FR2578424A1 - Mutant, vaccinal strains of bacteria, process for producing them and vaccinal preparations containing them - Google Patents

Abstract

Mutant bacteria which are hypovirulent, immunogenic and carry at least one character which is a genetic marker/selector, resulting from mutations independent of those having lead to the hypovirulence. The selection characters correspond to a resistance to substances which inhibit the growth of the corresponding wild-type strains. These inhibitory substances are antibacterials, antibiotics and the like. The hypovirulence and the immunogenicitiy can be obtained by a method for reversing the dependence on an antibiotic. Application to Salmonella and more particularly to Salmonella dublin, labelled with nalidixic acid and sodium azide.

Description

 The present invention relates to mutant, vaccinating strains of bacteria and more particularly of Salmonella, easily detectable in contaminated media, as well as their methods of obtaining and the vaccine preparations containing them.

Salmonellosis are infectious, bacterial, contagious diseases of worldwide distribution, generally transmissible between animals and humans. Salmonella abortus ovis is pathogenic for sheep but not for humans. In cattle, the two most frequently isolated serotypes currently are
Salmonella typhimurium and Salmonella dublin; they are pathogenic to humans. Mention may also be made of Salmonella cholerae suis, a serotype with partial adaptation to pigs.

 The prevention of these diseases is certainly based on hygiene measures; but these are difficult, costly to implement and of limited scope. Preventive, massive and systematic administration of antibiotics is not recommended: this practice increases the antibiotic resistance of wild strains. Vaccination is a means of prevention complementary to hygiene measures; it offers an alternative to antibiotic prevention; its aim is to reduce mortality and morbidity due to septicemia, enteritis, pneumonia or salmonella abortions, to limit the number of animals carrying salmonella and therefore to reduce the frequency and intensity of salmonella contamination of the environment, meat and milk.

 Different types of vaccines have been described.

Inactivated vaccines are known, based on killed bacteria and immunity adjuvants; but these are not very immunogenic, hence the need for repeated injections leading to a certain proportion of allergic reactions, sometimes fatal. Live vaccines are more effective and generally induce more durable protection. However, fully avirulent strains are not significantly more immunogenic than killed bacteria; as apparently any vaccine strain more effective than killed vaccines has a certain residual pathogenic power for sensitive species, it is necessary to be able to follow the vaccine bacteria in individuals and herds and also to be able to detect quickly, economically and with sensitivity possible persistence of Salmonella vaccines in food of animal origin.

 The live vaccines described to date contain strains which cannot be detected and counted simply within a large and complex bacterial population, with a detection threshold comparable to that of wild strains. This last point is particularly important in countries where the legislation in force imposes, in the interest of consumers, draconian measures of food hygiene vis-à-vis certain bacterial species, including Salmonella. In fact, the vaccine strains whose multiplication is a function of the addition of growth factors given in the usual culture media present, compared to the wild strains, higher cultural requirements and / or a reduction in their growth rate, this which clearly disadvantages them during enrichment and isolation operations on selective media.

 In addition, vaccine strains which are made hypovirulent simultaneously with the acquisition of a marker character, which will either be resistance or dependence on a given substance, risk, if they become virulent again by mutations, at the same time lose their character. marker, which removes the possibility of identifying the vaccine origin of virulent strains.

 Among these vaccines, mention may be made of the vaccine described by Meyer. and col. in: Archiv für Experimentelle Veterinàrmedizin, 1981, volume 34, p. 99 to 104. It is based on a mutant strain of Salmonella dublin whose multiplication requires the presence of streptomycin. This vaccine has been used in calves; large doses should be administered orally daily for 10 days to obtain satisfactory protection.

Another vaccine has been described by Hoiseth and
Stocker in Nature, 1981, volume 291, p. 238. The vaccine strain only multiplies in the presence of certain aromatic molecules and histidine. The dependence on aromatic molecules is linked to obtaining hypoviru] .ence. These two markers do not allow direct and sensitive isolation at the same time as the enumeration of the vaccine strain from naturally contaminated samples.

 Another vaccine strain has been partially described by Meyer et al. in Monatshefte für Veterinärmedizin, 1980, volume 35, p. 405 to 410. The two characters of dependence on the metabolites are also markers obtained during the obtaining of hypovirulence, and which do not allow the direct isolation of the vaccine strain.

