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Listeria monocytogenes-vaccine: Production and control
Zbl. Bakt. Hyg. A 259, 331-340 (1985)
Listeria monocytogenes-Vaccine: Production and Control!
J. POTEl and J. SCHULZE-LAMMERS Institute of Medical Microb iology, Medical School Hannover
With 2 Figures' Received March 15, 1984 . Accepted October 12, 1984
Abstract The experiments described in this paper were designed in order to develop the basis for the production and potency testing of a live vaccine against listeriosis. The vaccine contains the serovars 1/2 a and 4b of Listeria mon ocytogenes (L. m.). a) Production R-forms of both serovars with attenuated mice virulence were used as antigens. The vaccine strains can be kept stable by means of lyophilisation . The vaccine should contain at least 1 X 108 living bacteria of each serovar. During production, a cultivation temperarure of +22 °C is employed in order to guarantee the formation of somatic (0 -) and flagellar (H-)antigens. b) Potency testing The quantitative mice protection test is the only suitable method for potency testing: subcutaneous vaccination, followed ten days later by challenge with graded doses of sufficiently mice pathogenic, homologous forms of both serovars of L. m. The protection index calculating according to Karber should be at least 100. Due to the possible decline of the number of living bacteria during storage which could result in decreased potency of the vaccine, the expiration date of the vaccine should be one year after production. It was found that the antibody titres of vaccinated animals are without any value for estimating the protective potency of vaccines. Zusammenfassung Es wurde versucht, eine Arbeits- bzw. Diskussionsgrundlage fur die Herstellung und Prufung eines Lebend-Impfstoffes gegen Listeriose, der die Serovare 1/2a und 4b von Listeria monocytogenes (L. m.) enthalt, experimentell zu erarbeiten.
1
Dedicated
to
the honour of the 75 th anniversary of Prof. Dr. med. Georg Henneberg.
332
J. Potel and
J.Schulze-Lammers
a) Herstellung Als Antigen wurden die in ihrer Maus-Virulenz abgeschwachten R-Formen der beiden Serovare benutzt, die in stabiler Form als Impfstoffstarnme mittels Gefriertrocknung gehalten werden konnen, Der Impfstoff mug je Serovar mindestens eine Lebendkeimzahl von 1 x lOB/ml nach Herstellung enthalten; wobei durch Bebrutungstemperatur von +22°C die Ausbildung von Korper- (0-) und Geissel-(H-) Antigenen gewahrleistet ist,
b) Priifung und Bewertung Fur die Prufung eignet sich nur der quantitative Schutzversuch an der Maus: subkutane Impfung, 10 Tage danach Infektionsbelastung mit unterschiedlichen Keimzahlen Mausvirulenter homologer S-Formen der beiden Serovare von L. m, Nach der Flachenrnethode wird der Schutzindex K bestimmt. Er soll mindestens 102 betragen. Die Laufzeit (Verfallzeit) einer Impfstoff-Charge sollte wegen der Moglichkeit der Abnahme der Lebend-Keimzahl wahrend der Lagerung und als Folge der Verringerung der Schutzwirkung auf ein Jahr beschrankt werden. Eine Prufung des Antikorperverlaufes bei geimpften Tieren als Bewerrungsmafs eines Impfschutzes ist nach den vorliegenden Untersuchungen ohne Wert. Nicht gesondert diskutiert wurde, ob aufgrund von Erfahrungswerten der Beziehungen: Lebend-Keimzahl zu Schutzindex im Rahmen der Vorprufung auf den quantitativen Schutzversuch verzichtet werden kann. The detection of the spread of a "secondary zoonosis" (= infectious disease of animals with low spread) and its control by prophylactic measures presents basic problems. In this respect, sheep are of special importance, where listeriosis can be enzootic, resulting in CNS complications, sepsis and metrogenic forms. Such infections occur especially during the winter and spring season when animals are kept in stables (Biining, 1). Repeated outbreaks of listeriosis in sheep flocks suggest that the disease does not result in long lasting immunity. Serological investigations demonstrating humoral antibody seem to indicate shortterm immunity (Kuhlmann-Berger and Potel, 6). Obviously, cellular immune mechanisms are of greater importance for the intracellularly persisting Listeria monocytogenes (L. m.) as found by several workers (Hahn et a!', 3; Hof, et a!', 4, Emmerling et a!', 2) and is also shown in this paper. As specific prophylaxis vaccination is to be considered. However, Potel and Degen (to) demonstrated in 1962 that a killed L. m. vaccine failed to give significant protection in mice and this finding has been confirmed in 1982 by Wirsing v. Koenig et a!. (14). Vaccination with live germs is more effective. However, it is necessary to reduce the concentration of virulent germs in the vaccine to a level where immunity without disease is achieved. This difficulty can be overcome by the use of attenuated germs such as the R-forms of L. m. (loss mutation?). R-forms can be obtained starting with smooth (S) forms by the following procedures: 1. Spontaneous form change. 2. Cultivation in certain media, e. g. such containing tellurite (strain 46 J thereby transformed). 3. Treatment of fluid cultures at +56°C (water bath) and collection of germs at graded time intervals. Prior to inactivation at this temperature, surviving' germs show the growth of R-forms after cultivation.
