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Combination of vaccination and competitive exclusion to prevent Salmonella colonization in chickens: experimental studies
International Journal of Food Microbiology 49 (1999) 35–42
Combination of vaccination and competitive exclusion to prevent Salmonella colonization in chickens: experimental studies a, b a a U. Methner *, P.A. Barrow , A. Berndt , G. Steinbach a
Federal Institute for Health Protection of Consumers and Veterinary Medicine, Jena Branch, Naumburger Strasse 96 a, D-07743 Jena, Germany b Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7 NN, UK Received 2 October 1998; received in revised form 23 February 1999; accepted 31 March 1999
Abstract Vaccination and competitive exclusion (CE) represent accepted prophylactic measures to control Salmonella infections in chickens. To use the advantages of both the CE technique and vaccination with live Salmonella vaccines the combination of these methods was studied. In three experiments, SPF chickens were pre-treated using combined or unique administration of CE and vaccination with a live Salmonella typhimurium strain on days 1 and 2 of life and challenged with the antibiotic resistant, but otherwise isogenic mutant of this Salmonella typhimurium strain on days 3, 15 or 40 of life. The caecal colonization of both the vaccine and the challenge strain and the antibody response after infection were examined to evaluate the protective effects of the different combinations. The exclusion effect of CE cultures against Salmonella infection could be seen in very young chicks and was still considerable on day 40 of life of the birds. The Salmonella wild-type strain used as vaccine alone also resulted in a substantial protective effect against homologous challenge. The combined administration of competitive exclusion and immunization using the Salmonella wild-type strain as vaccine resulted in a considerable additional protective effect above the level of the respective exclusive application of these prophylactic measures. Administration of the Salmonella vaccine strain prior to or simultaneously with the CE culture produced the best protective effect, because such combinations ensure an adequate persistence of the vaccine strain as prerequisite for the expression of colonization inhibition effects and a strong immune response. The full exploitation of this potential using attenuated live Salmonella vaccines will require the presence of high inhibitory and immunogenic properties in the vaccine strain after attenuation of a selected parent strain. The combination of competitive exclusion and vaccination as a new measure in integrated control programmes against Salmonella infection in poultry could result in a considerable increase of protection in both very young and older chickens. 1999 Elsevier Science B.V. All rights reserved. Keywords: Salmonella; Live vaccines; Competitive exclusion; Vaccination; Inhibition; Chickens
1. Introduction *Corresponding author. Tel.: 1 49-3641-804-267; fax: 1 493641-804-228. E-mail address: [email protected] (U. Methner)
Different methods may be used to increase the resistance of both very young and older chickens
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00051-3
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U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
against Salmonella infection (Mead and Barrow, 1990). Using competitive exclusion (CE) cultures, the greater resistance of adult birds can be transferred to day-old chicks. Administration of live Salmonella vaccines to birds may also induce an exclusion or inhibition effect within a matter of hours in addition to simultaneously stimulating the development of a true immunity to infection, both of these effects resulting in a further increase of resistance to Salmonella infection. Exclusion effects (Nurmi and Rantala, 1973; Nuotio et al., 1992; Cameron et al., 1996) induced by complex intestinal flora and those induced by live Salmonella vaccines (Barrow et al., 1987; Berchieri and Barrow, 1990; Methner et al., 1997) may both be used in very young birds. After administration of Salmonella vaccines immunological effects may be used in immunocompetent chicks and adult birds with an established intestinal microflora (Hassan and Curtiss, 1994; Methner et al., 1994). To obtain maximum protective effects, CE cultures should be administered to chicks as soon as possible after hatching (Bolder et al., 1992). Live Salmonella vaccines given at 1 day of age ensure early intestinal colonization by the vaccinating strain (Cooper et al., 1994; Vielitz et al., 1995), thereby producing both an inhibitory effect and an early immunological response. Competitive exclusion is effective against different Salmonella serovars, while inhibition between salmonellas is probably most effective between strains of the same serovar. The possibility exists, therefore, that a combination of non-specific CE and more specific inhibition and vaccination with live Salmonella vaccines against serovars epidemiologically important for human beings may result in a higher protection against these serovars than use of either of these methods alone. One of the problems in studying different prophylactic measures to reduce or prevent Salmonella colonization of the alimentary tract of chickens lies in the terminology used. In this paper the following terminology is applied purely to facilitate discussion. Their use does not imply an understanding of the differences between, for example, exclusion and inhibition effects. Exclusion effect is the mechanism generated by a CE culture to prevent colonization of Salmonella challenge organisms as well as the colonization of the vaccine strain in the case of combined use. Inhibitory effects are defined as those occurring only between
Salmonella strains. Immunological effects after administration of Salmonella strains (attenuated or nonattenuated) are defined as those where some of the characteristics of classical immune response are evident. It has been the purpose of our experiments to study competitive exclusion, inhibitory and immunological effects after combined use of CE and vaccination against Salmonella infection in chickens of different ages. In order to evaluate the principal potential and limitations of a combination of these methods irrespective of the type and degree of attenuation of a live vaccine, a non-attenuated wildtype strain of Salmonella was used for immunization of the chicks.
2. Materials and methods
2.1. Chickens Specific pathogen-free (SPF) chickens (white leghorn) were hatched in the facilities of the institute from eggs received from Charles River Deutschland (Extertal, Germany). Experimental and control groups were kept in separate rooms. Commercial feed (powdered) without antibiotics or other additives and drinking water were both available ad libitum. Cleaning and feeding regimens were organized which prevented cross-contamination effectively throughout the course of the experiments.
