c) 60%
Day 6 Day 5
1 4 1
Day 2 Day 1 CFU Dilution
a
100 10 1 10 2 10 3 0 0
Day 0
3
Day 3
Day 4
(a) 16/20 (b) 11/15 (c) 6/10
LD50 Relative virulence Dead/testedb Mortality Dose per mouse
Mice per group Strain
Table 2 (continued)
The CFU (colony forming unit) was based on the actual plate counts obtained with a dilution of 10 7 . Three subsets were derived from the different combinations of dosage groups using the EGD mortality records as reference points. The first subset (a) included the mortality data for all four dosage groups (i.e., 100 , 10 1 , 10 2 and 10 3 ) at day six; the second subset (b) for three dosage groups (10 1 , 10 2 and 10 3 ) at day six; and the third subset (c) for two dosage groups (10 1 and 10 2 ) at day four.
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
5.2 108
162
with 0.1 ml of either 100 , 10 1 , 10 2 or 10 3 dilution of bacteria. One group of five mice was injected with 0.1 ml sterile saline, and one group of three mice was not injected. Observations were made daily and mortalities recorded until all of the mice inoculated with virulent strain EGD (NCTC7973) had died. One dead mouse per dose group was necropsied and spleen recovered for L. monocytogenes isolation in BHI agar and subsequent PCR confirmation. On the 15th day after inoculation, all surviving mice were euthanized, and one mouse per group was necropsied and cultured from the spleen for L. monocytogenes. The CFUs for individual L. monocytogenes strains were obtained by plating aliquats of diluted L. monocytogenes suspensions (at 10 7 and 10 9 ) on BHI agar. After overnight incubation, the resultant colonies were enumerated, and the CFUs in the original inoculations calculated. The 50% lethal dose (LD50 ) for each strain was then determined on the basis of mouse mortality data and CFU [7]. The relative virulence (%) was calculated by dividing the number of dead mice recorded with the number of mice tested per strain using the mortality data of the control strain L. monocytogenes EGD (NCTC7973) as reference points.
3. Results The mouse mortality resulting from i.p. injection of various L. monocytogenes strains was summarized in Table 2. The CFUs of individual L. monocytogenes strains were calculated using the colony counts of a 10 7 dilution of the relevant strains. With a seemingly similar number of bacteria being plated out (based on similarly adjusted OD540 nm values), the CFUs of different L. monocytogenes strains varied considerably (from 21 1010 for undiluted bacterial suspensions (100 ) of strains HCC8 and 1002 to 2 1010 for strain ATCC 15313 at the same dilution) (Table 2). In general, strains that caused more mouse mortalities displayed higher CFUs than those that produced less or no mouse mortalities. Based on these CFUs and accompanying mouse mortality data, the 50% lethal dose (LD50 ) for individual L. monocytogenes strains was determined (Table 2). After consideration of the PCR results obtained with the putative internalin gene lmo2821 primers (Table 1) [7], L. monocytogenes strains showing LD50 6109 were regarded as virulent and those showing LD50 P1010 as avirulent except for strain ATCC 15313 (Table 2). The relative virulence (%) was calculated using EGD mortality data as reference points. As mice inoculated with EGD at all four doses (i.e., 100 , 10 1 , 10 2 and 10 3 ) were dead by six days after inoculation, the mortality data from other strains (up to and including day six) were used to generate the first subset (a) of relative virulence values (for the combined dosage groups of 100 ,
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
10 1 , 10 2 and 10 3 ) (Table 2). The second subset (b) of relative virulence values for the dosage groups of 10 1 , 10 2 and 10 3 was also obtained by using the EGD mortality data at day six (Table 2). And the third subset (c) of relative virulence values for the dosage groups of 10 1 and 10 2 was determined by using EGD mortality data at day four as mice inoculated with 10 1 and 10 2 dilutions of EGD were all dead by that day (Table 2). Whereas the first subset (a) and to a lesser extent the second subset (b) of relative virulence values showed poor correlation with the PCR data (Table 1) [7], the third subset (c) of relative virulence (%) values was largely in agreement with the PCR results, with the exception of strain ATCC 15313 (Table 2). That is, strains with a relative virulence of 0% were negative (thus avirulent) by PCR, and those with a relative virulence of 30–100% were positive (thus virulent) by PCR [7]. The third subset of relative virulence values also showed good correlation with the LD50 data, as L. monocytogenes strains showing a 0% relative virulence were those having a LD50 value P1010 while L. monocytogenes strains showing a P30% relative virulence correlated to those having a LD50 value 6109 (Table 2). Interestingly, the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by LD50 values were in the hundreds (based on the LD50 values from 107 to 109 ) (Table 2). On the other hand, the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by relative virulence (%) were only 2–3 times (based on the relative virulence values from 100% to 30%) (Table 2).