 Finally, mutant strains of Salmonella abortus ovis have been described in FR-A-2,464,300. These strains are reverse strains of strains dependent on the presence of streptomycin, carrying two mutations.

The first, the dependent strain, is obtained by culture of a wild strain on a medium containing streptomycin; the second, the reverse strain, is obtained by seeding and culture of the dependent strain on a medium without streptomycin. The vaccine strain thus obtained does not carry a marker allowing its easy isolation from a complex bacterial population.

 The present invention does not have the drawbacks mentioned above. On the one hand, although being of attenuated virulence, the mutant vaccinating bacterial strains, according to the invention, have an immunogenic power such that they allow a simple vaccine protocol. In addition, thanks to a specific labeling of these vaccine strains, which gives them specific characters allowing their selection, called marker / selector characters, their detection threshold by direct seeding on selective culture medium is low, whatever the sample studied. , even rich in various contaminants and / or in wild strains of the same type as that from which the vaccine strains originate. Indeed, even if the multiplication of vaccine bacteria is less rapid than that of wild bacteria, their selective isolation allows at the same time their identification and their numbering can be obtained without prior enrichment, by direct seeding and culture on a sufficiently selective medium, chosen taking into account the marking of the vaccinating strains characteristic of the invention, and possibly the properties of the strain from which these strains are derived. immunizing. Finally, as this selective isolation of the vaccine strains is not based on markers linked to the mode of obtaining hypovirulence, we can effectively monitor the absence or possible occurrence of a reversion to virulence.

In what follows, we only refer to
Salmonella, but the process for obtaining vaccinating bacterial strains, that is to say useful for vaccinating animals against these bacteria and therefore immunogenic and hypovirulent, further carrying at least one chromosomal marker different from those obtained during the introduction of hypovirulence, and allowing the identification of bacteria after isolation and also their sensitive and quantitative detection by direct culture on an appropriate selective medium, can advantageously be applied to obtaining other bacterial vaccine strains and the strains mutants then obtained, as well as the vaccines which contain them, are another object of the invention. Among these bacteria, there may be mentioned:
Escherichia, Pasteurella, Listeria, Corynebacterium and Mvcobacterium.

 The bacteria of the invention are immunogenic and hypovirulent, which means that they can be used as vaccine strains; their administration results in good protection of animals against infections by virulent strains, without significant side effects. They include at least one marker character, which will allow their selection.

 These marker / selector characters can be resistance to agents inhibiting the growth of bacteria, such as certain antibacterials, antibiotics or toxic substances, against which chromosomal resistances can be selected without effects which are ultimately incompatible with immunogenicity and hypovirulence of the strain. Chromosomal resistance is preferred because plasmid resistance is relatively unstable and could be transmitted to other bacteria. Chromosomal resistance to antibacterials which may be used in the treatment or prevention of bacteria of the parental type is avoided, or to substances belonging to the families of disinfectants and / or antiseptics. In particular, the resistances that are rarely encountered in wild bacteria will be chosen. In addition, those skilled in the art will choose among the available antibacterials those which, for the bacteria in question, will give a reduction in virulence without significant loss. immunogenic properties. Among the antibiotics which may be suitable, mention may be made of beta-lactams, for example penicillin, ampicillin, cefalotin; cephalosporins; aminoglycosides, for example streptomycin, gentamicin; tetracyclines; macrolides such as erythromycin; rifamycins, such as rifampicin; quinolones, such as nalidixic acid or flumequin; isoniazids; fusidic acid; chloramphenicol; fosfomycin; the tetracalne; crital violet or sodium azide. For Salmonella, nalidixic acid or sodium azide is particularly preferred.

 Although a selection factor is in most cases sufficient to distinguish the bacteria according to the invention in a sample with little contamination, it is preferable to have at least two markers / selectors in order to ensure detection in a single step , the identification and the counting of the vaccine bacteria of the invention, from samples containing numerous bacteria of various species. These markers / selectors are chosen so that the detection threshold for the vaccine strain by direct seeding on solid medium containing the appropriate concentrations of selection factors is very low. Enrichment in a liquid medium containing the appropriate concentrations of selectors, carried out before inoculation on solid selective medium, is not essential but remains a modality achievable in parallel with direct inoculation. Among the pairs of marker selectors, we prefer that consisting of l nalidixic acid and sodium azide for Salmonella.

