624
4.
Ponglikitmongkol M, Green S, Chambon P. Genomic organization of human estrogen receptor gene. EMBO J 1988; 7: 3385-88.
the
5. Garcia T, Lehrer S, Bloomer WD, Schachter B. A variant estrogen receptor messenger ribonucleic acid is associated with reduced levels of estrogen binding in human mammary tumors. Molec Endocrinol 1988; 2: 785-91. 6. Winter E, Yamamoto F, Almoguera C, Perucho M. A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells. Proc Nat Acad Sci USA 1986; 82: 7575-79. 7. Garcia T, Sanchez M, Cox JL, et al. Identification of a variant form of human estrogen receptor with an aminoacid replacement. Nucleic Acids Res 1989; 17: 8364. 8. Mills JL, Simpson JL, Driscoll S, et al. Incidence of spontaneous abortion among normal women and insulin-dependent diabetic women whose pregnancies were identified within 21 days of conception. N Engl J Med 1988; 319: 1617-23. 9. Wilcox AJ, Weinberg CR, O’Connor JF, et al. Incidence of early loss of pregnancy. N Engl J Med 1988; 319: 189-94. 10. Ravindranath N, Moudgal NR. Use of tamoxifen, an anti-estrogen, in establishing a need for estrogen in early pregnancy in the bonnet monkey (Macaca radiata). J Reprod Fertil 1987; 81: 327-36. 11. Furr BJA, Valcaccia B, Challis JRG. The effects of Nolvadex (tamoxifen citrate; ICI 46,474) on pregnancy in rabbits. J Reprod Fertil 1976; 48: 367-69. 12. Kumar V, Green S, Stack G, Berry M, Jin J, Chambon P. Functional domains of the human estrogen receptor. Cell 1987; 51: 941-51.
Detection of
L, Gaub MP, Mader S, Dierich A, Bellard M, Chambon P. Cell specific activity of a GGTCA half palindromic estrogen responsive clement in the chicken ovalbumin gene promoter. EMBO J 1988; 7:
13. Tora
3771-78. 14. Koike S, Sakai M, Muramatsu M. Molecular cloning and characterization of rat estrogen receptor cDNA. Nucl Acids Res 1989; 15: 2499-513. 15. White R, Lees JA, Needham M, Ham J, Parker M. Structural organization and expression of the mouse estrogen receptor. Molec Endocrinol 1989; 1: 735-44. 16. Pike MC, Henderson BE, Casagrande JT, Rosario I, Gray GE. Oral contraceptive use and early abortion as risk factors for breast cancer in young women. Br J Cancer 1981; 43: 72-76. 17. Hadjimichael OC, Boyle CA, Meigs JW. Abortion before first livebirth and risk of breast cancer. Br J Cancer 1986; 53: 281-84. 18. Brinton LA, Hoover R, Fraumeni JF. Reproductive factors in the aetiology of breast cancer. Br J Cancer 1983; 47: 757-62. 19. Berkowitz GS. Risk Factors. In: Marchant DJ, Kase NG, Berkowitz RL, eds. Breast disease. New York: Churchill Livmgston, 1986: 13-41. 20. Salber EJ, Trichopoulos D, MacMahon B. Lactation and reproductive histories of breast cancer patients in Boston 1965-66. JNCI 1969; 43: 1013-24. 21. Paffenbarger RS, Kampert JB, Chang HG. Characteristics that predict risk of breast cancer before and after the menopause. Am J Epidemiol
1980; 112: 258-68. 22. Helmrich SP, Shapiro cancer.
S, Rosenberg L, et al. Risk factors for breast Am J Epidemiol 1983; 117: 35-45.
anti-listeriolysin O for serodiagnosis of human listeriosis
To
see whether detection of antibodies against listeriolysin O (LLO) could be used to diagnose human listeriosis, sera from 28 patients infected
with Listeria monocytogenes and 101 controls were tested by dot-blot titration with purified LLO. 27 patients (96·4%) with listeriosis produced specific anti-LLO. Anti-LLO was detected in 8 (15·6%) of 51 healthy controls and in 6 (12·0%) of 50 controls who had various bacterial, fungal, and viral infections. Anti-LLO titres did not exceed 100 in these two control groups. Anti-LLO could be detected soon after clinical onset of listeriosis, and antibodies persisted for at least several months. This test might be useful for epidemiological surveys and for serodiagnosis of listeriosis, especially when bacteria cannot be isolated.