2.2. Bacterial strains and culture A rifampicin-resistant variant (R) of the Salmonella typhimurium wild-type strain 421 (DT 204) was used for oral vaccination of the chicks on their first or second day of life with or without combined use of a commercial CE culture (Aviguard , Bayer AG, Germany). The viable numbers of the non-attenuated Salmonella typhimurium strain 421R (STM 421R) administered orally were 1–3 3 10 7 cfu / bird. The CE culture was dissolved in accordance with the manufacturer’s instructions and administered orally. Oral infection of the chickens was carried out using a nalidixic acid-resistant variant (N) of the wild-type strain Salmonella typhimurium 421. This challenge strain was already used earlier (Methner et al.,
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
1997). To enable the differentiation and the exact quantitative determination of both the vaccine and the challenge strain in the caeca, a rifampicin (R) and a nalidixic acid-(N)-resistant variant of Salmonella typhimurium 421 were used in the experiments. These strains are spontaneous mutants produced by the method of Smith and Tucker (1980). After purification and replating the mutants were checked for virulence in chicks and compared with the parent strain. The strains were stored in the Microbank system (Pro-Lab Diagnostics, Ontario, Canada) at 2 208C. Oral administration was performed by instillation into the crop of the birds, using syringes with an attached flexible tube. The volume of bacterial suspension used was 0.1 ml in birds until the age of 1 week and 1.0 ml in older birds. The Salmonella suspensions for inoculation were cultivated by shaking in nutrient broth I (SIFIN, Berlin, Germany). Counts were estimated by measuring extinction at 600 nm and comparing with a calibration graph determined for the strains used. Additionally, counts for the bacterial suspensions used were established by plate counting on nutrient agar (SIFIN, Berlin, Germany) after preparation of the suspensions.
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2.3. Experimental design and bacteriology To ensure that the chickens used in the experiments were free of salmonellas, hatching material (egg shells, faeces, organs from unhatched birds) was examined for their presence. The samples were preenriched in buffered (pH 7.2) peptone water (Merck, Darmstadt, Germany). Selective enrichment took place in Rappaport–Vassiliadis broth (Merck, Darmstadt, Germany) over 18–24 h at 428C. Loopfuls of material were spread on deoxycholate–citrate agar and xylose–lysine–deoxycholate agar (Merck, Darmstadt, Germany). The effect of pre-treatment of chicks with STM 421R with and without combined administration of CE culture against infection with STM 421N was studied in three different experiments (Table 1). The pre-treated chickens were challenged on day 3 (experiment 1), on day 15 (experiment 2) or on day 40 (experiment 3) of age. The challenge dose was 10 5 cfu / bird on day 3 of age and 10 9 cfu / bird on days 15 or 40 of age, respectively. Protective effects were evaluated by establishing the viable counts of the challenge strain STM 421N in whole caeca (with content) in experiment 1 (on
Table 1 Counts (x¯ log 10 cfu / g) of Salmonella typhimurium 421 N (STM 421N) in caeca of chickens given this strain after pre-treatment with live STM 421R wild-type strain and / or a competitive exclusion culture (CE) Age (days)
Group 1
2
3
4
5
6
1 2
STM 421 R –
CE –
STM 421 R CE
CE STM 421 R
CE1STM 421 R –
Control –
3 5a 9a
Experiment 1: Challenge: STM 421 N (10 5 cfu / bird) 3.7 6 3.6 6 3.3 6 6 6 4.1 3.9 3.6 6
2.5 6 1.1 1,2,6
1.6 1,2,6 1.2 1,2,6
7.6 6 8.7
15 18 b 22 b
Experiment 2: Challenge: STM 421 N (10 9 cfu / bird) 3.7 6 3.8 6 2.1 6 6 6 4.2 3.9 3.8 6
4.3 6 3.7 6
1.6 1,2,6 0.6 1,2,6
7.0 7.2
40 43 c 48 c
Experiment 3: Challenge: STM 421 N (10 9 cfu / bird) 2.9 6 2.3 6 0.8 1,2,6, 6 1,6 2.9 1.4 0.8 1,6
0.2 1,2,6, 0.2 1,2,6
0.5 1,2,6 0.4 1,2,6
4.7 4.3
a
Standard error: Standard error: c Standard error: 1,2,6 Significantly b
60.56. 60.56. 60.39. lower than group 1, 2, 6 (P,0.05).
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U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
days 2 and 6 after challenge) or a section of caecal mucosa with content in experiments 2 and 3 (on days 3 and 7 or 8 after challenge) from five chickens / group and day of examination. In all groups, using STM 421R as vaccine, the number of organisms of this strain in caeca was detected simultaneously. Bacterial counts in the caeca were estimated using a standard plating method as described previously (Methner et al., 1995b). Briefly, the caeca were removed aseptically, placed in sterile tubes and weighed. Phosphate-buffered (pH 7.2) saline was added to the material at a ratio of 1:3, afterwards the samples were homogenized using an Ultra-Turrax (Janke and Kunkel, Staufen, Germany) blender. Homogenized organ samples were diluted in PBS and plated on deoxycholate–citrate agar containing sodium nalidixate (50 mg / ml) or rifampicin (100 mg / ml) to determine the number of challenge and vaccine organisms, respectively. Buffered peptone water was used for pre-enrichment of the organ homogenates (1:10), incubated overnight at 378C and subcultured on the solid medium containing sodium nalidixate or rifampicin.
2.4. Antibody detection Detection of antibodies in serum was performed by means of an indirect ELISA on polystyrene microtiter plates (Greiner, Solingen, Germany). LPS (phenol–water extract) of Salmonella typhimurium served as antigen. To detect the IgG bound to the antigen, rabbit anti-chicken IgG (H 1 L) (ICN Pharmaceuticals, Amsterdam, The Netherlands) and the peroxidase (PO) conjugates, goat anti-rabbit / IgG PO (Nordic Immunological Laboratories, Tilburg, The Netherlands) or rabbit anti-goat IgG / PO (Sigma– Aldrich, Deisenhofen, Germany) were used. Orthophenylenediamine was used as substrate. In accordance with the expected antibody concentrations, the optical density for antibody of class IgG in serum (diluted 1:400) was determined.
2.5. Statistical analysis Viable counts were converted into logarithmic form. For the purposes of statistical analysis, a viable count of log 10 , 1.47 (limit for direct plate detection) obtained from a sample becoming positive only after enrichment was rated as log 10 5 1.0. A sample which
yielded no Salmonella growth after enrichment was rated as log 10 5 0. Data were evaluated by multifactorial variance analysis. The factors considered were group and time. The optical density expressing the parameter of the antibody concentration was converted into logarithmic form ( → normal distribution) and tested for differences. P values of , 0.05 were regarded as statistically significant (software: Statgraphics Plus, Rockville, MD, USA).