Listeria monocytogenes-Vaccine: Production and Control
333
Patel succeeded especially by means of the third method in obtaining R-form s of all epidemiologic important serovars of L. m. Since the Reform can be kept stable by means of special culture cond ition s (see 2.1, remarks), this allows the production of an effective live vaccine, as it is produced and marketed in the Federal Republic of Germany (Wirtschaftsgenossensch aft deutscher Tierarzte e. G. Hannover) since 1968. Th is finding can also be utizilized for formulating a test procedure for th is vaccine. Vaccine Production 1. Selection of strains The follow ing serova rs seem to have the greatest epidemiologic importan ce for the enzooti c spread of listeriosis in animals and should therefore be cont ained in a vaccine: 1I2a 4b
o antigen
I, II, (III) (III), V, VI
H antigen
AB ABC
and
The R-form s of the following serovars are currently recommended for vaccines: Serovar Serovar
1I2a 4b
strain 46 J stra in 1242
(see above) lyophil ised under No . 959 Iyophilised under No . 964
Both strains are from the culture collection of the Institut e of Medi cal Microbiology, Medic al School Hannover. Remarks The antig ens of R-form s cannot be analysed serolo gically by means of the Grub erreaction, since they agglut inate spontaneously in sodium chloride solution. - However, the two str ains can be distinguished by means of their colony morphology: 8 to 14 days cultivation at +20 "C to +22 °C: Strain 1I2a R: Strain 4b R:
Ground glass like colon y with rami fication Ground glass like colon y, slight ramificat ion only.
Onl y isolations from the edge of the colony should be further passaged in order to obtain Rvforms. - The lyophili sed vaccine strains are sto red separately. 2. Culture m edium (stated by the producer of the vaccine) Yeast powder Gelatine -peptone Na2HP04·H 20 Meat-broth Distilled water pH
5 15 25 150 750
g g g ml ml
~2
3 . Culture (stated by the producer of the vaccine) After open ing of the amp oules conta ining th e lyophilised seed cultures (sterile condition s), the respective cultures are added to 5- 10 ml of growth medium and cultivated at +22 °C for 48 h. Cont rol of purity, motility and R-form. Pure culture obt ained by
334
J. Potel and J.Schulze-Lammers
Fig. 1. Culture morphology (macrocolonies) of rough forms of L. monocytogenes.
fractionated inoculation on sheep blood agar, cultivation for 24 h at +3rC, 48 h at +22 -c Inoculation of the batch: Cultivation at + 22 °C for 48 h. Controls like that of preculture. The germ concentration of each vaccine strain is brought to 2 x 108/ml. Equal portions of both serovars are mixed, yielding concentrations of 1 x 10 8/ml for each serovar.
4. Batch-testing (stated by vaccine producer) Qualitative mice protection test. 10 mice are vaccinated with 0,5 ml vaccine i.p. Groups of 5 vaccinated and 5 unvaccinated animals are challenged 21 days after vaccination with 0.5 ml of the virulent S-form of serovar 1/2a and 4b (germ concentration 1 x 10 7/ml). Evaluation of test. All control animals must die within 3 days. Only 2 vaccinated animals may die. Vaccine testing: Mice Protection Test
1. Germ concentration 1.1. Total germ concentration. For example, the universal-colorimeter ("Jubilaumsmodell") produced by Dr. B. Lange is used. - The germ concentration is determined by means of a standard curve (Fig. 2), testing in parallel uninoculated medium for determining the background optical density. 1.2. Concentration of live germs. Dilution method, inoculation of the surface of
Listeria monocytogenes-Vaccine: Production and Control 0,9 O. D. reading 0,8
serovar y, a-R (strain 46 J)
335
serovar Y, a - S (strain 85)
0,7
0,6
0,5 0,4
0,3
0,2
0,1
2
2
4
6 8 10'0 2 germ concentration
4
Fig. 2. Relationship between 0, D. readings and concentration of L. monocytogenes. sheep-blood agar plates. Determination of the morphology of colonies, counting of Rcolonies and calculation per 1 m!.
2. Challenge-strains: S-forms Serovar 1/2a strain H 85 (Iyophilisation No. 1194) Serovar 4b strain 965 (Iyophilisation No. 289) Inoculate tryptone soya broth with previously tested cultures, cultivate at +37 °C for 24 h. - Arranging for the desired concentration of live germs (see 3.1) 1 X 10 9 germs / m!. Due to the different virulence of these two strains, use concentrations from 10 8 to 10 4 /ml (dilutions 10- 1 to 10-5 ) of serovar 1I2a and 10 9 to 105 /ml of serovar 4b (dilutions undiluted to 10--4) when challenging animals.
3. Vaccination and Challenge Use NMRI mice weighing from 18 to 22 g. Each animal is inoculated subcutaneously (dorsal skin ) with 0.5 ml of vaccine. Animals are controlled daily for death. Dead animals are dissected and liver and spleen examined for the pre sence of the S- or Rform of L. m.