Introduction Listeria monocytogenes is believed to be transmitted to mainly via contaminated food.! Although immunocompromised patients and pregnant women are
man
especially susceptible to listeriosis, the disease can also develop in seemingly healthy individuals. A vital stage of the infection process is the multiplication of the organism within the cytoplasm of host cells;2 genetic studies have shown that
extracellular haemolysin, listeriolysin 0 (LLO), is essential for this intracellular multiplication .3LLO is a 58 kDa protein belonging to the group of SH-activated haemolysins; it is antigenically related to streptolysin 0 (SLO), pneumolysin, and perfringolysin,8,9 and is produced by all pathogenic strains of L monocytogenes. We here investigate whether detection of specific anti-LLO could be used for serodiagnosis of human listeriosis. an
Patients and methods Patients 28 patients with severe listeriosis (septicaemia and/or meningoencephalitis) were studied. Three groups were assessed: (1) 13 newborn babies with listeriosis (12 at birth, 1 at age 11 days), and 2 pregnant women, aged 23 and 25 years, with bacteraemia
ADDRESSES Laboratoire de Microbiologie (Prof P Berche, MD, M. Bonnichon, J. L Beretti, J Raveneau, J L Gaillard, MD, M. Veron, MD) and Unité de Transplantation, Départment de Néphrologie (H. Kreis, MD), Hôpital Necker-Enfants Malades, Paris; Unité de Génie Microbiologique, Département des Biotechnologies (K A. Reich, PhD, P. Cossart, PhD), and Unité des Antigènes Bactériens (C. Geoffroy, PhD), Institut Pasteur, Paris; and Centre National de Référence des Pneumocoques (P. Geslin, MD), Creteil, France. Correspondence to Prof P. Berche, Laboratoire de Microbiologie, Hôpital Necker-Enfants Malades, 75730 Paris Cedex 15, France
625
LISTERIA ANTIBODIES IN PATIENTS AND CONTROLS
Titres
are
for first available
serum
tested
(1 abortion, 1 neonatal infection); (2) 9 immunocompromised patients, aged 20-50 years (7 renal transplant patients treated with
immunosuppressive drugs, 1 AIDS patient treated with zidovudine, and 1 patient with chronic lymphoid leukaemia who had been receiving chemotherapy for the previous 2 years; and (3) 4 previously healthy patients, aged 14, 55, 58, and 84 years who had not had any treatment or underlying disease before infection. The diagnosis of listeriosis in each patient was confirmed by isolation of L monocytogenes from at least
one clinical specimen (blood, cerebrospinal fluid, placenta, meconium, gastric fluid, or, in 1 patient, arthritic exudate). Bacteria were grown on blood agar (5 % horse blood in ’Brain Heart Infusion’ agar [Difco, Detroit, Michigan, USA]) with or without nalidixic acid (1 ug/ml). Identification was based on the usual criteria-namely, gram stain, catalase activity, motility, CAMP test, serotyping, and typical sugar fermentation pattem.12 Sera were obtained within the first week of life (12 infected newborn babies), before day 14 of the disease (2 pregnant mothers and 4 previously healthy patients), and before day 30 of the disease (6 immunocompromised patients) and between 2 and 5 months (3 immunocompromised patients).
Controls We assessed two control groups: (1) 51 healthy people (17 newborn babies from whom cord blood was sampled at birth, and 34 blood donors); and (2) 50 patients with various active infections, including 15 with acute pulmonary infections due to Streptococcus pnezernoniae; 19 with recent bacterial and fungal infections (3 with septicaemia due to Staphylococcus aureus, Pseudomonas aeruginosa, or the association Escherichia coli with Enterococcus faecalis; 2 with brucellosis; 3 with urinary tract infections due to either E coli [2 patients] or P aeruginosa [1patient],2with meningitis due to either Haemophilus influenzae or E coli; 1 with enteric infection due to Yersinia enterocolitica; 3 with active pulmonary tuberculosis; 5 cystic fibrosis patients superinfected with P aeruginosa; and 1 with septicaemia due to Candida parapsilopsis); and 16 with active viral infections (6 cytomegalovirus, 5 Epstein-Barr virus, and 5 HIV). Sera from controls with S pneumoniae infection and with recent bacterial and fungal infections were collected within 1-2 weeks after onset of infection.