3. Results
3.1. Colonization of the challenge strain Studies on CE and inhibition (challenge on day 3 of life) using the non-attenuated strain STM 421R as vaccine (Table 1) clearly demonstrated the protective effect of the CE culture (group 2) against Salmonella challenge in comparison to untreated controls (group 6). A difference of about 3–4 log 10 units in the number of challenge organisms was seen between these groups. The inhibitory effect of STM 421R (group 1) against STM 421N was nearly identical with the exclusion effect produced by the CE culture and amounted also to about 4 log 10 units. A combination of both led to a further significant increase in protection on day 9 of life in groups 4 and 5. Studies on CE and vaccination using the nonattenuated STM wild-type strain (challenge on days 15 or 40 of life) showed that the protective effect of a CE culture against Salmonella infection was still substantial in 15-day-old chickens (Table 1). The difference in the number of STM 421 N in caeca between birds pre-treated with the CE culture and untreated controls was about 3 log 10 units. The protection level generated by the STM 421 R wildtype strain equalled that obtained with the CE culture. A considerable increase of protection was found after administration of CE culture and STM 421R simultaneously to day-old chicks as compared to the exclusive application of either of these methods alone. A strong exclusion effect produced by the CE culture alone was found also after challenge of 40day-old birds in experiment 3 (Table 1). These chickens and the birds vaccinated with STM 421R only showed a reduction in the number of challenge
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
organisms compared to untreated controls of about 2–3 log 10 units. Combined pre-treatment of chickens with the CE culture and the vaccine strain 421R almost completely prevented the intestinal colonization by the challenge strain 421N administered in a dose of 10 9 cfu / bird. There were no significant differences between groups 3, 4 and 5.
3.2. Colonization of the vaccine strain Colonization by the Salmonella vaccine strain was affected to a varying degree, depending on the mode of administration of the CE culture (Table 2). In experiments 1 and 2 the administration of the CE culture 24 h after the vaccine strain had no significant impact on the intestinal colonization of the vaccine. The application of the CE culture both before and simultaneously with the vaccine strain 421R resulted in a considerably reduced colonization of the vaccine in comparison to single administration. The STM 421R wild-type strain applied as vaccine to day-old chicks persisted in the caecum until 6 weeks of life in numbers of about 5.0 log 10 cfu / g (data not shown). In all groups with combined administration of a CE culture, the caecal colonization by strain 421R was considerably suppressed (to about 1.2–2.5 log 10 cfu / g), but not completely eliminated during this additional period. The different modes of combination of CE culture and STM
39
421R vaccine strain did not result in significant differences in the caecal concentration of this strain as found in younger chicks.
3.3. Antibody response The IgG antibody response in the serum measured after challenge of the chicks on day 15 and 40 of age (Table 3) exhibited a high positive correlation with the persistence of the vaccine strain (Table 2). The intensity of the antibody response after infection is determined by the extent of caecal colonization of the vaccine strain which depends on the mode of administration of the CE culture and the live vaccine strain. The group administered the vaccine alone showed, in fact, the highest colonization of the vaccine strain, but not the strongest IgG response in serum.
4. Discussion In these studies, the potential and limitations of a combination of immunization with Salmonella live vaccines and CE have been tested for the first time. Attenuated live vaccines which have already been approved for use in poultry, or are still being tested, vary in their properties on account of their different type of attenuation. Therefore, no attenuated vaccine strain was used for the immunization, but a non-
Table 2 Counts (x¯ log 10 cfu / g) of Salmonella typhimurium 421R (STM 421R) in caeca of chickens given this strain as vaccine alone or in combination with a competitive exclusion culture (CE) Age (days)
Group 1
3
4
5
1 2
STM 421 R –
STM 421 R CE
CE STM 421 R
CE1STM 421 R –
3 5a 9a
Experiment 1: Challenge: STM 421 N (10 5 cfu / bird) 8.8 8.2 8.5 7.8
4.5 1,3,5 4.2 1,3,5
6.6 1,3 5.7 1,3
15 18 b 22 b
Experiment 2: Challenge: STM 421 N (10 9 cfu / bird) 6.8 6.5 6.9 5.9
2.5 1,3 1.4 1,3
3.6 1,3 2.1 1,3
a
Standard error: 60.21. Standard error: 60.44. 1,3,5 Significantly lower than group 1, 3, 5 (P,0.05). b
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
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Table 3 Mean optical density of Salmonella antibodies of isotype lgG in serum of pre-treated chickens after challenge with Salmonella typhimurium 421 N (STM 421N) on days 15 or 40 of life Age (days)
Group 1
2
3
4
5
6
1 2
STM 421R –
CE –
STM 421R CE
CE STM 421R
CE1STM 421R –
Control –
15 18 a 22 a
Challenge: STM 421 N (10 9 cfu / bird) 1.19 01 0.87 1.35 01 1.40
01 0.76
0.26 1 1.03
01 01
40 43 b 48 b
Challenge: STM 421 N (10 9 cfu / bird) 0.80 0.02 1 1.01 1.06 0.41 1 1.40
0.44 0.57
1.03 1.24
0.14 1 0.83
a
Standard error: 60.29. Standard error: 60.20. 1 Significantly lower than group 1 (P,0.05). b
attenuated Salmonella wild-type strain. The reason for this was to draw conclusions concerning the maximum protective effects to be achieved as it can be assumed that the protective properties of an attenuated vaccine strain will not be superior to those of a non-attenuated wild-type strain. The colonization characteristics of the strain STM 421 have been well documented (Methner et al., 1995a, 1997). Using a dose of 1–3 3 10 7 cfu / bird of STM 421 in day-old chicks a strong colonization of the intestine and invasion into liver is induced, but there will be no clinical manifestations among the animals. Only some birds show a slight inflammation of the intestine in the first week after administration (Methner and Barrow, 1997). In order to consider different stages of development of the host, the oral challenge of chickens pre-treated in different ways was performed in three independent experiments on days 3, 15, or 40 of life. The exclusion effect produced within a short time after application of the CE culture in chicks which are not completely immunocompetent, was nearly identical with the inhibitory effect of STM 421R against STM 421N (experiment 1). The combination of both resulted in an additional increase of protection in the groups given the CE culture before or simultaneously with the vaccine. It can be assumed that in chicks aged only a few days which are not completely immunocompetent no specific immune response occurs which could result in such considerable protective effect within 2 days after challenge (Barrow et al., 1987; Methner and