4. Discussion The main purpose of this study was to examine if mouse virulence assay could be interpreted by an easyto-use alternative to the well established medium lethal dose (LD50 ) [10,11]. As estimation of CFU is a corner stone for calculation of the LD50 values, obtaining a consistent CFU is vital for correct interpretation of mouse virulence assay for L. monocytogenes bacteria. Unfortunately, being easily affected by minute changes in handling procedures, CFU estimation is a notably delicate task. This may partly explain for the fact that LD50 values for a given L. monocytogenes strain can vary between different runs and between individual laboratories. The observations that L. monocytogenes strains with varied level of virulence demonstrate vastly different growth rates on many selective media further exacerbate the problem [13]. In the present studdy, even with a non-selective medium (i.e., BHI agar), L. monocytogenes strains causing more mouse mortalities tended to show higher CFUs than those producing less or no mouse mortalities.
163
By omiting the estimation of CFU, the relative virulence (%) provides a practical and more direct interpretation for L. monocytogenes mouse virulence assay. In this study, the third subset (c) of relative virulence (%) values derived from the dosage groups of 10 1 and 10 2 showed excellent correlation with LD50 values. That is, L. monocytogenes strains showing a 0% relative virulence were those having a LD50 value P1010 whereas L. monocytogenes strains showing a P30% relative virulence were those having a LD50 value 6109 (Table 2). As these results were generated with inoculation of two mouse dosage groups (10 1 and 10 2 dilutions of overnight L. monocytogenes culture with a uniformly adjusted OD value), inclusion of additional dosage groups (such as 100 and 10 3 dilutions) was unnecessary, and indeed reduced the usefulness of the relative virulence values. Therefore, using the relative virulence (%) to interpret the results, the cost of conducting mouse virulence assay for L. monocytogenes can be decreased in comparsion for LD50 which may require three to four instead of two dosage groups. More importantly, as the relative virulence (%) is independent of CFU, it eliminates a potential source of variability in the interpretation of mouse virulence assay. Although A/J mice were used in this study for their enhanced sensitivity to L. monocytogenes bacteria in comparison with BDF mice [5], other mouse strains should be equally applicable. In the end, the mortality data of an accompanying control strain (e.g., EGD) determine the reference point for a given mouse virulence assay, upon which the relative virulence (%) is calculated. As the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by LD50 values were in the hundreds (based on the LD50 values from 107 to 109 ) (Table 2), it implies that a virulent strain with a LD50 value around 107 could be potentially 100 times or more virulent than a moderately virulent strain with a LD50 value around 109 . On the other hand, the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by relative virulence (%) were only 2–3 times (based on the relative virulence values from 100% to 30%) (Table 2). The actual mouse mortality data suggest that the relative virulence (%) reflects more closely to the virulence differentials between highly virulent and moderately virulent strains of L. monocytogenes (Table 2), while LD50 values may exagerate these differences to some extent. In addition, a close correlation between the relative virulence (%) of L. monocytogenes and PCR detection of the putative internalin gene lmo2821 [7] was also noted. For example, L. monocytogenes avirulent strains with a relative virulence of 0% were shown to be negative by PCR, whereas L. monocytogenes virulent strains with a relative virulence of 30–100% were invariably positive by