4. Evaluation Determination of the Protection Index K Method: Area-method as described by (Spearmant-Kdrber (13 ). This method offers the advantage that the results are within limits independent from the number of ani-
336
]. Potel and]. Schulze-Lammers
mals per group and from irregular distribution of dead animals (see Bonin , 1973 ). The dlso is calculated for vaccinated and for control animals. The protection index K is the ratio of dlso of controls to that of vaccinated animals. 5. Serological Investigations 5.1. Bacterial agglutination test (Patel and Degen, 11). The complete antigen (0 and H-antigen) is produced by cultivation of the respective serovar of L. m. in soya broth at + 22 °C. Subsequentl y, the concentration is adjusted to 5 x lOs/ml (see 3.11 ). Positive and negative control sera are tested in like manner, control of antigen and antisera. The serum antigen mixtures are incubated at +3rc for 24 h. The test is read by means of an agglutinoscope. 5.2. Growth-test (Patel and Degen, 8). Tube test: Serum dilut ions in soya broth, sterility testing for 24 h at + 3 r c. Inoculation with the respective strain (serovar) cultivated for 24 h at +37 °C. Readings are taken after 24 h incubation at +3rc. Final titre so-called bracket value, at least 2/2. 5.3. Agglutination-immobilization test (Patel, 7). Microtiter Method: Graded serum dilutions are prepared in soya broth , one drop of culture is added derived from an inoculated blood agar dish with broth overlay (1 x 10 9/ml , 24 h at +22°C), moist chamber for 10 min at +3rc. Then , 1 loop of the antigen-serum mixture is placed on slides and read for motility and agglutination by means of a phase contrast microscope (objective No. 40). Titres are expressed in terms of the serum dilution where at least 50 per cent of germs are agglutinated and immobile. Results 1. Dose of vaccine 1.1. Recovery ofgerms from the vaccinated animals in relationship to the number of germs inoculated. Each animal received 0.5 ml subcutaneously containing between 10 6 to 10 9 L. m. strain 46 J (serovar 1/2a-R). One animal per day was tested: Cardiac blood , liver and spleen, 1 to 7 days after vaccination. It was found that the inoculated germs could be recovered within few days after vaccination, only, and that the concentration recovered depended on the concentration of germs inoculated. This finding agree with previous reports by Potel and Degen (10). Animals inoculated with 10 9 germs/ml showed a weight reduction of 10 per cent in comparison to the controls. Two animals of the group given 10 9/ml died on days 4 and 5. L. m. of the R-form was cultivated. Evaluation: The inoculation with 109/ml of the strain 46 J resulted in the persistence of viable germs for some days. Inapparent and even lethal apparent courses of the disease were observed, the latter with weight loss. It is to be excepted that "disease" results in immunity which should not be considered as vaccine induced immunity, but as immunity caused by disease. 1.2. Dependency of protection index K on the dose of vaccine (Table 1). Values of the protection index K increased with increasing doses of vaccine: A protection index K of 10 2 = 100 was recorded for germ concentrations 2,5 x lOs and 1 x 107/m!. The germ concentration present in the commercial vaccine is within this range. The protection index depends on the concentration of viable germs in the vaccine. This conclusion is confirmed by the results obtained when testing vaccines stored for
Listeria monocytogenes-Vaccine: Production and Control
337
Table 1. Protection index K in relationship to inoculated dose (mice experiment) L. m. serovar 1/2a R (46 J): Inoculated doselml
i.oxio' i.oxro' i.oxio-
Control Lux lu" 7,5Xl08 5,Oxl08 2,5 X108 dlso
10-4*
Protection index K;/.
10- 1,5
10-1,66
10- 1,75
10- 2,0
10-2 ,0
10-2 ,0
102,67
102 •4
102•28
102,0
102,0
102,0
10-3•0
* = dilution of starting material containing 109/ml
graded periods of time after production (T able 2) and is emphasized by the results of experiments where live and killed vaccines of equal germ concentration were tested comparatively (Table 3).
2. SpeCificity of vaccine-induced protection The homologous protection expressed in terms of the protection index K is higher than the heterologous protection (Table 4). This finding agrees with the results of Patel and Degen (10). Therefore, a listeriosis vaccine should contain the epidemiologically most important serovars (e. g. 1/2a and 4b ). Table 2. Protection index K (L.m. 1/2a) of vaccines stored for graded time intervals Time vaccine batch has been stored
Concentration of live germs at time of production
A : 1 year
1,14
B : 1/2 year
1,5 x 108
C: 1 month
8
1
X
X
10 8 10
Concentration of live germs found after storage
Protection index K Serovar 1/2a 101
1,8
X
107
3,5
X
107
102,5
7
104 ,5
9
X
10
Table 3. Comparison of protection index K of killed and live vaccines Vaccine L.m. 1/2a R: 1 x 10 8/ml inactivated live Protection index K
o
Table 4. Serovar-specificity after L.m. vaccination (mice experiment) Protection index K after infection
1/2a
1/2a
178
100
20
79
4b
Vaccination 4b
338
J. Potel and J.Schulze-Lammers
3. Humoral antibody response to 1. m. vaccination Methods: Bacterial agglutination reaction (listeriosis Widal), growth test (GT) and agglutination immobilization test (AIT). The results presented in Table 5 indicate that independently from the type of vaccine (killed vaccine yielding low and live vaccine yielding high protection) only low antibody titres were recovered. This finding is consistent with the results of Kuhlmann-Berger and Potel (6) obtained in sheep. The antibody titres were sensitive to rnercapto-ethanol, indicating the presence of antibody of the IgM class. The results obtained indicate that the potency of L. rn. vaccines cannot be determined by means of recording the antibody response in mice. Table 5. Detection of humoral antibody (mice: 7 animals/group/bleeding day) after Lrn. vaccination Type of vaccine and time intervals after vaccination sera have drawn (pooled sera titer 1:) Serological method Agglutination reaction