Steinbach, 1997). The occurrence of an additive protective effect leads to the conclusion that the inhibitory effect and the exclusion effect are caused by at least partly different mechanisms. CE organisms compete with salmonellas for essential nutrients and produce volatile fatty acids at low pH levels that are bacteriostatic for Salmonella strains (Mead and Barrow, 1990; Corrier et al., 1995; Scanlan, 1997; Schneitz et al., 1998). Evidence on the specificity of inhibition between Salmonella strains is given by the facts that the most profound level of inhibition is produced between isogenic Salmonella strains and that the inhibitory effect does not occur between unrelated organisms (Barrow et al., 1987; Berchieri and Barrow, 1990). Barrow et al. (1997) identified a number of genes (uncH, cya, oxrA, fliM, tsr, ftn) whose expression is necessary for the induction of the inhibitory effect in chickens. Irrespective of the expression of certain genes, the extent of the caecal colonization by the vaccine strain at the time of challenge is of great importance for the formation of the inhibitory phenomenon (Barrow et al., 1987; Methner et al., 1997). Although exact data from the day of infection are not available the counts of the vaccine strain on days 5 and 9 of age should reflect the differences existing between the groups on day of challenge. In spite of the high number of vaccine organisms in the group administered the vaccine strain one day before the CE culture no additive protection was seen in this group. An additional protective effect occurred only in the groups given
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
the CE culture before or simultaneously with the vaccine strain on day 1 of age. This result might be due to the longer time available for the CE culture to colonize the gut in these groups before challenge. Therefore, in chicks of this age, it seems that the exclusion effect has a higher quantitative share of the additive protection than the inhibitory effect of the vaccine strain used. In numerous studies, the efficacy of CE cultures against various Salmonella serovars was examined under a variety of rearing conditions and different modes of administration. (Nuotio et al., 1992; Schneitz et al., 1992; Corrier et al., 1994; Cameron et al., 1996). In these studies, however, efficacy was mostly tested in chicks that were only a few days old because that age group is the key one for prophylactic use of the CE preparations. In our experiments, it could be shown that administration of these preparations resulted in a considerably reduced caecal colonization of the Salmonella challenge strain until week 6 of life of the chicks. These findings support the observation that the complete establishment of the normal intestinal flora occurs only at an age between 3 and 6 weeks (Barnes et al., 1972) and that the establishment of the intestinal flora will vary depending on rearing and feeding conditions for the animals. The use of a Salmonella wild-type strain as vaccine with the detected difference of about 2.5–3.0 log 10 units in the number of challenge organisms in the caecum between control group and the immunized group represent a model to measure the efficacy of live Salmonella vaccines since the immunogenic properties of an attenuated vaccine can hardly be better than those of a virulent wild-type strain. The caecal colonization of the challenge strain STM 421N after application on day 15 or 40 of life is influenced by an inhibitory and exclusion effect as well as the immune response induced by the vaccine. It is important to emphasize that an inhibition effect on day 15 of age is expected to occur only in the groups given STM 421R alone or before CE because caecal counts of the vaccine strain below 10 5 –10 6 cfu / g at time of challenge are not sufficient to produce high inhibitory effects (Barrow et al., 1987; Berchieri and Barrow, 1990). Therefore, apart from the group given the vaccine strain 421R alone an inhibitory effect is not likely to be involved in protection after challenge in the seventh week of life. The results of the experiments with challenge on days 15 and 40 of life clearly demonstrate that
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administration of the vaccine strain 421R 1 day prior, and especially simultaneously, to the CE culture resulted in a considerable additive protection as compared to the respective single administration. Administration of the vaccine strain 1 day after the CE culture (experiment 2) resulted in a significantly reduced colonization of the vaccine strain and thus in the considerably reduced antibody response after infection, so that in these animals no additive protection occurred. The appearance of an additive protection in this group in experiment 3 may be attributed to the fact that the persistence reached by the wild-type Salmonella strain used as vaccine was sufficient to trigger a corresponding immune response. Use of an attenuated live Salmonella vaccine with reduced ability to colonize the intestinal tract may result in a reduction of efficacy if combined with a CE culture in this way. The level of the antibody response after challenge on days 15 and 40 showed a very close relationship with both the extent of the caecal colonization of the vaccine strain until the day of challenge and the protective effect. Therefore, an effective combination of immunization and CE has to guarantee a correspondingly high persistence of the vaccine strain in the animals, ensured by the administration of the vaccine prior to, or simultaneously with, the CE culture. There is a probability that the exposure of chicks to the Salmonella strain (vaccine strain or challenge strain) prior to or simultaneously with the CE culture does not lead to a prevention of the colonization, but that the Salmonella strain becomes an integrated component of the developing intestinal flora. This is supported by the observation that exposure to salmonellas before CE treatment, which is particularly important in hatcheries, can substantially reduce the subsequent protective effect of CE (Mead and Barrow, 1990; Bailey et al., 1998). The results of this model study demonstrate principally that a combination of CE and immunization against Salmonella infection result in a degree of protection considerably beyond that afforded by single use of the two methods. Regarding all three experiments, the administration of the live Salmonella vaccine strain prior to or simultaneously with the CE culture revealed the best protective effect, because such combinations ensure an adequate persistence of the vaccine strain as prerequisite for the expression of inhibition effects in very young chicks and the development of a strong immune response
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U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
protective in older birds. Further investigations will demonstrate to what extent the results of these model studies using a non-attenuated Salmonella wild-type strain as vaccine may be reached with an attenuated Salmonella vaccine.
Acknowledgements The authors wish to thank S. Keiling and P. Schweinitz for their excellent technical assistance.