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PCR for lmo2821 [7]. The only exception was L. monocytogenes type strain ATCC 15313 as the results obtained from the mouse virulence assay and PCR for this particular strain were contradictory. Originated from an infected rabbit, ATCC 15313 was initially hemolytic, but later became non-hemolytic and avirulent after successive laboratory subculturing [14]. Previously, it was shown that ATCC 15313 is negative for listeriolysin (LLO), a known virulence factor [5]. Further PCR analysis indicates that this strain harbors intact prfA, mpl and plcB genes, but is defective in hlyA and plcA genes [15]. Despite the presence of many intact virulence associated genes as confirmed by PCR, a defective LLO synthesis in ATCC 15313 has rendered it avirulent in mouse. On the whole, this study presents additional evidence on the usefulness of detecting lmo2821 gene for L. monocytogenes virulence. As the putative internalin gene lmo2821 is identified in L. monocytogenes strains/ serotypes that are capable of causing human listerial outbreaks and mouse mortality, but absent in avirulent, non-pathogenic strains/serotypes [7,12], it offers a more specific target for L. monocytogenes virulence in comparison with other previously described virulence-associated genes such as hlyA (encoding LLO), plcA (encoding PI-PLC) and plcB (encoding PC-PLC), that are found in both virulent and avirulent strains of L. monocytogenes [15]. In conclusion, the relative virulence (%) described in this study represents a comparable and easy-to-use alternative to the conventional LD50 values for interpreting mouse virulence assay for L. monocytogenes. As the relative virulence (%) is independent of CFU, it eliminates a potentially variable step that is fundamental to the calculation of LD50 . Moreover, with only two instead of commonly used three to four dosage groups, the relative viurlence (%) offers a more economical means of conducting mouse virulence assay for L. monocytogenes.
Acknowledgements Research in author’s laboratory is supported by US Department of Agriculture Agricultural Research Service (Agreement No. 58-6202-5-083). The author is grateful to Drs. M.L. Lawrence, A.J. Ainsworth and F.W. Austin for their interest in and support of this project. In addition, the author thanks Dr. Catherine W. Donnelly of Department of Nutrition and Food Sci-
ences, University of Connecticut for providing Listeria food and environmental isolates examined in this study. References [1] Robinson, R.K., Batt, C.A. and Patel, P.D. (2000) Encyclopedia of Food Microbiology. Academic Press, San Diego, CA. [2] Fleming, D.W., Cochi, S.L., MacDonald, K.L., Brondum, J., Hayes, P.S., Plikaytis, B.D., Holmes, M.B., Audurier, A., Broome, C.V. and Reingold, A.L. (1985) Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312, 404–407. [3] Linnan, M.J., Mascola, L., Lou, X.D., Goulet, V., May, S., Salminen, C., Hird, D.W., Yonekura, M.L., Hayes, P. and Weaver, R., et al. (1988) Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 319, 823–828. [4] Altekruse, S.F., Cohen, M.L. and Swerdlow, D.L. (1997) Emerging foodborne diseases. Emerg. Infect. Dis. 3, 285–293. [5] Erdenlig, S., Ainsworth, A.J. and Austin, F.W. (2000) Pathogenicity and production of virulence factors by Listeria monocytogenes isolates from channel catfish. J. Food Protect. 63, 613–619. [6] Barbour, A.H., Rampling, A. and Hormaeche, C.E. (2001) Variation in the infectivity of Listeria monocytogenes isolates following intragastric inoculation of mice. Infect. Immun. 69, 4657–4660. [7] Liu, D., Austin, F.W., Ainsworth, A.J. and Lawrence, M. (2003) Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes. J. Med. Microbiol. 52, 1066–1070. [8] Olier, M., Pierre, F., Lemaitre, J.-P., Davies, C., Rousset, A. and Guzzo, J. (2002) Assessment of the pathogenic potential of two Listeria monocytogenes human faecal carriage isolates. Microbiology 148, 1855–1862. [9] Mackaness, G.B. (1962) Cellular resistance to infection. J. Exp. Med. 116, 381–406. [10] Reed, L.J. and Muench, H. (1938) A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493–497. [11] Welkos, S. and O’Brien, A. (1994) Determination of medium lethal and infections doses in animal model systems. Methods Enzymol. 235, 29–39. [12] Doumith, M., Cazalet, C., Simoes, N., Frangeul, L., Jacquet, C., Kunst, F., Martin, P., Cossart, P., Glaser, P. and Buchrieser, C. (2004) New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays. Infect. Immun. 72, 1072–1083. [13] Gracieux, P., Roche, S.M., Pardon, P. and Velge, P. (2003) Hypovirulent Listeria monocytogenes strains are less frequently recovered than virulent strains on PALCAM and Rapid’ L. mono media. Int. J. Food Microbiol. 83, 133–145. [14] Kathariou, S. and Pine, L. (1991) The type strain(s) of Listeria monocytogenes: a source of continuing difficulties. Int. J. Syst. Bacteriol. 41, 328–330. [15] Nishibori, T., Cooray, K., Xiong, H., Kawamura, I., Fujita, M. and Mitsuyama, M. (1995) Correlation between the presence of virulence-associated genes as determined by PCR and actual virulence to mice in various strains of Listeria spp. Microbiol. Immunol. 39, 343–349.