+ 22 °C, inactivated
7
14
50
400
Growth test
< 50
< 50
Agglutinationimmobilization test (AIT)
< 50
< 50
7
+ 22°C, live 14
200
21
200
100
7
+ 37°C, live 14
50
21
100
100
control 7 50
< 50 < 50 < 50 < 50 < 50 < 50 < 50 50
100
50 < 50 < 50 < 50 < 50
Discussion A marked protection of mice against listeriosis can be achieved by the use of live attenuated (e. g. R-forms) L. m. strains only, as found in mice (Potel and Degen, 10) and domestic cattle (Ivanov et al., 5). Such vaccines are commercially available and are practically used. Therefore, it is necessary to define the optimal composition of such vaccines and to formulate the conditions of official vaccine testing. The results presented in this paper could be a helpful basis for solving these problems. The results of the mice protection tests confirmed the conclusion that killed vaccines fail to confer protection (Table 3). The protection expressed in terms of the protection index K was found to depend on the concentration of viable germs of suitable strains in the vaccines. Such strains are for example the R-forms of L. m. (Table 2). The use of too high a vaccine dose (> 10 9/ml) can result in inapparent disease (sometimes lethal). The resulting immunity may not be considered as vaccine-induced immunity. The results obtained suggest that a concentration of viable germs of 1 X lO B/ml yielding a protection index K of 10 2 (dlso = 10-4 using a starting concentration of the challenge strain of 10 9/ml = 105 germs/ml) is desirable. The smaller virulence (dlso) of the selected serovar 4b (average 10 7 germs/ml) does not allow to draw a similar conclusion for this serovar. Until now, attempts to increase
Listeria monocytogenes-Vaccine: Production and Control
339
Table 6. Protection index K in relationship to the incubation temperature Incubation temperature
L. m. serovar lI2a R (46 J): Vaccine dose 1 X 109/ml 1 X 108/ml
+22°C
102,583
+37°C
102,42
(0- and H-antigen)
102,25
the virulence of this strain by means of mice passages have failed. Generally, no mouse died after infection with serovar 4b, preventing determination of the dl so in this group. Attempts to find a more vilurent strain in clinical specimens are in progress. Furthermore, the survival rate of germs during storage of vaccines is a problem, connected with that of defining the expiration dates of the vaccines. The results presented in Table 2 indicate that a decrease in the concentration of viable germs yielded a decrease of the protection index. Empirically, it is suggested that vaccines produced according to the principles described in this paper should be used within one year after production, only. The homologous protection was more effective than the heterologous protection (Table 4). Since the serovars lI2a and 4b differ in their 0 antigen, this result was to be expected. The vaccines should contain both the 0 and the H antigens of L. m. yielding optimal protection (Table 6). The results presented in Table 5 indicate that recording of the humoral antibody response does not reflect the potency of L. m. vaccines.
References 1. Buning, U.: Auswertung der im Tiergesundheitsamt der Landwirtschaftskammer Hannover bei Schafen diagnostizierten Listeriosefalle, Inaugur.-Diss., Tierarztl, Hochschule Hannover (1975) 2. Emmerling, P., H. Finger und ]. Bockemiihl: Die Bedeutung der T-Zell-vermittelten Reaktion bei der Abwehr einer Infektion mit Listeria monocytogenes. Zbl. Bakt. Hyg., I. Abt, Orig. A 235 (1976) 142-146 3. Hahn, H. and S. H. E. Kaufmann: The role of cell-mediated immunity in bacterial infections. Rev. infect. Dis. 3 (1981) 1221-1250 4. Hof, H., P. Emmerling, H. Finger und C. H. Wirsing: Einfluf von abgetoteten Zellen von B. pertussis auf die Infektionsabwehr gegen Listeria monocytogenes. Zbl. Bakt. Hyg., I. Abt. Orig. A 240 (1978) 208-214 5. Ivanov, 1., M. Draganov, and Tz. Dikova: Studies on the active immunprophylaxis of listeriosis. VII. Internat. Sympos. on the Problems of Listeriosis. Varna 1977 6. Kuhlmann-Berget, S. and]. Potel: On the serology and immunobiology of listeriosis, VIII. Communication: Antibody against Listeria monocytogenes in sheep without and after vaccination against listeriosis. Zbl. Bakt. Hyg. A 259 (1985) 51-58 7. Potel, ].: Zur Serologie und Immunologie der Listeriose. VI. Mitt.: AgglutinationsImmobilisationstest (AIT). Zbl. Bakt., I. Abt. Orig. 207 (1968) 63-68 8. Potel, ]. und 1. Degen: Zur Serologie und Immunologie der Listeriose. I. Mitt.: Die Wachstumsprobe. Zbl. Bakt., I. Abt, Orig. 180 (1960) 61-67
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J.Potel and J.Schulze-Lammers
10. Patel, J. und L. Degen: Zur Serologie und Immunologie der Listeriose. V. Mitt.: Spezifische Therapie und Prophylace der Listeriose beim Versuchstier. Z. Immun.-Forsch. 123 (1962) 432-446 11. Patel, J. und L. Degen: Zur Serologie und Immunologie der Listeriose. III. Mitt.: Vergleichende Untersuchung verschiedener serodiagnostischer Methoden. Zbl. Bakt., I. Abt. Orig. 185 (1962) 204-214 12. Schulze-Lammers, ].: Erstellung einer Priifvorschrift fiir Listeria-Lebendimpfstoffe. Inaugur.-Diss., Tieriirztliche Hochschule Hannover (1983) 13. Spearman-Karber, in: O. Bonin, Quantitative virologische Methodik. Georg Thieme, Stuttgart (1973) 14. Wirsing v. Konig, C. H., H. Fischer, and H. Hof: Failure of killed Listeria monocytogenes vaccine to produce protective immunity. Nature 297 (1982) 233-234
Prof. Dr. med. J. Patel, Konstanty-Gutschow-Str. 8, 0-3000 Hannover 61
Listeria monocytogenes-Vaccine: Production and Control!