Methods
Adsorption of anti-SLO. Because SLO and LLO are antigenically related,8,9 the detection of specific anti-LLO requires previous adsorption of anti-SLO, which is often present at low titre in human sera. Preliminary assays with a selected human hyperimmune anti-SLO serum (2000 IU/ml) from a patient infected with Streptococcus pyogenes, allowed us to defme the optimum conditions to completely remove anti-SLO from the tested sera. Semi-purified SLO (60 !J.g/ml) was coated by incubation for 1 h at 22°C on nitrocellulose filters (200 µl per well) with a dot-blot microfiltration apparatus (Biorad Laboratories, Richmond, California). Each human serum (05 ml), diluted 1/100 in PBS (pH 7-2) containing ’Tween-20’ (0-1 %) and ’Regilait’ (5%), then incubated for 1 h at room temperature with SLOadsorbed nitrocellulose filters. We used dot-blot analysis to confirm that anti-SLO had been removed from each serum. Detection and titration of anti- L L 0. Purified LLO was used as antigen for dot-blot titration of anti-LLO. This protein was purified from the culture supernatant of L monocytogenes strain L02813 by concentration on a ’ZETA-Prep’ filter cartridge (FLOT-CUNO, Boissy-St-Leger, France), followed by gel filtration and FPLC ion exchange chromatography, details of which will be published elsewhere. The purity of the protein was monitored by SDS-PAGE silver staining. 0-11 [ig of antigen (LLO, semi-purified SLO, or bovine serum albumin [Sigma Chemical Co, St Louis, Missouri, USA]) were adsorbed onto 01 µm nitrocellulose filters (Schleicher and Schull, Dasnel, Federal Republic of Germany) with a dot-blot SF apparatus (Biorad). Filters were then incubated for 1 h at 22°C with PBS-tween-20regilait and then incubated for 1 h at 22°C with doubling dilutions of SLO-adsorbed human sera (1/100 to 1/1600). After extensive washings in PBS-tween-20 (0 15%), filters were further incubated for 1 h at 22°C with a peroxidase-labelled affinity-purified goat anti-human IgG (Fc fragment, y chain specific, Cappel, Malvern, Pennsylvania, USA) diluted 1/500 and revealed with 3.3’ diaminobenzidine tetrachloride, 006% (Sigma)-hydrogen peroxide, 0-075%. The titre was the highest dilution giving a visible We on nitrocellulose filters. determined precipitate immunoglobulin class by testing diluted sera (1/100) with peroxidase-labelled affinity purified goat anti-human IgM (Fc fragment, p chain specific, Cappel) diluted 1/500. Controls consisted of normal rabbit serum or rabbit anti-LLO antiserum diluted 1/100 prepared as previously described,10 and revealed with a peroxidase-labelled goat anti-rabbit antibody (Cappel) diluted was
1/500.
Listeria agglutinating antibodies and anti-SLO. Bacterial 0 and suspensions (serovars 1/2 and 4b, Behring, Marburg, Federal Republic of Germany) were used as described by the manufacturer to detect agglutinating antibodies against L monocytogenes 0 and H antigens. Doubling dilutions of sera in phosphate buffered saline (PBS) pH 72 were incubated for 18 h at 37°C; the titre was the highest dilution giving a visible agglutination. Anti-SLO was titrated with a neutralising procedure (bioMerieux, Marcy l’Etoile, France). Briefly, SLO was incubated for 15 min at 37°C with increasing dilutions of serum. Rabbit red blood cells were added and incubated for 45 min at 37°C. The titre was the highest dilution
Listeria 0 and H antibodies were detected in 10 (35-7%) and 3 (10-7%) patients, respectively (table). By contrast, anti-LLO was detected in 26 patients (92-8%), with titres ranging from 100 to 800. There were genuine seroconversions (ie, anti-LLO detected in previously
inhibiting haemolysis (IU/ml).
seronegative patients)
H
Results Patients
as
follows: 2 kidney transplant
626
Anti-LLO titres during the
course
of infection.
35 sera from 28 patients with listeriosis A =pregnant
4.