Listeria monocytogenes: comparative interpretation of mouse virulence assay Dongyou Liu
*
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, P.O. Box 6100, Mississippi State, MS 39762, USA Received 9 January 2004; received in revised form 4 February 2004; accepted 6 February 2004 First published online 21 February 2004
Abstract Being an opportunistic bacterial pathogen, Listeria monocytogenes demonstrates significant strain variations in virulence and pathogenicity. The availability of laboratory procedures to ascertain the pathogenic potential of L. monocytogenes bacteria would greatly enhance the control and prevention of listerial infections. As a method that measures all virulent determinants, mouse virulence assay has been frequently used for assessing L. monocytogenes virulence. The pathogenic potential of a given L. monocytogenes strain as determined by mouse virulence assay is often calculated from mouse mortality data in combination with colony forming units (CFUs) derived from plate counts, and expressed by medium lethal dose (LD50 ). In this report, we describe an alternative method [i.e., relative virulence (%)] that does not involve CFU estimation, and is comparable to LD50 for interpretation of mouse virulence assay for L. monocytogenes. The relative virulence (%) is obtained by dividing the number of dead mice with the total number of mice tested for a particular strain using a known virulent strain (e.g., L. monocytogenes EGD) as reference. Besides providing a more direct interpretation in comparison with LD50 values for mouse virulence assay, this method requires fewer dosage groups per L. monocytogenes strain, and eliminates CFU estimation that is step subject to variations between runs and also between laboratories. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Listeria monocytogenes; Mouse virulence assay; Medium lethal dose; Colony forming units; Relative virulence
1. Introduction Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that is capable of causing severe illness in man and animals. With its ubiquitous distribution and its tolerance to many manufacturing processes such as high concentrations of salt, extreme pH and temperature, L. monocytogenes has accounted for an increasing proportion of human foodborne diseases worldwide [1]. Indeed, with mortality rates on average approaching 30% [2,3], this bacterium far exceeds the mortality caused by other common foodborne pathogens such as Salmonella enteritidis (with a mortality of 0.38%), Campylobacter species (0.02–0.1%) and Vibrio species (0.005–0.01%) [4]. *
Tel.: +1-662-325-1286; fax: +1-662-325-1031. E-mail address: [email protected] (D. Liu).
Despite being pathogenic at the species level, L. monocytogenes encompasses a diversity of strains or genotypes with varying pathogenic potential. While many L. monocytogenes strains are highly pathogenic and sometimes deadly, others are relatively avirulent and produce little harm in the host [5–8]. The availability of methods to accurately assess the pathogenic potential of L. monocytogenes strains would therefore contribute to the improved control and prevention strategies for listeriosis. Over the years, a number of methods have been developed for gauging the virulence of L. monocytogenes. These include mouse virulence assay, in vitro culture technique using established cell lines or chicken embryos, detection of virulence associated genes or proteins, and genetic typing [1,5–8]. Among these procedures, only mouse virulence assay provides an in vivo measurement of all virulent determinants and is
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.02.005
160
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
often used as a reference standard for any newly developed tests for L. monocytogenes virulence. Since its first application to L. monocytogenes over 40 years ago [9], the mouse virulence assay has played an essential role in the determination of virulence potential and pathological effects as well as elucidation of invasion mechanisms of L. monocytogenes bacteria. In general, mouse virulence assay is carried out by inoculating groups of mice with various doses of L. monocytogenes bacteria via oral, nostril, intraperitoneal, intravenous or subcutaneous route. The virulence of a given L. monocytogenes strain is then determined by the mouse mortality resulting from the infection of this bacterium after estimation of corresponding colony forming unit (CFU) through plate counts, and is commonly expressed as medium lethal dose (LD50 ) [10,11]. Being a vital step in LD50 calculation, estimation of CFU through plate counts can be a delicate task with a narrow margin of error. Not surprisingly, CFUs for a given L. monocytogenes strain may vary from run to run and from laboratory to laboratory, resulting in somewhat different LD50 values for an identical test strain. In this report, we describe the use of relative virulence (%) as an alternative to LD50 for interpretation of mouse virulence assay for L. monocytogenes. The relative virulence (%) is calculated by dividing the number of dead mice with the total number of mice tested per L. monocytogenes strain using the mortality data from a control strain [L. monocytogenes EGD (NCTC 7973) in this study] as reference. As the relative virulence (%) is independent of CFU, it represents a more direct measurement of L. monocytogenes virulence, and eliminates a potential source of variability in the interpretation of mouse virulence assay. Furthermore, with two instead of three to four dosage groups per strain commonly used in the LD50 calculation, the relative virulence (%) offers a practical and economical approach to the assessment of L. monocytogenes virulence based on mouse virulence assay.
