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Combination of competitive exclusion and immunisation with a live Salmonella vaccine in newly hatched chickens: Immunological and microbiological effects
Research in Veterinary Science 107 (2016) 34–41
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Combination of competitive exclusion and immunisation with a live Salmonella vaccine in newly hatched chickens: Immunological and microbiological effects M. Braukmann a, P.A. Barrow c, A. Berndt b, U. Methner a,⁎ a b c
Institute of Bacterial Infections and Zoonoses at the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Naumburger Str. 96a, D-07743 Jena, Germany Institute of Molecular Pathogenesis at the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Naumburger Str. 96a, D-07743 Jena, Germany School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, United Kingdom
a r t i c l e
i n f o
Article history: Received 28 January 2016 Received in revised form 25 April 2016 Accepted 7 May 2016 Available online xxxx Keywords: Chicken Competitive exclusion Immune response Inhibition Salmonella live vaccine
a b s t r a c t In addition to evaluating the efficacy potential of a combined use of vaccination and competitive exclusion (CE) against Salmonella exposure in chicks at 3-days of age, a live Salmonella Enteritidis vaccine (SE-LV) and a CE culture were tested for their ability to induce parameters of the innate immunity. Whereas the invasive SE-LV induced an influx of granulocytes and macrophages as well as an increased transcription of several cytokines in the caecal mucosa, the CE culture did not demonstrate any differences in these parameters compared to controls. It is therefore highly probable that the effects observed with CE cultures are not due to the rapid stimulation of the immune system. The combined use of both preparations did not result in an additive intestinal exclusion effect of the challenge strain more pronounced than that after single administration of the CE culture. The combined use of the Salmonella live vaccine and the CE culture resulted in an additive protective effect and prevented completely the systemic dissemination of the Salmonella challenge strain. To exploit the potential of combined use of CE and vaccination further and most effectively, live Salmonella vaccines are needed that are despite their attenuation in virulence still capable to induce both intestinal colonisation- and invasioninhibition effects against Salmonella exposure. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Poultry meat and eggs are considered to be the major source of Salmonella infection for humans (EFSA, 2013). In addition to efficient hygiene regimes at all stages of production, immunisation with both live and inactivated Salmonella vaccines and competitive exclusion (CE) represent the most important methods to increase the resistance of both very young and adult chickens (EFSA, 2004). CE cultures are effective against different serovars (Mead, 2000); however, the mechanisms of protection are not fully understood. Effects like the creation of a restrictive physiological environment, the competition for enteric receptor sites or with other microbes for nutrients, the production of antimicrobial compounds and the stimulation of the immune system are discussed (La Ragione and Mead, 2013). Protective effects induced by vaccination of birds include the reduced intestinal colonisation and the diminished systemic invasion of
⁎ Corresponding author. E-mail address: ulrich.methner@fli.bund.de (U. Methner).
http://dx.doi.org/10.1016/j.rvsc.2016.05.001 0034-5288/© 2016 Elsevier Ltd. All rights reserved.