It is preferable to add the hereditary marker characters to bacteria made previously hypovirulent and which are always good immunogenic agents. These can be obtained by a known process known as reversing dependence on an antibacterial. This process has already been applied to the preparation of vaccine strains and in particular to
Salmonella abortus ovis, as described in FR-A2.464.300 using streptomycin. However, the operating conditions of this reversion process are not general; they must be adapted by the specialist to the bacterial species considered, and in addition, a population of reverse strains is obtained which are heterogeneous as regards both their hypovirulence, their immunogenicity and their sensitivity to streptomycin and to other aminoglycosides; therefore the selection of the suitable mutant strain is important.

Thus, in the case of a search for suitable reverse strains from a clone dependent on Salmonella dublin, almost half of the isolated reverse strains had a virulence still too high to be acceptable vaccine strains.

As antibacterials in this reversion process, it is possible to use antibiotics and other toxic substances from bacteria, mentioned above as usable for the introduction of a marker / selector character, according to the invention. It is understood that the antibacterial which will be chosen for obtaining hypovirulent and immunogenic mutant strains by the "reversion" process must be clearly different from that which will be used to introduce any marker / selector character into the bacterial strain according to the 'invention. In the case of
Salmonella, the aminoglycosides and in particular streptomycin make it possible to obtain, during mutations of the "reversion" process, a virulence spectrum which is particularly suitable.

 It is possible to use bacterial clones having undergone a slight reduction in their virulence, and to seek in subsequent steps to further reduce their virulence with or without the addition of markers allowing, after isolation, to distinguish the strains obtained from the wild strains. . The other, preferred, possibility is to immediately ensure the vaccine acceptability of certain strains and then to mark them by modifying as little as possible their virulence and their immunogenicity, by choosing as markers / s-voters , those that facilitate the isolation, identification and counting of vaccine strains.

 Another subject of the invention is a process for obtaining strains of hypovirulent and immunogenic bacteria, vaccinating and carrying at least one hereditary marker which can serve as a selector. This process consists in particular in obtaining strains of bacteria resistant to an antibacterial by culture of hypovirulent and immunogenic strains, and not resistant on a medium comprising this antibacterial at a concentration such that colonies of mutant bacteria, resistant to this antibacterial can be isolated.

After cloning, the strains with the least modified level of virulence and immunogenicity are retained. According to one aspect of the invention, the hypovirulent and immunogenic strains are mutant strains, previously obtained by a "reversion" method, but any other bacterial strain sufficiently immunogenic and weakly virulent could be used, whatever its method of obtaining.

 In the case where it is chosen to also introduce a second selector marker to facilitate the direct quantitative isolation of the vaccine bacteria from various contaminated media, the method of the invention comprises an additional step, namely the culture of the clones previously chosen on a medium comprising another antibacterial, of structure and mechanism of action sufficiently different from ceudqutiigseseSdans the previous steps, and in sufficient quantity so that mutant bacteria resistant to this other antibacterial can be isolated. After cloning, the strains having retained the first marker character and of the same level of virulence and immunogenicity are retained.

 It has been found in certain cases that the order of use of the antibacterials used to modify the virulence or to mark the strains is to be taken into consideration at the same time as the nature of these antibacterials in order to arrive at suitable strains as vaccine agents and it is best to perform preliminary experiments to determine which order is best for a given couple.

 As parental strain, use is preferably made of strains sensitive to the antibiotics usually used against bacteria of the same species, in particular those not containing antimicrobial resistance with plasmid support, in order to avoid possible dissemination of this trait by administration. of the vaccine. Among wild strains, highly immunogenic and virulent strains are preferred, and especially those carrying, moreover, a characteristic present on a minority of wild strains.

 Among the Salmonella, there may be used in particular the Salmonella tvphimurium, Salmonella dublin, which are particularly useful for preparing a bovine vaccine, or the Salmonella abortus ovis for preparing a vaccine for sheep; Salmonella dublins are preferred for cattle; mixed live vaccines containing strains from several serotypes are another object of the invention.

 The culture media used in the various stages of the process of the invention are those known to be suitable for the development of bacterial colonies of the species considered, to which the appropriate antibiotic or antibacterial agents are added according to the stages. In the case of Salmonella, an agar-trypticase-soy medium is used in particular, marketed by BioMérieux, (Lyon, France).