Ponglikitmongkol M, Green S, Chambon P. Genomic organization of human estrogen receptor gene. EMBO J 1988; 7: 3385-88.
the
5. Garcia T, Lehrer S, Bloomer WD, Schachter B. A variant estrogen receptor messenger ribonucleic acid is associated with reduced levels of estrogen binding in human mammary tumors. Molec Endocrinol 1988; 2: 785-91. 6. Winter E, Yamamoto F, Almoguera C, Perucho M. A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells. Proc Nat Acad Sci USA 1986; 82: 7575-79. 7. Garcia T, Sanchez M, Cox JL, et al. Identification of a variant form of human estrogen receptor with an aminoacid replacement. Nucleic Acids Res 1989; 17: 8364. 8. Mills JL, Simpson JL, Driscoll S, et al. Incidence of spontaneous abortion among normal women and insulin-dependent diabetic women whose pregnancies were identified within 21 days of conception. N Engl J Med 1988; 319: 1617-23. 9. Wilcox AJ, Weinberg CR, O’Connor JF, et al. Incidence of early loss of pregnancy. N Engl J Med 1988; 319: 189-94. 10. Ravindranath N, Moudgal NR. Use of tamoxifen, an anti-estrogen, in establishing a need for estrogen in early pregnancy in the bonnet monkey (Macaca radiata). J Reprod Fertil 1987; 81: 327-36. 11. Furr BJA, Valcaccia B, Challis JRG. The effects of Nolvadex (tamoxifen citrate; ICI 46,474) on pregnancy in rabbits. J Reprod Fertil 1976; 48: 367-69. 12. Kumar V, Green S, Stack G, Berry M, Jin J, Chambon P. Functional domains of the human estrogen receptor. Cell 1987; 51: 941-51.
Detection of
L, Gaub MP, Mader S, Dierich A, Bellard M, Chambon P. Cell specific activity of a GGTCA half palindromic estrogen responsive clement in the chicken ovalbumin gene promoter. EMBO J 1988; 7:
13. Tora
3771-78. 14. Koike S, Sakai M, Muramatsu M. Molecular cloning and characterization of rat estrogen receptor cDNA. Nucl Acids Res 1989; 15: 2499-513. 15. White R, Lees JA, Needham M, Ham J, Parker M. Structural organization and expression of the mouse estrogen receptor. Molec Endocrinol 1989; 1: 735-44. 16. Pike MC, Henderson BE, Casagrande JT, Rosario I, Gray GE. Oral contraceptive use and early abortion as risk factors for breast cancer in young women. Br J Cancer 1981; 43: 72-76. 17. Hadjimichael OC, Boyle CA, Meigs JW. Abortion before first livebirth and risk of breast cancer. Br J Cancer 1986; 53: 281-84. 18. Brinton LA, Hoover R, Fraumeni JF. Reproductive factors in the aetiology of breast cancer. Br J Cancer 1983; 47: 757-62. 19. Berkowitz GS. Risk Factors. In: Marchant DJ, Kase NG, Berkowitz RL, eds. Breast disease. New York: Churchill Livmgston, 1986: 13-41. 20. Salber EJ, Trichopoulos D, MacMahon B. Lactation and reproductive histories of breast cancer patients in Boston 1965-66. JNCI 1969; 43: 1013-24. 21. Paffenbarger RS, Kampert JB, Chang HG. Characteristics that predict risk of breast cancer before and after the menopause. Am J Epidemiol
1980; 112: 258-68. 22. Helmrich SP, Shapiro cancer.
S, Rosenberg L, et al. Risk factors for breast Am J Epidemiol 1983; 117: 35-45.
anti-listeriolysin O for serodiagnosis of human listeriosis
To
see whether detection of antibodies against listeriolysin O (LLO) could be used to diagnose human listeriosis, sera from 28 patients infected
with Listeria monocytogenes and 101 controls were tested by dot-blot titration with purified LLO. 27 patients (96·4%) with listeriosis produced specific anti-LLO. Anti-LLO was detected in 8 (15·6%) of 51 healthy controls and in 6 (12·0%) of 50 controls who had various bacterial, fungal, and viral infections. Anti-LLO titres did not exceed 100 in these two control groups. Anti-LLO could be detected soon after clinical onset of listeriosis, and antibodies persisted for at least several months. This test might be useful for epidemiological surveys and for serodiagnosis of listeriosis, especially when bacteria cannot be isolated.