2. Materials and methods 2.1. Bacteria and culture conditions Twelve L. monocytogenes strains were examined in the study (Table 1). These strains were either acquired from the American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC), or isolated from catfish or food samples [5,7]. The virulence of these strains was previously determined by PCR using several novel virulence specific gene markers of L. monocytogenes including a putative internalin gene lmo2821 (Table 1) [7]. The benefit of using the putative internalin gene lmo2821 for L. monocytogenes virulence assessment was further demonstrated in a recent DNA array study, as this gene was only found in L. monocytogenes strains/serotypes that are responsible for listerial outbreaks in human populations [12]. L. monocytogenes bacteria were initially grown on 5% sheep blood agar plates (TSA II, Becton–Dickinson Microbiology Systems, Cockeysville, MD). Several colonies from each strain were transferred to flasks containing 25 ml of brain heart infusion (BHI) broth (Difco Laboratories, Detroit, MI) and incubated at 37°C for 18 h with aeration prior to use in mouse virulence assay. 2.2. Mouse virulence assay Six- to eight-week-old female A/J mice (Jackson Laboratory, Bar Harbor, ME) were housed five per cage and allowed to acclimate for 1 week. On the day of inoculation, the optical densities (at OD540 nm ) of L. monocytogenes BHI broth cultures were adjusted to 1.35, and about 1 ml from each strain was pelleted by centrifugation, washed twice, and resuspended in 1 ml of sterile saline (0.9% NaCl). Serial dilutions (100 , 10 1 , 10 2 and 10 3 ) of L. monocytogenes suspensions were prepared in saline. For each L. monocytogenes strain, four groups of mice were inoculated intraperitoneally
Table 1 Listeria monocytogenes strains examined in this study No.
Strain
1 2 3 4 5 6 7 8 9 10 11 12
L. L. L. L. L. L. L. L. L. L. L. L.
monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes
ATCC 19112 ATCC 19114 ATCC 19115 ATCC 19116 ATCC 19117 ATCC 19118 ATCC 15313 EGD (NCTC7973) HCC8 HCC25 874 1002
Serovar
Source
Lmo2821PCRa
2 4a 4b 4c 4d 4e 1 1/2a 1 4
Human Human Human Chicken Sheep Chicken Rabbit Guinea pig Catfish brain Catfish kidney Cow brain Pork sausage
+ ) + + + + + + + ) + +
a The virulence of these strains was evaluated in PCR using virulence specific primers derived from a L. monocytogenes putative internalin gene lmo2821 [7].