J. POTEl and J. SCHULZE-LAMMERS Institute of Medical Microb iology, Medical School Hannover
With 2 Figures' Received March 15, 1984 . Accepted October 12, 1984
Abstract The experiments described in this paper were designed in order to develop the basis for the production and potency testing of a live vaccine against listeriosis. The vaccine contains the serovars 1/2 a and 4b of Listeria mon ocytogenes (L. m.). a) Production R-forms of both serovars with attenuated mice virulence were used as antigens. The vaccine strains can be kept stable by means of lyophilisation . The vaccine should contain at least 1 X 108 living bacteria of each serovar. During production, a cultivation temperarure of +22 °C is employed in order to guarantee the formation of somatic (0 -) and flagellar (H-)antigens. b) Potency testing The quantitative mice protection test is the only suitable method for potency testing: subcutaneous vaccination, followed ten days later by challenge with graded doses of sufficiently mice pathogenic, homologous forms of both serovars of L. m. The protection index calculating according to Karber should be at least 100. Due to the possible decline of the number of living bacteria during storage which could result in decreased potency of the vaccine, the expiration date of the vaccine should be one year after production. It was found that the antibody titres of vaccinated animals are without any value for estimating the protective potency of vaccines. Zusammenfassung Es wurde versucht, eine Arbeits- bzw. Diskussionsgrundlage fur die Herstellung und Prufung eines Lebend-Impfstoffes gegen Listeriose, der die Serovare 1/2a und 4b von Listeria monocytogenes (L. m.) enthalt, experimentell zu erarbeiten.
1
Dedicated
to
the honour of the 75 th anniversary of Prof. Dr. med. Georg Henneberg.
332
J. Potel and
J.Schulze-Lammers
a) Herstellung Als Antigen wurden die in ihrer Maus-Virulenz abgeschwachten R-Formen der beiden Serovare benutzt, die in stabiler Form als Impfstoffstarnme mittels Gefriertrocknung gehalten werden konnen, Der Impfstoff mug je Serovar mindestens eine Lebendkeimzahl von 1 x lOB/ml nach Herstellung enthalten; wobei durch Bebrutungstemperatur von +22°C die Ausbildung von Korper- (0-) und Geissel-(H-) Antigenen gewahrleistet ist,
b) Priifung und Bewertung Fur die Prufung eignet sich nur der quantitative Schutzversuch an der Maus: subkutane Impfung, 10 Tage danach Infektionsbelastung mit unterschiedlichen Keimzahlen Mausvirulenter homologer S-Formen der beiden Serovare von L. m, Nach der Flachenrnethode wird der Schutzindex K bestimmt. Er soll mindestens 102 betragen. Die Laufzeit (Verfallzeit) einer Impfstoff-Charge sollte wegen der Moglichkeit der Abnahme der Lebend-Keimzahl wahrend der Lagerung und als Folge der Verringerung der Schutzwirkung auf ein Jahr beschrankt werden. Eine Prufung des Antikorperverlaufes bei geimpften Tieren als Bewerrungsmafs eines Impfschutzes ist nach den vorliegenden Untersuchungen ohne Wert. Nicht gesondert diskutiert wurde, ob aufgrund von Erfahrungswerten der Beziehungen: Lebend-Keimzahl zu Schutzindex im Rahmen der Vorprufung auf den quantitativen Schutzversuch verzichtet werden kann. The detection of the spread of a "secondary zoonosis" (= infectious disease of animals with low spread) and its control by prophylactic measures presents basic problems. In this respect, sheep are of special importance, where listeriosis can be enzootic, resulting in CNS complications, sepsis and metrogenic forms. Such infections occur especially during the winter and spring season when animals are kept in stables (Biining, 1). Repeated outbreaks of listeriosis in sheep flocks suggest that the disease does not result in long lasting immunity. Serological investigations demonstrating humoral antibody seem to indicate shortterm immunity (Kuhlmann-Berger and Potel, 6). Obviously, cellular immune mechanisms are of greater importance for the intracellularly persisting Listeria monocytogenes (L. m.) as found by several workers (Hahn et a!', 3; Hof, et a!', 4, Emmerling et a!', 2) and is also shown in this paper. As specific prophylaxis vaccination is to be considered. However, Potel and Degen (to) demonstrated in 1962 that a killed L. m. vaccine failed to give significant protection in mice and this finding has been confirmed in 1982 by Wirsing v. Koenig et a!. (14). Vaccination with live germs is more effective. However, it is necessary to reduce the concentration of virulent germs in the vaccine to a level where immunity without disease is achieved. This difficulty can be overcome by the use of attenuated germs such as the R-forms of L. m. (loss mutation?). R-forms can be obtained starting with smooth (S) forms by the following procedures: 1. Spontaneous form change. 2. Cultivation in certain media, e. g. such containing tellurite (strain 46 J thereby transformed). 3. Treatment of fluid cultures at +56°C (water bath) and collection of germs at graded time intervals. Prior to inactivation at this temperature, surviving' germs show the growth of R-forms after cultivation.