Introduction Listeria monocytogenes is believed to be transmitted to mainly via contaminated food.! Although immunocompromised patients and pregnant women are
man
especially susceptible to listeriosis, the disease can also develop in seemingly healthy individuals. A vital stage of the infection process is the multiplication of the organism within the cytoplasm of host cells;2 genetic studies have shown that
extracellular haemolysin, listeriolysin 0 (LLO), is essential for this intracellular multiplication .3LLO is a 58 kDa protein belonging to the group of SH-activated haemolysins; it is antigenically related to streptolysin 0 (SLO), pneumolysin, and perfringolysin,8,9 and is produced by all pathogenic strains of L monocytogenes. We here investigate whether detection of specific anti-LLO could be used for serodiagnosis of human listeriosis. an
Patients and methods Patients 28 patients with severe listeriosis (septicaemia and/or meningoencephalitis) were studied. Three groups were assessed: (1) 13 newborn babies with listeriosis (12 at birth, 1 at age 11 days), and 2 pregnant women, aged 23 and 25 years, with bacteraemia
ADDRESSES Laboratoire de Microbiologie (Prof P Berche, MD, M. Bonnichon, J. L Beretti, J Raveneau, J L Gaillard, MD, M. Veron, MD) and Unité de Transplantation, Départment de Néphrologie (H. Kreis, MD), Hôpital Necker-Enfants Malades, Paris; Unité de Génie Microbiologique, Département des Biotechnologies (K A. Reich, PhD, P. Cossart, PhD), and Unité des Antigènes Bactériens (C. Geoffroy, PhD), Institut Pasteur, Paris; and Centre National de Référence des Pneumocoques (P. Geslin, MD), Creteil, France. Correspondence to Prof P. Berche, Laboratoire de Microbiologie, Hôpital Necker-Enfants Malades, 75730 Paris Cedex 15, France
625
LISTERIA ANTIBODIES IN PATIENTS AND CONTROLS
Titres
are
for first available
serum
tested
(1 abortion, 1 neonatal infection); (2) 9 immunocompromised patients, aged 20-50 years (7 renal transplant patients treated with
immunosuppressive drugs, 1 AIDS patient treated with zidovudine, and 1 patient with chronic lymphoid leukaemia who had been receiving chemotherapy for the previous 2 years; and (3) 4 previously healthy patients, aged 14, 55, 58, and 84 years who had not had any treatment or underlying disease before infection. The diagnosis of listeriosis in each patient was confirmed by isolation of L monocytogenes from at least
one clinical specimen (blood, cerebrospinal fluid, placenta, meconium, gastric fluid, or, in 1 patient, arthritic exudate). Bacteria were grown on blood agar (5 % horse blood in ’Brain Heart Infusion’ agar [Difco, Detroit, Michigan, USA]) with or without nalidixic acid (1 ug/ml). Identification was based on the usual criteria-namely, gram stain, catalase activity, motility, CAMP test, serotyping, and typical sugar fermentation pattem.12 Sera were obtained within the first week of life (12 infected newborn babies), before day 14 of the disease (2 pregnant mothers and 4 previously healthy patients), and before day 30 of the disease (6 immunocompromised patients) and between 2 and 5 months (3 immunocompromised patients).
Controls We assessed two control groups: (1) 51 healthy people (17 newborn babies from whom cord blood was sampled at birth, and 34 blood donors); and (2) 50 patients with various active infections, including 15 with acute pulmonary infections due to Streptococcus pnezernoniae; 19 with recent bacterial and fungal infections (3 with septicaemia due to Staphylococcus aureus, Pseudomonas aeruginosa, or the association Escherichia coli with Enterococcus faecalis; 2 with brucellosis; 3 with urinary tract infections due to either E coli [2 patients] or P aeruginosa [1patient],2with meningitis due to either Haemophilus influenzae or E coli; 1 with enteric infection due to Yersinia enterocolitica; 3 with active pulmonary tuberculosis; 5 cystic fibrosis patients superinfected with P aeruginosa; and 1 with septicaemia due to Candida parapsilopsis); and 16 with active viral infections (6 cytomegalovirus, 5 Epstein-Barr virus, and 5 HIV). Sera from controls with S pneumoniae infection and with recent bacterial and fungal infections were collected within 1-2 weeks after onset of infection.