 For obtaining hypovirulence, it is preferred to use an antibiotic from the group of aminoglycosides, particularly streptomycin. Streptomycin is introduced into the culture medium in an amount greater than the minimum inhibitory concentration of wild strains carrying resistance plasmids and at a concentration of between 5 and 2000 pg / ml, preferably at a rate of 500, ug / ml of streptomycin .

To obtain useful markers as selectors, the selected antibacterials are introduced into the culture media at a concentration at least equal to the minimum inhibitory concentration for the wild strains of the species considered. For the
Salmonella, nalidixic acid is preferred to which little
Wild Salmonella are resistant, and sodium azide to which no wild Salmonella is resistant; the first is introduced into the culture medium at a concentration between 5 and 1000 pg / ml, the second between 50 and 1500 pg / ml.

 At each stage of the process, the bacteria which have acquired the desired characteristics will be selected and classified, which multiply rapidly on the culture medium and retain the acquired characteristics.

The study of residual virulence, both of
So-called reverse Salmonella as reverse and labeled Salmonella can be carried out using mice, previously inoculated with the Salmonella strains to be studied; the plantar subcutaneous inoculation of approximately 106 viable bacteria per animal is preferably used. The virulence of the mutant strains and of the parental strain is compared, by studying the weight of the spleen of the mice and the number of viable Salmonella per spleen , 6 days after the test injection.

 The study of immunogenicity can also be carried out in mice. The animals are vaccinated by injecting each animal with 106 bacteria of the strain to be studied in solution in 0.2 ml of physiological saline, by dorsal subcutaneous route. On a batch of vaccinated mice, the persistence of vaccine bacteria in the spleen and liver is studied after one month. Another batch receives a test injection of a virulent strain of Salmonella by plantar subcutaneous route and 6 days later, the number of Salmonella present in the spleen of each animal is determined.

 Another subject of the invention consists of vaccines comprising, in combination with a pharmaceutically acceptable vehicle, hypovirulent, immunogenic bacteria carrying at least one marker / selector character according to the invention. These vaccines can be administered prophylactically to animals likely to suffer from infection with virulent bacteria of the corresponding species. These vaccines can be administered to animals by oral, intramuscular, subcutaneous, conjunctival or nasal route. The conjunctival route is preferred, since it ensures the best compromise between ease and safety of use, minimal contamination of the material. culture and stimulation of the immune system by a route close to the most likely contamination route.

 Unit doses of vaccine are generally).

preserved in lyophilized form, dilutable at the time of use, in which are found the vaccinating bacteria associated or not with the usual preservatives.

 The safety of vaccines has been studied in animal breeds susceptible to vaccination. The animals were administered a dose corresponding to at least the dose considered to be vaccine and studied the serological reaction to bacterial antigens, the persistence and excretion of vaccine bacteria and noted any rise in temperature or possible clinical sign which could be the 'a certain pathogenic activity of the bacteria injected.

In the case of bacteria of the Salmonella dublin type, objects of the invention, a reference sample of which has been deposited in the National Collection of
Culture of Microorganisms (CNCM), Paris, France, January 31, 1985 under nOI-419, the vaccine dose for conjunctival administration in cattle will contain from 107 to 1010 bacteria; for administration by subcutaneous or intramuscular route, the doses will be from 106 to 109 and by oral or nasal route from 107 to 101 viable bacteria per animal.

Such a vaccine increases the resistance of animals to infections due to several serotypes of
Salmonella and in particular those with Salmonella dublin and
Salmonella typhymurium, which is another advantage of the invention.

In the following, an embodiment of the invention is described
Example: Obtaining vaccine strains of Salmonella dublin
The parental bacteria are wild type Salmonella dublin bacteria, carrying the Vi antigen, as defined in an agglutination test on a slide with a specific anti-serum, marketed by the Pasteur Institute.

 The parental strain is the Salmonella dublin strain, reference 136-69 (Institut Pasteur).

a) the bacteria were cultured on 20 boxes of trypticase-soy medium, marketed by
Bio-Mérieux (France) under the reference nO 5146, supplemented by 500, ug / ml of streptomycin. The bacteria were seeded at the rate of 1.5 × 10 9 per box of
90 mm diameter petri dish. After 7 days of incubation at
T = 370C, the 60 colonies appeared; after cloning, only one of the 60 clones was found to be completely streptomycin-dependent. This clone has been retained.

 b) the strain was then cultured on a medium without streptomycin.