Table 2 Summary of the mouse virulence assay results Strain
19112
19114
19115
19117
19118
15313
EGD
HCC8
HCC25
874
Dilution 0
10 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3 100 10 1 10 2 10 3
Mortality
CFUa
Day 0
Day 1
10
3 10 3 109 3 108 3 107 4.4 1010 4.4 109 4.4 108 4.4 107 11 1010 11 109 11 108 11 107 6 1010 6 109 6 108 6 107 1.6 1010 1.6 109 1.6 108 1.6 107 18 1010 18 109 18 108 18 107 2 1010 2 109 2 108 2 107 18 1010 18 109 18 108 18 107 21 1010 21 109 21 108 21 107 6.4 1010 6.4 109 6.4 108 6.4 107 12 1010 12 109 12 108 12 107
Day 2
Day 3
Day 6
1
1
(a) 10/20 (b) 5/15 (c) 3/10
(a) 50% (b) 33% (c)30%
1.6 109
3
(a) 6/20 (b) 1/15 (c) 0/10
(a) 30% (b) 7% (c) 0%
1.9 1010
1
(a) 14/20 (b) 9/15 (c) 7/10
(a) 70% (b) 60% (c) 70%
6.0 108
(a) 16/20 (b) 10/15 (c) 10/10
(a) 80% (b) 73% (c) 100%
2.6 108
(a) 10/20 (b) 5/15 (c) 4/10
(a) 50% (b) 33% (c) 40%
8.8 108
(a) 10/20 (b) 6/15 (c) 5/10
(a) 50% (b) 40% (c) 50%
7.8 109
(a) 0/20 (b) 0/15 (c) 0/10
(a) 0% (b) 0% (c) 0%
>1.2 1011
(c) 20/20 (b) 15/15 (c) 10/10
(a) 100% (b) 100% (c) 100%
<1.1 107
(a) 18/20 (b) 13/15 (c) 7/10
(a) 90% (b) 87% (c) 70%
<7 108
(a) 5/20 (b) 1/15 (c) 0/10
(a) 40% (b) 7% (c) 0%
3.5 1010
(a) 18/20 (b) 14/15 (c) 10/10
(a) 90% (b) 93% (c) 100%
<8.0 107
1 3
2 2
1
1
5 4
1 2
LD50
Day 5
4
4
Relative virulence
Day 4
5
1
Dead/testedb
3 1
5 3
1
1
4 5 1
5 5 3 5 3
2 4
1
2 2
1 4
1
4 1
2
1 4
2 1 3 1
2 3
161
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
Dose per mouse
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
19116
Mice per group
Saline Not treated
b
0/5 0/3
21 1010 21 109 21 108 21 107 0 0 5 5 5 5 5 3 1002
a
1 1 1 1 1 2
(a) 80% (b) 77% (c) 60%
Day 6 Day 5
1 4 1
Day 2 Day 1 CFU Dilution
a
100 10 1 10 2 10 3 0 0
Day 0
3
Day 3
Day 4
(a) 16/20 (b) 11/15 (c) 6/10
LD50 Relative virulence Dead/testedb Mortality Dose per mouse
Mice per group Strain
Table 2 (continued)
The CFU (colony forming unit) was based on the actual plate counts obtained with a dilution of 10 7 . Three subsets were derived from the different combinations of dosage groups using the EGD mortality records as reference points. The first subset (a) included the mortality data for all four dosage groups (i.e., 100 , 10 1 , 10 2 and 10 3 ) at day six; the second subset (b) for three dosage groups (10 1 , 10 2 and 10 3 ) at day six; and the third subset (c) for two dosage groups (10 1 and 10 2 ) at day four.
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
5.2 108
162
with 0.1 ml of either 100 , 10 1 , 10 2 or 10 3 dilution of bacteria. One group of five mice was injected with 0.1 ml sterile saline, and one group of three mice was not injected. Observations were made daily and mortalities recorded until all of the mice inoculated with virulent strain EGD (NCTC7973) had died. One dead mouse per dose group was necropsied and spleen recovered for L. monocytogenes isolation in BHI agar and subsequent PCR confirmation. On the 15th day after inoculation, all surviving mice were euthanized, and one mouse per group was necropsied and cultured from the spleen for L. monocytogenes. The CFUs for individual L. monocytogenes strains were obtained by plating aliquats of diluted L. monocytogenes suspensions (at 10 7 and 10 9 ) on BHI agar. After overnight incubation, the resultant colonies were enumerated, and the CFUs in the original inoculations calculated. The 50% lethal dose (LD50 ) for each strain was then determined on the basis of mouse mortality data and CFU [7]. The relative virulence (%) was calculated by dividing the number of dead mice recorded with the number of mice tested per strain using the mortality data of the control strain L. monocytogenes EGD (NCTC7973) as reference points.