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Combination of vaccination and competitive exclusion to prevent Salmonella colonization in chickens: experimental studies a, b a a U. Methner *, P.A. Barrow , A. Berndt , G. Steinbach a
Federal Institute for Health Protection of Consumers and Veterinary Medicine, Jena Branch, Naumburger Strasse 96 a, D-07743 Jena, Germany b Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7 NN, UK Received 2 October 1998; received in revised form 23 February 1999; accepted 31 March 1999
Abstract Vaccination and competitive exclusion (CE) represent accepted prophylactic measures to control Salmonella infections in chickens. To use the advantages of both the CE technique and vaccination with live Salmonella vaccines the combination of these methods was studied. In three experiments, SPF chickens were pre-treated using combined or unique administration of CE and vaccination with a live Salmonella typhimurium strain on days 1 and 2 of life and challenged with the antibiotic resistant, but otherwise isogenic mutant of this Salmonella typhimurium strain on days 3, 15 or 40 of life. The caecal colonization of both the vaccine and the challenge strain and the antibody response after infection were examined to evaluate the protective effects of the different combinations. The exclusion effect of CE cultures against Salmonella infection could be seen in very young chicks and was still considerable on day 40 of life of the birds. The Salmonella wild-type strain used as vaccine alone also resulted in a substantial protective effect against homologous challenge. The combined administration of competitive exclusion and immunization using the Salmonella wild-type strain as vaccine resulted in a considerable additional protective effect above the level of the respective exclusive application of these prophylactic measures. Administration of the Salmonella vaccine strain prior to or simultaneously with the CE culture produced the best protective effect, because such combinations ensure an adequate persistence of the vaccine strain as prerequisite for the expression of colonization inhibition effects and a strong immune response. The full exploitation of this potential using attenuated live Salmonella vaccines will require the presence of high inhibitory and immunogenic properties in the vaccine strain after attenuation of a selected parent strain. The combination of competitive exclusion and vaccination as a new measure in integrated control programmes against Salmonella infection in poultry could result in a considerable increase of protection in both very young and older chickens. 1999 Elsevier Science B.V. All rights reserved. Keywords: Salmonella; Live vaccines; Competitive exclusion; Vaccination; Inhibition; Chickens
1. Introduction *Corresponding author. Tel.: 1 49-3641-804-267; fax: 1 493641-804-228. E-mail address: [email protected] (U. Methner)
Different methods may be used to increase the resistance of both very young and older chickens
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00051-3
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against Salmonella infection (Mead and Barrow, 1990). Using competitive exclusion (CE) cultures, the greater resistance of adult birds can be transferred to day-old chicks. Administration of live Salmonella vaccines to birds may also induce an exclusion or inhibition effect within a matter of hours in addition to simultaneously stimulating the development of a true immunity to infection, both of these effects resulting in a further increase of resistance to Salmonella infection. Exclusion effects (Nurmi and Rantala, 1973; Nuotio et al., 1992; Cameron et al., 1996) induced by complex intestinal flora and those induced by live Salmonella vaccines (Barrow et al., 1987; Berchieri and Barrow, 1990; Methner et al., 1997) may both be used in very young birds. After administration of Salmonella vaccines immunological effects may be used in immunocompetent chicks and adult birds with an established intestinal microflora (Hassan and Curtiss, 1994; Methner et al., 1994). To obtain maximum protective effects, CE cultures should be administered to chicks as soon as possible after hatching (Bolder et al., 1992). Live Salmonella vaccines given at 1 day of age ensure early intestinal colonization by the vaccinating strain (Cooper et al., 1994; Vielitz et al., 1995), thereby producing both an inhibitory effect and an early immunological response. Competitive exclusion is effective against different Salmonella serovars, while inhibition between salmonellas is probably most effective between strains of the same serovar. The possibility exists, therefore, that a combination of non-specific CE and more specific inhibition and vaccination with live Salmonella vaccines against serovars epidemiologically important for human beings may result in a higher protection against these serovars than use of either of these methods alone. One of the problems in studying different prophylactic measures to reduce or prevent Salmonella colonization of the alimentary tract of chickens lies in the terminology used. In this paper the following terminology is applied purely to facilitate discussion. Their use does not imply an understanding of the differences between, for example, exclusion and inhibition effects. Exclusion effect is the mechanism generated by a CE culture to prevent colonization of Salmonella challenge organisms as well as the colonization of the vaccine strain in the case of combined use. Inhibitory effects are defined as those occurring only between
Salmonella strains. Immunological effects after administration of Salmonella strains (attenuated or nonattenuated) are defined as those where some of the characteristics of classical immune response are evident. It has been the purpose of our experiments to study competitive exclusion, inhibitory and immunological effects after combined use of CE and vaccination against Salmonella infection in chickens of different ages. In order to evaluate the principal potential and limitations of a combination of these methods irrespective of the type and degree of attenuation of a live vaccine, a non-attenuated wildtype strain of Salmonella was used for immunization of the chicks.
2. Materials and methods
2.1. Chickens Specific pathogen-free (SPF) chickens (white leghorn) were hatched in the facilities of the institute from eggs received from Charles River Deutschland (Extertal, Germany). Experimental and control groups were kept in separate rooms. Commercial feed (powdered) without antibiotics or other additives and drinking water were both available ad libitum. Cleaning and feeding regimens were organized which prevented cross-contamination effectively throughout the course of the experiments.