 Among the clones that have become capable of multiplying again in the absence of streptomycin, 26 clones sensitive to streptomycin were selected, giving rise to colonies morphologically similar to those of the parental strain and having retained the Vi antigen, and having at least 1 mm in diameter after 36 hours. Their virulence was checked on mice. Half of them having a virulence too close to the wild strain was rejected. The 5 most virulent strains among the group of attenuated strains were selected, and the persistence of an immunogenic power and of the Vi antigen was verified.

c) bacteria from each clone were then seeded at the rate of 2.5 × 10 9 bacteria per 90 mm diameter petri dish containing trypticase-soy medium, supplemented with 100 μg / ml of nalidixic acid. After incubation for 3 days at T = 370C, mutant bacteria were cloned and the clones still having the Vi antigen were preserved and hypovirulence and immunogenicity comparable to those of the parental reverse clones;
d) the 5 clones thus retained were seeded, at the rate of 2.5 × 10 9 bacteria per ml, on a trypticase-soy medium, supplemented with 100 μg / ml of sodium azide.

 After incubation for 3 days at T = 370C, the properties of the mutant bacterial colonies which appeared have been studied in order to select those which can enter into a vaccine composition.

 The study concerned the virulence, immunogenicity and stability over time of the acquired marker characters.

 Among the 26 reverse clones studied, half had virulence very close to that of the parental strain. Indeed, while after plantar subcutaneous injection of 106 bacteria of the parental strain, the spleens of OF1 mice contained a number of viable bacteria of 5.53 (in log decimal), after injection under the same conditions of bacteria of 12 other strains, the rats contained 4.9 to 4.0 (log) bacteria, while the other group of strains only infected 2.7 to 1.8 (log) viable bacteria.

 The immunogenic activity of the latter was studied by the method previously described. The vaccine strain studied V, injected at a dose of 106 viable bacteria into OF1 mice, not vaccinated, according to the virulence test previously described, infects the spleen at an average height of 2.96 (in log), against 5.59 ( in log) (one mouse died in the 6 mice having received the parental wild strain under the same conditions. The mice vaccinated with 106 viable bacteria of strain V and tested with the parental strain, are infected at an average height of 2.28 ( in log) of the test strain per spleen, against 4.86 (in log) in the spleen of controlled, tested, but not vaccinated mice.

 A sample of the strain V, lyophilized, was deposited with the CNCM under the nO I-419 after verification of the conservation of its characteristics of hypovirulence and immunogenicity after lyophilization.

We have also studied other important characteristics of strain V and in particular:
1) its stability: no significant change in its virulence and its immunogenicity after 10 passages in vitro or in mice; resistance to the two antibacterials is retained after these passages.

 2) its development on a selective medium, such as the Salmonella-Shiqella medium, marketed by the Pasteur Institute, to which were added 100 μg / ml of nalidixic acid and 100 μg / ml of sodium azide.

After inoculation of aliquots of the same bacterial suspension, the number of colonies expressed in log decimal is substantially the same after culture on normal medium (log q = 2.19 and log q = 2.40) "or on supplemented medium ( log q = 2.29 and 2.53 respectively) whereas the parental wild strain does not give colonies on this supplemented medium;
3) the duration of the persistence of vaccine bacteria in the spleens of mice which have been injected by the plantar subcutaneous route 106 strain V bacteria. In the spleen of mice which have been immunized, the presence of viable vaccine bacteria is not detected than in one of the seven mice killed eight weeks after vaccination.In mice vaccinated and then tested eight weeks after vaccination, the resistance to challenge infection was comparable to that of mice tested earlier after vaccination, which then housed more frequently vaccine bacteria.

 4) the specificity of the immunity conferred in mice. There is no protection against infection by Listeria, but protection against infections with Salmonella dublin and Salmonella typhymurium, however slightly lower against bacteria of the second type, whether or not the vaccine bacteria of strain V are lyophilized.