Listeria monocytogenes-Vaccine: Production and Control
333
Patel succeeded especially by means of the third method in obtaining R-form s of all epidemiologic important serovars of L. m. Since the Reform can be kept stable by means of special culture cond ition s (see 2.1, remarks), this allows the production of an effective live vaccine, as it is produced and marketed in the Federal Republic of Germany (Wirtschaftsgenossensch aft deutscher Tierarzte e. G. Hannover) since 1968. Th is finding can also be utizilized for formulating a test procedure for th is vaccine. Vaccine Production 1. Selection of strains The follow ing serova rs seem to have the greatest epidemiologic importan ce for the enzooti c spread of listeriosis in animals and should therefore be cont ained in a vaccine: 1I2a 4b
o antigen
I, II, (III) (III), V, VI
H antigen
AB ABC
and
The R-form s of the following serovars are currently recommended for vaccines: Serovar Serovar
1I2a 4b
strain 46 J stra in 1242
(see above) lyophil ised under No . 959 Iyophilised under No . 964
Both strains are from the culture collection of the Institut e of Medi cal Microbiology, Medic al School Hannover. Remarks The antig ens of R-form s cannot be analysed serolo gically by means of the Grub erreaction, since they agglut inate spontaneously in sodium chloride solution. - However, the two str ains can be distinguished by means of their colony morphology: 8 to 14 days cultivation at +20 "C to +22 °C: Strain 1I2a R: Strain 4b R:
Ground glass like colon y with rami fication Ground glass like colon y, slight ramificat ion only.
Onl y isolations from the edge of the colony should be further passaged in order to obtain Rvforms. - The lyophili sed vaccine strains are sto red separately. 2. Culture m edium (stated by the producer of the vaccine) Yeast powder Gelatine -peptone Na2HP04·H 20 Meat-broth Distilled water pH
5 15 25 150 750
g g g ml ml
~2
3 . Culture (stated by the producer of the vaccine) After open ing of the amp oules conta ining th e lyophilised seed cultures (sterile condition s), the respective cultures are added to 5- 10 ml of growth medium and cultivated at +22 °C for 48 h. Cont rol of purity, motility and R-form. Pure culture obt ained by
334
J. Potel and J.Schulze-Lammers
Fig. 1. Culture morphology (macrocolonies) of rough forms of L. monocytogenes.
fractionated inoculation on sheep blood agar, cultivation for 24 h at +3rC, 48 h at +22 -c Inoculation of the batch: Cultivation at + 22 °C for 48 h. Controls like that of preculture. The germ concentration of each vaccine strain is brought to 2 x 108/ml. Equal portions of both serovars are mixed, yielding concentrations of 1 x 10 8/ml for each serovar.
4. Batch-testing (stated by vaccine producer) Qualitative mice protection test. 10 mice are vaccinated with 0,5 ml vaccine i.p. Groups of 5 vaccinated and 5 unvaccinated animals are challenged 21 days after vaccination with 0.5 ml of the virulent S-form of serovar 1/2a and 4b (germ concentration 1 x 10 7/ml). Evaluation of test. All control animals must die within 3 days. Only 2 vaccinated animals may die. Vaccine testing: Mice Protection Test
1. Germ concentration 1.1. Total germ concentration. For example, the universal-colorimeter ("Jubilaumsmodell") produced by Dr. B. Lange is used. - The germ concentration is determined by means of a standard curve (Fig. 2), testing in parallel uninoculated medium for determining the background optical density. 1.2. Concentration of live germs. Dilution method, inoculation of the surface of
Listeria monocytogenes-Vaccine: Production and Control 0,9 O. D. reading 0,8
serovar y, a-R (strain 46 J)
335
serovar Y, a - S (strain 85)
0,7
0,6
0,5 0,4
0,3
0,2
0,1
2
2
4
6 8 10'0 2 germ concentration
4
Fig. 2. Relationship between 0, D. readings and concentration of L. monocytogenes. sheep-blood agar plates. Determination of the morphology of colonies, counting of Rcolonies and calculation per 1 m!.
2. Challenge-strains: S-forms Serovar 1/2a strain H 85 (Iyophilisation No. 1194) Serovar 4b strain 965 (Iyophilisation No. 289) Inoculate tryptone soya broth with previously tested cultures, cultivate at +37 °C for 24 h. - Arranging for the desired concentration of live germs (see 3.1) 1 X 10 9 germs / m!. Due to the different virulence of these two strains, use concentrations from 10 8 to 10 4 /ml (dilutions 10- 1 to 10-5 ) of serovar 1I2a and 10 9 to 105 /ml of serovar 4b (dilutions undiluted to 10--4) when challenging animals.
3. Vaccination and Challenge Use NMRI mice weighing from 18 to 22 g. Each animal is inoculated subcutaneously (dorsal skin ) with 0.5 ml of vaccine. Animals are controlled daily for death. Dead animals are dissected and liver and spleen examined for the pre sence of the S- or Rform of L. m.