2.2. Bacterial strains and culture A rifampicin-resistant variant (R) of the Salmonella typhimurium wild-type strain 421 (DT 204) was used for oral vaccination of the chicks on their first or second day of life with or without combined use of a commercial CE culture (Aviguard , Bayer AG, Germany). The viable numbers of the non-attenuated Salmonella typhimurium strain 421R (STM 421R) administered orally were 1–3 3 10 7 cfu / bird. The CE culture was dissolved in accordance with the manufacturer’s instructions and administered orally. Oral infection of the chickens was carried out using a nalidixic acid-resistant variant (N) of the wild-type strain Salmonella typhimurium 421. This challenge strain was already used earlier (Methner et al.,
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
1997). To enable the differentiation and the exact quantitative determination of both the vaccine and the challenge strain in the caeca, a rifampicin (R) and a nalidixic acid-(N)-resistant variant of Salmonella typhimurium 421 were used in the experiments. These strains are spontaneous mutants produced by the method of Smith and Tucker (1980). After purification and replating the mutants were checked for virulence in chicks and compared with the parent strain. The strains were stored in the Microbank system (Pro-Lab Diagnostics, Ontario, Canada) at 2 208C. Oral administration was performed by instillation into the crop of the birds, using syringes with an attached flexible tube. The volume of bacterial suspension used was 0.1 ml in birds until the age of 1 week and 1.0 ml in older birds. The Salmonella suspensions for inoculation were cultivated by shaking in nutrient broth I (SIFIN, Berlin, Germany). Counts were estimated by measuring extinction at 600 nm and comparing with a calibration graph determined for the strains used. Additionally, counts for the bacterial suspensions used were established by plate counting on nutrient agar (SIFIN, Berlin, Germany) after preparation of the suspensions.
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2.3. Experimental design and bacteriology To ensure that the chickens used in the experiments were free of salmonellas, hatching material (egg shells, faeces, organs from unhatched birds) was examined for their presence. The samples were preenriched in buffered (pH 7.2) peptone water (Merck, Darmstadt, Germany). Selective enrichment took place in Rappaport–Vassiliadis broth (Merck, Darmstadt, Germany) over 18–24 h at 428C. Loopfuls of material were spread on deoxycholate–citrate agar and xylose–lysine–deoxycholate agar (Merck, Darmstadt, Germany). The effect of pre-treatment of chicks with STM 421R with and without combined administration of CE culture against infection with STM 421N was studied in three different experiments (Table 1). The pre-treated chickens were challenged on day 3 (experiment 1), on day 15 (experiment 2) or on day 40 (experiment 3) of age. The challenge dose was 10 5 cfu / bird on day 3 of age and 10 9 cfu / bird on days 15 or 40 of age, respectively. Protective effects were evaluated by establishing the viable counts of the challenge strain STM 421N in whole caeca (with content) in experiment 1 (on
Table 1 Counts (x¯ log 10 cfu / g) of Salmonella typhimurium 421 N (STM 421N) in caeca of chickens given this strain after pre-treatment with live STM 421R wild-type strain and / or a competitive exclusion culture (CE) Age (days)
Group 1
2
3
4
5
6
1 2
STM 421 R –
CE –
STM 421 R CE
CE STM 421 R
CE1STM 421 R –
Control –
3 5a 9a
Experiment 1: Challenge: STM 421 N (10 5 cfu / bird) 3.7 6 3.6 6 3.3 6 6 6 4.1 3.9 3.6 6
2.5 6 1.1 1,2,6
1.6 1,2,6 1.2 1,2,6
7.6 6 8.7
15 18 b 22 b
Experiment 2: Challenge: STM 421 N (10 9 cfu / bird) 3.7 6 3.8 6 2.1 6 6 6 4.2 3.9 3.8 6
4.3 6 3.7 6
1.6 1,2,6 0.6 1,2,6
7.0 7.2
40 43 c 48 c
Experiment 3: Challenge: STM 421 N (10 9 cfu / bird) 2.9 6 2.3 6 0.8 1,2,6, 6 1,6 2.9 1.4 0.8 1,6
0.2 1,2,6, 0.2 1,2,6
0.5 1,2,6 0.4 1,2,6
4.7 4.3
a
Standard error: Standard error: c Standard error: 1,2,6 Significantly b
60.56. 60.56. 60.39. lower than group 1, 2, 6 (P,0.05).
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U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
days 2 and 6 after challenge) or a section of caecal mucosa with content in experiments 2 and 3 (on days 3 and 7 or 8 after challenge) from five chickens / group and day of examination. In all groups, using STM 421R as vaccine, the number of organisms of this strain in caeca was detected simultaneously. Bacterial counts in the caeca were estimated using a standard plating method as described previously (Methner et al., 1995b). Briefly, the caeca were removed aseptically, placed in sterile tubes and weighed. Phosphate-buffered (pH 7.2) saline was added to the material at a ratio of 1:3, afterwards the samples were homogenized using an Ultra-Turrax (Janke and Kunkel, Staufen, Germany) blender. Homogenized organ samples were diluted in PBS and plated on deoxycholate–citrate agar containing sodium nalidixate (50 mg / ml) or rifampicin (100 mg / ml) to determine the number of challenge and vaccine organisms, respectively. Buffered peptone water was used for pre-enrichment of the organ homogenates (1:10), incubated overnight at 378C and subcultured on the solid medium containing sodium nalidixate or rifampicin.
2.4. Antibody detection Detection of antibodies in serum was performed by means of an indirect ELISA on polystyrene microtiter plates (Greiner, Solingen, Germany). LPS (phenol–water extract) of Salmonella typhimurium served as antigen. To detect the IgG bound to the antigen, rabbit anti-chicken IgG (H 1 L) (ICN Pharmaceuticals, Amsterdam, The Netherlands) and the peroxidase (PO) conjugates, goat anti-rabbit / IgG PO (Nordic Immunological Laboratories, Tilburg, The Netherlands) or rabbit anti-goat IgG / PO (Sigma– Aldrich, Deisenhofen, Germany) were used. Orthophenylenediamine was used as substrate. In accordance with the expected antibody concentrations, the optical density for antibody of class IgG in serum (diluted 1:400) was determined.
2.5. Statistical analysis Viable counts were converted into logarithmic form. For the purposes of statistical analysis, a viable count of log 10 , 1.47 (limit for direct plate detection) obtained from a sample becoming positive only after enrichment was rated as log 10 5 1.0. A sample which
yielded no Salmonella growth after enrichment was rated as log 10 5 0. Data were evaluated by multifactorial variance analysis. The factors considered were group and time. The optical density expressing the parameter of the antibody concentration was converted into logarithmic form ( → normal distribution) and tested for differences. P values of , 0.05 were regarded as statistically significant (software: Statgraphics Plus, Rockville, MD, USA).