3. Results The mouse mortality resulting from i.p. injection of various L. monocytogenes strains was summarized in Table 2. The CFUs of individual L. monocytogenes strains were calculated using the colony counts of a 10 7 dilution of the relevant strains. With a seemingly similar number of bacteria being plated out (based on similarly adjusted OD540 nm values), the CFUs of different L. monocytogenes strains varied considerably (from 21 1010 for undiluted bacterial suspensions (100 ) of strains HCC8 and 1002 to 2 1010 for strain ATCC 15313 at the same dilution) (Table 2). In general, strains that caused more mouse mortalities displayed higher CFUs than those that produced less or no mouse mortalities. Based on these CFUs and accompanying mouse mortality data, the 50% lethal dose (LD50 ) for individual L. monocytogenes strains was determined (Table 2). After consideration of the PCR results obtained with the putative internalin gene lmo2821 primers (Table 1) [7], L. monocytogenes strains showing LD50 6109 were regarded as virulent and those showing LD50 P1010 as avirulent except for strain ATCC 15313 (Table 2). The relative virulence (%) was calculated using EGD mortality data as reference points. As mice inoculated with EGD at all four doses (i.e., 100 , 10 1 , 10 2 and 10 3 ) were dead by six days after inoculation, the mortality data from other strains (up to and including day six) were used to generate the first subset (a) of relative virulence values (for the combined dosage groups of 100 ,
D. Liu / FEMS Microbiology Letters 233 (2004) 159–164
10 1 , 10 2 and 10 3 ) (Table 2). The second subset (b) of relative virulence values for the dosage groups of 10 1 , 10 2 and 10 3 was also obtained by using the EGD mortality data at day six (Table 2). And the third subset (c) of relative virulence values for the dosage groups of 10 1 and 10 2 was determined by using EGD mortality data at day four as mice inoculated with 10 1 and 10 2 dilutions of EGD were all dead by that day (Table 2). Whereas the first subset (a) and to a lesser extent the second subset (b) of relative virulence values showed poor correlation with the PCR data (Table 1) [7], the third subset (c) of relative virulence (%) values was largely in agreement with the PCR results, with the exception of strain ATCC 15313 (Table 2). That is, strains with a relative virulence of 0% were negative (thus avirulent) by PCR, and those with a relative virulence of 30–100% were positive (thus virulent) by PCR [7]. The third subset of relative virulence values also showed good correlation with the LD50 data, as L. monocytogenes strains showing a 0% relative virulence were those having a LD50 value P1010 while L. monocytogenes strains showing a P30% relative virulence correlated to those having a LD50 value 6109 (Table 2). Interestingly, the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by LD50 values were in the hundreds (based on the LD50 values from 107 to 109 ) (Table 2). On the other hand, the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by relative virulence (%) were only 2–3 times (based on the relative virulence values from 100% to 30%) (Table 2).
4. Discussion The main purpose of this study was to examine if mouse virulence assay could be interpreted by an easyto-use alternative to the well established medium lethal dose (LD50 ) [10,11]. As estimation of CFU is a corner stone for calculation of the LD50 values, obtaining a consistent CFU is vital for correct interpretation of mouse virulence assay for L. monocytogenes bacteria. Unfortunately, being easily affected by minute changes in handling procedures, CFU estimation is a notably delicate task. This may partly explain for the fact that LD50 values for a given L. monocytogenes strain can vary between different runs and between individual laboratories. The observations that L. monocytogenes strains with varied level of virulence demonstrate vastly different growth rates on many selective media further exacerbate the problem [13]. In the present studdy, even with a non-selective medium (i.e., BHI agar), L. monocytogenes strains causing more mouse mortalities tended to show higher CFUs than those producing less or no mouse mortalities.