4. Evaluation Determination of the Protection Index K Method: Area-method as described by (Spearmant-Kdrber (13 ). This method offers the advantage that the results are within limits independent from the number of ani-
336
]. Potel and]. Schulze-Lammers
mals per group and from irregular distribution of dead animals (see Bonin , 1973 ). The dlso is calculated for vaccinated and for control animals. The protection index K is the ratio of dlso of controls to that of vaccinated animals. 5. Serological Investigations 5.1. Bacterial agglutination test (Patel and Degen, 11). The complete antigen (0 and H-antigen) is produced by cultivation of the respective serovar of L. m. in soya broth at + 22 °C. Subsequentl y, the concentration is adjusted to 5 x lOs/ml (see 3.11 ). Positive and negative control sera are tested in like manner, control of antigen and antisera. The serum antigen mixtures are incubated at +3rc for 24 h. The test is read by means of an agglutinoscope. 5.2. Growth-test (Patel and Degen, 8). Tube test: Serum dilut ions in soya broth, sterility testing for 24 h at + 3 r c. Inoculation with the respective strain (serovar) cultivated for 24 h at +37 °C. Readings are taken after 24 h incubation at +3rc. Final titre so-called bracket value, at least 2/2. 5.3. Agglutination-immobilization test (Patel, 7). Microtiter Method: Graded serum dilutions are prepared in soya broth , one drop of culture is added derived from an inoculated blood agar dish with broth overlay (1 x 10 9/ml , 24 h at +22°C), moist chamber for 10 min at +3rc. Then , 1 loop of the antigen-serum mixture is placed on slides and read for motility and agglutination by means of a phase contrast microscope (objective No. 40). Titres are expressed in terms of the serum dilution where at least 50 per cent of germs are agglutinated and immobile. Results 1. Dose of vaccine 1.1. Recovery ofgerms from the vaccinated animals in relationship to the number of germs inoculated. Each animal received 0.5 ml subcutaneously containing between 10 6 to 10 9 L. m. strain 46 J (serovar 1/2a-R). One animal per day was tested: Cardiac blood , liver and spleen, 1 to 7 days after vaccination. It was found that the inoculated germs could be recovered within few days after vaccination, only, and that the concentration recovered depended on the concentration of germs inoculated. This finding agree with previous reports by Potel and Degen (10). Animals inoculated with 10 9 germs/ml showed a weight reduction of 10 per cent in comparison to the controls. Two animals of the group given 10 9/ml died on days 4 and 5. L. m. of the R-form was cultivated. Evaluation: The inoculation with 109/ml of the strain 46 J resulted in the persistence of viable germs for some days. Inapparent and even lethal apparent courses of the disease were observed, the latter with weight loss. It is to be excepted that "disease" results in immunity which should not be considered as vaccine induced immunity, but as immunity caused by disease. 1.2. Dependency of protection index K on the dose of vaccine (Table 1). Values of the protection index K increased with increasing doses of vaccine: A protection index K of 10 2 = 100 was recorded for germ concentrations 2,5 x lOs and 1 x 107/m!. The germ concentration present in the commercial vaccine is within this range. The protection index depends on the concentration of viable germs in the vaccine. This conclusion is confirmed by the results obtained when testing vaccines stored for
Listeria monocytogenes-Vaccine: Production and Control
337
Table 1. Protection index K in relationship to inoculated dose (mice experiment) L. m. serovar 1/2a R (46 J): Inoculated doselml
i.oxio' i.oxro' i.oxio-
Control Lux lu" 7,5Xl08 5,Oxl08 2,5 X108 dlso
10-4*
Protection index K;/.
10- 1,5
10-1,66
10- 1,75
10- 2,0
10-2 ,0
10-2 ,0
102,67
102 •4
102•28
102,0
102,0
102,0
10-3•0
* = dilution of starting material containing 109/ml
graded periods of time after production (T able 2) and is emphasized by the results of experiments where live and killed vaccines of equal germ concentration were tested comparatively (Table 3).
2. SpeCificity of vaccine-induced protection The homologous protection expressed in terms of the protection index K is higher than the heterologous protection (Table 4). This finding agrees with the results of Patel and Degen (10). Therefore, a listeriosis vaccine should contain the epidemiologically most important serovars (e. g. 1/2a and 4b ). Table 2. Protection index K (L.m. 1/2a) of vaccines stored for graded time intervals Time vaccine batch has been stored
Concentration of live germs at time of production
A : 1 year
1,14
B : 1/2 year
1,5 x 108
C: 1 month
8
1
X
X
10 8 10
Concentration of live germs found after storage
Protection index K Serovar 1/2a 101
1,8
X
107
3,5
X
107
102,5
7
104 ,5
9
X
10
Table 3. Comparison of protection index K of killed and live vaccines Vaccine L.m. 1/2a R: 1 x 10 8/ml inactivated live Protection index K
o
Table 4. Serovar-specificity after L.m. vaccination (mice experiment) Protection index K after infection
1/2a
1/2a
178
100
20
79
4b
Vaccination 4b
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J. Potel and J.Schulze-Lammers
3. Humoral antibody response to 1. m. vaccination Methods: Bacterial agglutination reaction (listeriosis Widal), growth test (GT) and agglutination immobilization test (AIT). The results presented in Table 5 indicate that independently from the type of vaccine (killed vaccine yielding low and live vaccine yielding high protection) only low antibody titres were recovered. This finding is consistent with the results of Kuhlmann-Berger and Potel (6) obtained in sheep. The antibody titres were sensitive to rnercapto-ethanol, indicating the presence of antibody of the IgM class. The results obtained indicate that the potency of L. rn. vaccines cannot be determined by means of recording the antibody response in mice. Table 5. Detection of humoral antibody (mice: 7 animals/group/bleeding day) after Lrn. vaccination Type of vaccine and time intervals after vaccination sera have drawn (pooled sera titer 1:) Serological method Agglutination reaction