3. Results
3.1. Colonization of the challenge strain Studies on CE and inhibition (challenge on day 3 of life) using the non-attenuated strain STM 421R as vaccine (Table 1) clearly demonstrated the protective effect of the CE culture (group 2) against Salmonella challenge in comparison to untreated controls (group 6). A difference of about 3–4 log 10 units in the number of challenge organisms was seen between these groups. The inhibitory effect of STM 421R (group 1) against STM 421N was nearly identical with the exclusion effect produced by the CE culture and amounted also to about 4 log 10 units. A combination of both led to a further significant increase in protection on day 9 of life in groups 4 and 5. Studies on CE and vaccination using the nonattenuated STM wild-type strain (challenge on days 15 or 40 of life) showed that the protective effect of a CE culture against Salmonella infection was still substantial in 15-day-old chickens (Table 1). The difference in the number of STM 421 N in caeca between birds pre-treated with the CE culture and untreated controls was about 3 log 10 units. The protection level generated by the STM 421 R wildtype strain equalled that obtained with the CE culture. A considerable increase of protection was found after administration of CE culture and STM 421R simultaneously to day-old chicks as compared to the exclusive application of either of these methods alone. A strong exclusion effect produced by the CE culture alone was found also after challenge of 40day-old birds in experiment 3 (Table 1). These chickens and the birds vaccinated with STM 421R only showed a reduction in the number of challenge
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
organisms compared to untreated controls of about 2–3 log 10 units. Combined pre-treatment of chickens with the CE culture and the vaccine strain 421R almost completely prevented the intestinal colonization by the challenge strain 421N administered in a dose of 10 9 cfu / bird. There were no significant differences between groups 3, 4 and 5.
3.2. Colonization of the vaccine strain Colonization by the Salmonella vaccine strain was affected to a varying degree, depending on the mode of administration of the CE culture (Table 2). In experiments 1 and 2 the administration of the CE culture 24 h after the vaccine strain had no significant impact on the intestinal colonization of the vaccine. The application of the CE culture both before and simultaneously with the vaccine strain 421R resulted in a considerably reduced colonization of the vaccine in comparison to single administration. The STM 421R wild-type strain applied as vaccine to day-old chicks persisted in the caecum until 6 weeks of life in numbers of about 5.0 log 10 cfu / g (data not shown). In all groups with combined administration of a CE culture, the caecal colonization by strain 421R was considerably suppressed (to about 1.2–2.5 log 10 cfu / g), but not completely eliminated during this additional period. The different modes of combination of CE culture and STM
39
421R vaccine strain did not result in significant differences in the caecal concentration of this strain as found in younger chicks.
3.3. Antibody response The IgG antibody response in the serum measured after challenge of the chicks on day 15 and 40 of age (Table 3) exhibited a high positive correlation with the persistence of the vaccine strain (Table 2). The intensity of the antibody response after infection is determined by the extent of caecal colonization of the vaccine strain which depends on the mode of administration of the CE culture and the live vaccine strain. The group administered the vaccine alone showed, in fact, the highest colonization of the vaccine strain, but not the strongest IgG response in serum.
4. Discussion In these studies, the potential and limitations of a combination of immunization with Salmonella live vaccines and CE have been tested for the first time. Attenuated live vaccines which have already been approved for use in poultry, or are still being tested, vary in their properties on account of their different type of attenuation. Therefore, no attenuated vaccine strain was used for the immunization, but a non-
Table 2 Counts (x¯ log 10 cfu / g) of Salmonella typhimurium 421R (STM 421R) in caeca of chickens given this strain as vaccine alone or in combination with a competitive exclusion culture (CE) Age (days)
Group 1
3
4
5
1 2
STM 421 R –
STM 421 R CE
CE STM 421 R
CE1STM 421 R –
3 5a 9a
Experiment 1: Challenge: STM 421 N (10 5 cfu / bird) 8.8 8.2 8.5 7.8
4.5 1,3,5 4.2 1,3,5
6.6 1,3 5.7 1,3
15 18 b 22 b
Experiment 2: Challenge: STM 421 N (10 9 cfu / bird) 6.8 6.5 6.9 5.9
2.5 1,3 1.4 1,3
3.6 1,3 2.1 1,3
a
Standard error: 60.21. Standard error: 60.44. 1,3,5 Significantly lower than group 1, 3, 5 (P,0.05). b
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
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Table 3 Mean optical density of Salmonella antibodies of isotype lgG in serum of pre-treated chickens after challenge with Salmonella typhimurium 421 N (STM 421N) on days 15 or 40 of life Age (days)
Group 1
2
3
4
5
6
1 2
STM 421R –
CE –
STM 421R CE
CE STM 421R
CE1STM 421R –
Control –
15 18 a 22 a
Challenge: STM 421 N (10 9 cfu / bird) 1.19 01 0.87 1.35 01 1.40
01 0.76
0.26 1 1.03
01 01
40 43 b 48 b
Challenge: STM 421 N (10 9 cfu / bird) 0.80 0.02 1 1.01 1.06 0.41 1 1.40
0.44 0.57
1.03 1.24
0.14 1 0.83
a
Standard error: 60.29. Standard error: 60.20. 1 Significantly lower than group 1 (P,0.05). b
attenuated Salmonella wild-type strain. The reason for this was to draw conclusions concerning the maximum protective effects to be achieved as it can be assumed that the protective properties of an attenuated vaccine strain will not be superior to those of a non-attenuated wild-type strain. The colonization characteristics of the strain STM 421 have been well documented (Methner et al., 1995a, 1997). Using a dose of 1–3 3 10 7 cfu / bird of STM 421 in day-old chicks a strong colonization of the intestine and invasion into liver is induced, but there will be no clinical manifestations among the animals. Only some birds show a slight inflammation of the intestine in the first week after administration (Methner and Barrow, 1997). In order to consider different stages of development of the host, the oral challenge of chickens pre-treated in different ways was performed in three independent experiments on days 3, 15, or 40 of life. The exclusion effect produced within a short time after application of the CE culture in chicks which are not completely immunocompetent, was nearly identical with the inhibitory effect of STM 421R against STM 421N (experiment 1). The combination of both resulted in an additional increase of protection in the groups given the CE culture before or simultaneously with the vaccine. It can be assumed that in chicks aged only a few days which are not completely immunocompetent no specific immune response occurs which could result in such considerable protective effect within 2 days after challenge (Barrow et al., 1987; Methner and