163
By omiting the estimation of CFU, the relative virulence (%) provides a practical and more direct interpretation for L. monocytogenes mouse virulence assay. In this study, the third subset (c) of relative virulence (%) values derived from the dosage groups of 10 1 and 10 2 showed excellent correlation with LD50 values. That is, L. monocytogenes strains showing a 0% relative virulence were those having a LD50 value P1010 whereas L. monocytogenes strains showing a P30% relative virulence were those having a LD50 value 6109 (Table 2). As these results were generated with inoculation of two mouse dosage groups (10 1 and 10 2 dilutions of overnight L. monocytogenes culture with a uniformly adjusted OD value), inclusion of additional dosage groups (such as 100 and 10 3 dilutions) was unnecessary, and indeed reduced the usefulness of the relative virulence values. Therefore, using the relative virulence (%) to interpret the results, the cost of conducting mouse virulence assay for L. monocytogenes can be decreased in comparsion for LD50 which may require three to four instead of two dosage groups. More importantly, as the relative virulence (%) is independent of CFU, it eliminates a potential source of variability in the interpretation of mouse virulence assay. Although A/J mice were used in this study for their enhanced sensitivity to L. monocytogenes bacteria in comparison with BDF mice [5], other mouse strains should be equally applicable. In the end, the mortality data of an accompanying control strain (e.g., EGD) determine the reference point for a given mouse virulence assay, upon which the relative virulence (%) is calculated. As the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by LD50 values were in the hundreds (based on the LD50 values from 107 to 109 ) (Table 2), it implies that a virulent strain with a LD50 value around 107 could be potentially 100 times or more virulent than a moderately virulent strain with a LD50 value around 109 . On the other hand, the differences in pathogenic potential between highly virulent and moderately virulent strains of L. monocytogenes as measured by relative virulence (%) were only 2–3 times (based on the relative virulence values from 100% to 30%) (Table 2). The actual mouse mortality data suggest that the relative virulence (%) reflects more closely to the virulence differentials between highly virulent and moderately virulent strains of L. monocytogenes (Table 2), while LD50 values may exagerate these differences to some extent. In addition, a close correlation between the relative virulence (%) of L. monocytogenes and PCR detection of the putative internalin gene lmo2821 [7] was also noted. For example, L. monocytogenes avirulent strains with a relative virulence of 0% were shown to be negative by PCR, whereas L. monocytogenes virulent strains with a relative virulence of 30–100% were invariably positive by
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PCR for lmo2821 [7]. The only exception was L. monocytogenes type strain ATCC 15313 as the results obtained from the mouse virulence assay and PCR for this particular strain were contradictory. Originated from an infected rabbit, ATCC 15313 was initially hemolytic, but later became non-hemolytic and avirulent after successive laboratory subculturing [14]. Previously, it was shown that ATCC 15313 is negative for listeriolysin (LLO), a known virulence factor [5]. Further PCR analysis indicates that this strain harbors intact prfA, mpl and plcB genes, but is defective in hlyA and plcA genes [15]. Despite the presence of many intact virulence associated genes as confirmed by PCR, a defective LLO synthesis in ATCC 15313 has rendered it avirulent in mouse. On the whole, this study presents additional evidence on the usefulness of detecting lmo2821 gene for L. monocytogenes virulence. As the putative internalin gene lmo2821 is identified in L. monocytogenes strains/ serotypes that are capable of causing human listerial outbreaks and mouse mortality, but absent in avirulent, non-pathogenic strains/serotypes [7,12], it offers a more specific target for L. monocytogenes virulence in comparison with other previously described virulence-associated genes such as hlyA (encoding LLO), plcA (encoding PI-PLC) and plcB (encoding PC-PLC), that are found in both virulent and avirulent strains of L. monocytogenes [15]. In conclusion, the relative virulence (%) described in this study represents a comparable and easy-to-use alternative to the conventional LD50 values for interpreting mouse virulence assay for L. monocytogenes. As the relative virulence (%) is independent of CFU, it eliminates a potentially variable step that is fundamental to the calculation of LD50 . Moreover, with only two instead of commonly used three to four dosage groups, the relative viurlence (%) offers a more economical means of conducting mouse virulence assay for L. monocytogenes.
Acknowledgements Research in author’s laboratory is supported by US Department of Agriculture Agricultural Research Service (Agreement No. 58-6202-5-083). The author is grateful to Drs. M.L. Lawrence, A.J. Ainsworth and F.W. Austin for their interest in and support of this project. In addition, the author thanks Dr. Catherine W. Donnelly of Department of Nutrition and Food Sci-
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