+ 22 °C, inactivated
7
14
50
400
Growth test
< 50
< 50
Agglutinationimmobilization test (AIT)
< 50
< 50
7
+ 22°C, live 14
200
21
200
100
7
+ 37°C, live 14
50
21
100
100
control 7 50
< 50 < 50 < 50 < 50 < 50 < 50 < 50 50
100
50 < 50 < 50 < 50 < 50
Discussion A marked protection of mice against listeriosis can be achieved by the use of live attenuated (e. g. R-forms) L. m. strains only, as found in mice (Potel and Degen, 10) and domestic cattle (Ivanov et al., 5). Such vaccines are commercially available and are practically used. Therefore, it is necessary to define the optimal composition of such vaccines and to formulate the conditions of official vaccine testing. The results presented in this paper could be a helpful basis for solving these problems. The results of the mice protection tests confirmed the conclusion that killed vaccines fail to confer protection (Table 3). The protection expressed in terms of the protection index K was found to depend on the concentration of viable germs of suitable strains in the vaccines. Such strains are for example the R-forms of L. m. (Table 2). The use of too high a vaccine dose (> 10 9/ml) can result in inapparent disease (sometimes lethal). The resulting immunity may not be considered as vaccine-induced immunity. The results obtained suggest that a concentration of viable germs of 1 X lO B/ml yielding a protection index K of 10 2 (dlso = 10-4 using a starting concentration of the challenge strain of 10 9/ml = 105 germs/ml) is desirable. The smaller virulence (dlso) of the selected serovar 4b (average 10 7 germs/ml) does not allow to draw a similar conclusion for this serovar. Until now, attempts to increase
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Table 6. Protection index K in relationship to the incubation temperature Incubation temperature
L. m. serovar lI2a R (46 J): Vaccine dose 1 X 109/ml 1 X 108/ml
+22°C
102,583
+37°C
102,42
(0- and H-antigen)
102,25
the virulence of this strain by means of mice passages have failed. Generally, no mouse died after infection with serovar 4b, preventing determination of the dl so in this group. Attempts to find a more vilurent strain in clinical specimens are in progress. Furthermore, the survival rate of germs during storage of vaccines is a problem, connected with that of defining the expiration dates of the vaccines. The results presented in Table 2 indicate that a decrease in the concentration of viable germs yielded a decrease of the protection index. Empirically, it is suggested that vaccines produced according to the principles described in this paper should be used within one year after production, only. The homologous protection was more effective than the heterologous protection (Table 4). Since the serovars lI2a and 4b differ in their 0 antigen, this result was to be expected. The vaccines should contain both the 0 and the H antigens of L. m. yielding optimal protection (Table 6). The results presented in Table 5 indicate that recording of the humoral antibody response does not reflect the potency of L. m. vaccines.
References 1. Buning, U.: Auswertung der im Tiergesundheitsamt der Landwirtschaftskammer Hannover bei Schafen diagnostizierten Listeriosefalle, Inaugur.-Diss., Tierarztl, Hochschule Hannover (1975) 2. Emmerling, P., H. Finger und ]. Bockemiihl: Die Bedeutung der T-Zell-vermittelten Reaktion bei der Abwehr einer Infektion mit Listeria monocytogenes. Zbl. Bakt. Hyg., I. Abt, Orig. A 235 (1976) 142-146 3. Hahn, H. and S. H. E. Kaufmann: The role of cell-mediated immunity in bacterial infections. Rev. infect. Dis. 3 (1981) 1221-1250 4. Hof, H., P. Emmerling, H. Finger und C. H. Wirsing: Einfluf von abgetoteten Zellen von B. pertussis auf die Infektionsabwehr gegen Listeria monocytogenes. Zbl. Bakt. Hyg., I. Abt. Orig. A 240 (1978) 208-214 5. Ivanov, 1., M. Draganov, and Tz. Dikova: Studies on the active immunprophylaxis of listeriosis. VII. Internat. Sympos. on the Problems of Listeriosis. Varna 1977 6. Kuhlmann-Berget, S. and]. Potel: On the serology and immunobiology of listeriosis, VIII. Communication: Antibody against Listeria monocytogenes in sheep without and after vaccination against listeriosis. Zbl. Bakt. Hyg. A 259 (1985) 51-58 7. Potel, ].: Zur Serologie und Immunologie der Listeriose. VI. Mitt.: AgglutinationsImmobilisationstest (AIT). Zbl. Bakt., I. Abt. Orig. 207 (1968) 63-68 8. Potel, ]. und 1. Degen: Zur Serologie und Immunologie der Listeriose. I. Mitt.: Die Wachstumsprobe. Zbl. Bakt., I. Abt, Orig. 180 (1960) 61-67
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10. Patel, J. und L. Degen: Zur Serologie und Immunologie der Listeriose. V. Mitt.: Spezifische Therapie und Prophylace der Listeriose beim Versuchstier. Z. Immun.-Forsch. 123 (1962) 432-446 11. Patel, J. und L. Degen: Zur Serologie und Immunologie der Listeriose. III. Mitt.: Vergleichende Untersuchung verschiedener serodiagnostischer Methoden. Zbl. Bakt., I. Abt. Orig. 185 (1962) 204-214 12. Schulze-Lammers, ].: Erstellung einer Priifvorschrift fiir Listeria-Lebendimpfstoffe. Inaugur.-Diss., Tieriirztliche Hochschule Hannover (1983) 13. Spearman-Karber, in: O. Bonin, Quantitative virologische Methodik. Georg Thieme, Stuttgart (1973) 14. Wirsing v. Konig, C. H., H. Fischer, and H. Hof: Failure of killed Listeria monocytogenes vaccine to produce protective immunity. Nature 297 (1982) 233-234
Prof. Dr. med. J. Patel, Konstanty-Gutschow-Str. 8, 0-3000 Hannover 61