 5) the safety of the vaccine strain V vis-à-vis adult cows and calves, after administration by conjunctival route at a dose of 1010 bacteria per animal, in 2 drops of 33 l of isotonic saline solution. A frequent and lasting rise in the rectal temperature of the animals was not observed after vaccination, nor any ocular reaction, nor any significant attack of diarrhea, despite the transient, but low concentration, presence of vaccine bacteria in the faeces. The increase in antisalmonella antibody titers in the blood was most marked in adults.

Claims (21)

 1. Mutant, vaccinating bacteria, characterized in that they carry at least one hereditary marker / selector character which appears independently of any mutation which has led to hypovirulence and to immunogenicity.  2. Bacteria according to claim 1, characterized in that hypovirulence and immunogenicity are obtained by reversion of a phenotype dependent on an antibacterial.  3. Bacteria according to one of claims 1 or 2, which have as a marker / selector character increased resistance to an antibacterial.  4. Bacteria according to any one of claims 1 to 3, characterized in that the antibacterials are chosen from antibiotics, such as beta-lactams, aminoglycosides, tetracyclines, cephalosporins, macrolides, rifamycins, quinolones, isoniazids, chloramphenicol, fusidic acid , fosfamycin and tetracalne, violet crystal or sodium azide.  5. Mutant, vaccinating salmonella having the characteristics mentioned in any one of the preceding claims.  6. Salmonella, according to claim 5, reverses of an antibiotic-dependent phenotype and resistant to nalidixic acid.  7. Salmonella according to claim 6, further resistant to sodium azide.  8. Salmonella according to any one of the preceding claims, reverses of a phenotype dependent on streptomycin.  9. Salmonella according to claim 8 of the dublin type.  10. Salmonella dublin, according to claim 9, carrying the Vi antigen.  11. Salmonella dublin according to claim 10, having the characteristics of the strain, a sample of which has been deposited in the National Collection of Culture of Microorganisms (CNCM) under nO I-419.  12. Salmonella dublin according to one of claims 9 to 11, in lyophilized form.  13. Process for obtaining immunogenic hypovirulent bacteria, marked with a character not linked to obtaining hypovirulence, characterized in that it consists:  1) cultivating immunogenic bacteria on a medium containing an antibiotic in a quantity such that mutant bacteria resistant to this antibiotic can be isolated;  2) to select from resistant bacteria those which are dependent on this antibiotic;  3) to inoculate these resistant and dependent bacteria on a medium lacking this antibiotic and to isolate the reverse and hypovirulent bacteria which have multiplied on this medium;  4) to cultivate these reverse, hypovirulent bacteria on a medium containing another antibacterial in a quantity such that mutant bacteria resistant to the antibacterial and hypovirulent can be isolated.  14. A process for obtaining labeled, vaccinating bacteria, characterized in that the bacteria obtained by the process according to claim 13 are then cultivated on a medium containing another antibacterial in an amount such that mutant bacteria resistant to the latter can be isolated. and hypovirulent.  15. A method of obtaining according to claim 13 of Salmonella hypovirulent, immunogenic, labeled, characterized in that the antibiotic is chosen from aminoglycosides and the antibacterial from quinolones.  16. The method of claim 14, for obtaining Salmonella, characterized in that the second antibacterial is sodium azide.  17. The method of claim 16, for obtaining Salmonella dublin, characterized in that the first culture is carried out in the presence of streptomycin at a concentration in the culture medium of between 5 and 2000 pg / ml, as the culture of bacteria reverses is carried out in the presence of nalidixic acid at a concentration between 5 and 1000 lug / ml and that the last culture is carried out in the presence of sodium azide at a concentration between 50 and 1500 fg / ml.  18. Method according to "claim 17, characterized in that streptomycin is introduced in an amount of 500 ug / ml, nalidixic acid in an amount of 100 ug / ml and sodium azide in an amount of 100 fig / ml.  19. Live attenuated vaccine comprising hypovirulent, immunogenic bacteria and carrying at least one marker / sel.ector character according to one of claims 1 to 12, associated with a pharmaceutically acceptable excipient.  20. Vaccine according to claim 19 for protection against Salmonella infections, characterized in that the immunogenic agent consists of mutant Salmonella dublins according to claims 9 to 12.  21. Vaccine sel.onla claim 20 for the protection of cattle, characterized in that the unit dose per animal for administration by the conjunctival route, comprises from 106 to 1010 Salmonella dublin according to claim 11, in lyophilized form.