Methods
Adsorption of anti-SLO. Because SLO and LLO are antigenically related,8,9 the detection of specific anti-LLO requires previous adsorption of anti-SLO, which is often present at low titre in human sera. Preliminary assays with a selected human hyperimmune anti-SLO serum (2000 IU/ml) from a patient infected with Streptococcus pyogenes, allowed us to defme the optimum conditions to completely remove anti-SLO from the tested sera. Semi-purified SLO (60 !J.g/ml) was coated by incubation for 1 h at 22°C on nitrocellulose filters (200 µl per well) with a dot-blot microfiltration apparatus (Biorad Laboratories, Richmond, California). Each human serum (05 ml), diluted 1/100 in PBS (pH 7-2) containing ’Tween-20’ (0-1 %) and ’Regilait’ (5%), then incubated for 1 h at room temperature with SLOadsorbed nitrocellulose filters. We used dot-blot analysis to confirm that anti-SLO had been removed from each serum. Detection and titration of anti- L L 0. Purified LLO was used as antigen for dot-blot titration of anti-LLO. This protein was purified from the culture supernatant of L monocytogenes strain L02813 by concentration on a ’ZETA-Prep’ filter cartridge (FLOT-CUNO, Boissy-St-Leger, France), followed by gel filtration and FPLC ion exchange chromatography, details of which will be published elsewhere. The purity of the protein was monitored by SDS-PAGE silver staining. 0-11 [ig of antigen (LLO, semi-purified SLO, or bovine serum albumin [Sigma Chemical Co, St Louis, Missouri, USA]) were adsorbed onto 01 µm nitrocellulose filters (Schleicher and Schull, Dasnel, Federal Republic of Germany) with a dot-blot SF apparatus (Biorad). Filters were then incubated for 1 h at 22°C with PBS-tween-20regilait and then incubated for 1 h at 22°C with doubling dilutions of SLO-adsorbed human sera (1/100 to 1/1600). After extensive washings in PBS-tween-20 (0 15%), filters were further incubated for 1 h at 22°C with a peroxidase-labelled affinity-purified goat anti-human IgG (Fc fragment, y chain specific, Cappel, Malvern, Pennsylvania, USA) diluted 1/500 and revealed with 3.3’ diaminobenzidine tetrachloride, 006% (Sigma)-hydrogen peroxide, 0-075%. The titre was the highest dilution giving a visible We on nitrocellulose filters. determined precipitate immunoglobulin class by testing diluted sera (1/100) with peroxidase-labelled affinity purified goat anti-human IgM (Fc fragment, p chain specific, Cappel) diluted 1/500. Controls consisted of normal rabbit serum or rabbit anti-LLO antiserum diluted 1/100 prepared as previously described,10 and revealed with a peroxidase-labelled goat anti-rabbit antibody (Cappel) diluted was
1/500.
Listeria agglutinating antibodies and anti-SLO. Bacterial 0 and suspensions (serovars 1/2 and 4b, Behring, Marburg, Federal Republic of Germany) were used as described by the manufacturer to detect agglutinating antibodies against L monocytogenes 0 and H antigens. Doubling dilutions of sera in phosphate buffered saline (PBS) pH 72 were incubated for 18 h at 37°C; the titre was the highest dilution giving a visible agglutination. Anti-SLO was titrated with a neutralising procedure (bioMerieux, Marcy l’Etoile, France). Briefly, SLO was incubated for 15 min at 37°C with increasing dilutions of serum. Rabbit red blood cells were added and incubated for 45 min at 37°C. The titre was the highest dilution
Listeria 0 and H antibodies were detected in 10 (35-7%) and 3 (10-7%) patients, respectively (table). By contrast, anti-LLO was detected in 26 patients (92-8%), with titres ranging from 100 to 800. There were genuine seroconversions (ie, anti-LLO detected in previously
inhibiting haemolysis (IU/ml).
seronegative patients)
H
Results Patients
as
follows: 2 kidney transplant
626
Anti-LLO titres during the
course
of infection.
35 sera from 28 patients with listeriosis A =pregnant