Steinbach, 1997). The occurrence of an additive protective effect leads to the conclusion that the inhibitory effect and the exclusion effect are caused by at least partly different mechanisms. CE organisms compete with salmonellas for essential nutrients and produce volatile fatty acids at low pH levels that are bacteriostatic for Salmonella strains (Mead and Barrow, 1990; Corrier et al., 1995; Scanlan, 1997; Schneitz et al., 1998). Evidence on the specificity of inhibition between Salmonella strains is given by the facts that the most profound level of inhibition is produced between isogenic Salmonella strains and that the inhibitory effect does not occur between unrelated organisms (Barrow et al., 1987; Berchieri and Barrow, 1990). Barrow et al. (1997) identified a number of genes (uncH, cya, oxrA, fliM, tsr, ftn) whose expression is necessary for the induction of the inhibitory effect in chickens. Irrespective of the expression of certain genes, the extent of the caecal colonization by the vaccine strain at the time of challenge is of great importance for the formation of the inhibitory phenomenon (Barrow et al., 1987; Methner et al., 1997). Although exact data from the day of infection are not available the counts of the vaccine strain on days 5 and 9 of age should reflect the differences existing between the groups on day of challenge. In spite of the high number of vaccine organisms in the group administered the vaccine strain one day before the CE culture no additive protection was seen in this group. An additional protective effect occurred only in the groups given
U. Methner et al. / International Journal of Food Microbiology 49 (1999) 35 – 42
the CE culture before or simultaneously with the vaccine strain on day 1 of age. This result might be due to the longer time available for the CE culture to colonize the gut in these groups before challenge. Therefore, in chicks of this age, it seems that the exclusion effect has a higher quantitative share of the additive protection than the inhibitory effect of the vaccine strain used. In numerous studies, the efficacy of CE cultures against various Salmonella serovars was examined under a variety of rearing conditions and different modes of administration. (Nuotio et al., 1992; Schneitz et al., 1992; Corrier et al., 1994; Cameron et al., 1996). In these studies, however, efficacy was mostly tested in chicks that were only a few days old because that age group is the key one for prophylactic use of the CE preparations. In our experiments, it could be shown that administration of these preparations resulted in a considerably reduced caecal colonization of the Salmonella challenge strain until week 6 of life of the chicks. These findings support the observation that the complete establishment of the normal intestinal flora occurs only at an age between 3 and 6 weeks (Barnes et al., 1972) and that the establishment of the intestinal flora will vary depending on rearing and feeding conditions for the animals. The use of a Salmonella wild-type strain as vaccine with the detected difference of about 2.5–3.0 log 10 units in the number of challenge organisms in the caecum between control group and the immunized group represent a model to measure the efficacy of live Salmonella vaccines since the immunogenic properties of an attenuated vaccine can hardly be better than those of a virulent wild-type strain. The caecal colonization of the challenge strain STM 421N after application on day 15 or 40 of life is influenced by an inhibitory and exclusion effect as well as the immune response induced by the vaccine. It is important to emphasize that an inhibition effect on day 15 of age is expected to occur only in the groups given STM 421R alone or before CE because caecal counts of the vaccine strain below 10 5 –10 6 cfu / g at time of challenge are not sufficient to produce high inhibitory effects (Barrow et al., 1987; Berchieri and Barrow, 1990). Therefore, apart from the group given the vaccine strain 421R alone an inhibitory effect is not likely to be involved in protection after challenge in the seventh week of life. The results of the experiments with challenge on days 15 and 40 of life clearly demonstrate that
41
administration of the vaccine strain 421R 1 day prior, and especially simultaneously, to the CE culture resulted in a considerable additive protection as compared to the respective single administration. Administration of the vaccine strain 1 day after the CE culture (experiment 2) resulted in a significantly reduced colonization of the vaccine strain and thus in the considerably reduced antibody response after infection, so that in these animals no additive protection occurred. The appearance of an additive protection in this group in experiment 3 may be attributed to the fact that the persistence reached by the wild-type Salmonella strain used as vaccine was sufficient to trigger a corresponding immune response. Use of an attenuated live Salmonella vaccine with reduced ability to colonize the intestinal tract may result in a reduction of efficacy if combined with a CE culture in this way. The level of the antibody response after challenge on days 15 and 40 showed a very close relationship with both the extent of the caecal colonization of the vaccine strain until the day of challenge and the protective effect. Therefore, an effective combination of immunization and CE has to guarantee a correspondingly high persistence of the vaccine strain in the animals, ensured by the administration of the vaccine prior to, or simultaneously with, the CE culture. There is a probability that the exposure of chicks to the Salmonella strain (vaccine strain or challenge strain) prior to or simultaneously with the CE culture does not lead to a prevention of the colonization, but that the Salmonella strain becomes an integrated component of the developing intestinal flora. This is supported by the observation that exposure to salmonellas before CE treatment, which is particularly important in hatcheries, can substantially reduce the subsequent protective effect of CE (Mead and Barrow, 1990; Bailey et al., 1998). The results of this model study demonstrate principally that a combination of CE and immunization against Salmonella infection result in a degree of protection considerably beyond that afforded by single use of the two methods. Regarding all three experiments, the administration of the live Salmonella vaccine strain prior to or simultaneously with the CE culture revealed the best protective effect, because such combinations ensure an adequate persistence of the vaccine strain as prerequisite for the expression of inhibition effects in very young chicks and the development of a strong immune response
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protective in older birds. Further investigations will demonstrate to what extent the results of these model studies using a non-attenuated Salmonella wild-type strain as vaccine may be reached with an attenuated Salmonella vaccine.
Acknowledgements The authors wish to thank S. Keiling and P. Schweinitz for their excellent technical assistance.
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