Salmonella wild-type organisms by mechanisms of the adaptive immunity (EFSA, 2004). In general it is accepted that live Salmonella vaccines are more effective against intestinal and systemic infection than are inactivated vaccine preparations (Lillehoj et al., 2000). Moreover, live Salmonella vaccines are also capable of inducing protective mechanisms effective during the ‘immunity gap’, the time between administration of the vaccine and development of the adaptive immune response. The i) intestinal colonisation-inhibition effect (Barrow et al., 1987; Methner et al., 2011) and the ii) invasion-inhibition effect (Methner et al., 2010) have not been considered until now in the development of live Salmonella vaccines for chickens. The combined use of a CE culture and a live Salmonella vaccine which is able to induce both a colonisation-inhibition and an invasion-inhibition effect, may produce an additive protective impact more pronounced than the single use of either of these methods. A registered live Salmonella Enteritidis vaccine is capable to stimulate a protective adaptive immune response in juvenile chickens (Springer et al., 2000); however, the vaccine has not yet been tested for its potential to induce colonisation- or invasion-inhibition effects. Apart from evaluating the possible protective effects after single and
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combined use of this live Salmonella Enteritidis vaccine and a commercial CE culture against Salmonella Enteritidis exposure in newly hatched chicks, the study also aims to identify parameters of the innate immune response which might bring about the effects observed. 2. Materials and methods 2.1. Chickens Specific pathogen-free White Leghorn chickens were hatched at the facilities of the Friedrich-Loeffler-Institute from eggs obtained from Charles River Deutschland GmbH. Experimental and control groups were kept in cages in separate negative pressure rooms. Commercial feed (coarse meal without antibacterial additives) and public drinking water were both available ad libitum. The single groups were managed separately (including cleaning and feeding regimes) to prevent crosscontamination between the groups effectively throughout the trials. Animal experiments were performed in accordance with the German Animal Protection Act and approved by an ethical committee (Animal Ethics approval number: 04-005/11- 01 December 2011). 2.2. Bacterial strains and culture Salmovac SE (IDT Biologika), a registered Salmonella Enteritidis live vaccine (SE-LV) strain (phage type PT4) was used for oral immunisation of the chickens on day 1 of life with or without combined use of a commercial CE culture (Aviguard, Microbial Developments Ltd.). To facilitate accurate enumeration of the vaccine strain in caecal contents and liver, a spontaneous nalidixic acid-resistant (N) mutant was produced for immunisation (Smith and Tucker, 1980). The resistance has no perceptible impact on the in vivo results (Barrow et al., 1987; Methner et al., 2010, 2011). To confirm this assumption, the original nonresistant SE-LV and the nalidixic acid-resistant SE-LV were compared using in vitro experiments for both, their adhesion and invasion in a cell-culture model as well as for their ability to inhibit the growth of other Salmonella organisms in nutrient broth (Methner and Barrow, 1997). Both variants of the SE-LV did not differ in these characteristics (data not shown). The viable count of the attenuated SE-LV administered PO via crop instillation was 2 × 108 colony forming units (cfu) per bird. The CE culture was dissolved in accordance with the manufacturer's instruction and administered via crop instillation. Oral infection of the chicken was carried out with a rifampicin (R) resistant variant (Methner et al., 2011) of the comprehensively characterised strain Salmonella enterica subspecies enterica serovar Enteritids 147 (SE 147R, phage type PT4) (Methner et al., 2010, 2011) at a dose of 2 × 105 cfu/bird. All strains had been stored in a Cryobank system (Mast Diagnostica) at − 20 °C. 2.3. Experimental design and bacteriology Caecal colonisation and systemic invasion of the attenuated SE-LV after single administration on day 1 of life as well as in combination with a CE culture (SE-LV on day 1 followed by the CE culture on day 2 of age) without Salmonella challenge was studied in experiment 1 (Table 1). SE-LV was enumerated in caecal contents and in liver at days 3, 4, 7, 8, 9, 10, 11, and 14 of life from 4 birds/group, respectively, by a standard plating method (Methner et al., 2001, 2010). Homogenised organ samples were diluted and plated on brilliantgreen phenol red agar (SIFIN) with sodium nalidixate (50 μg/mL) and incubated at 37 °C for 18–24 h. Caecal contents and liver samples from all birds in groups A–D (Table 1) were also pre-enriched in buffered peptone water (SIFIN), incubated at 37 °C for 18–24 h and streaked onto brilliant-green phenol red agar with sodium nalidixate (SIFIN). Additionally, caeca from each animal of the groups administered the SE-LV alone or in combination with the CE culture, a control group and a group given the CE culture only were taken and frozen in
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Table 1 Number (mean log10 cfu/g of 4 birds) of Salmonella Enteritidis live vaccine (SE-LV) in liver and caecal contents of chickens after oral administration of 2 × 108 cfu/bird at 1 day of age without or with subsequent application of a competitive exclusion (CE) culture at day 2 of age (experiment 1). Day of age
Group A
Group B
Group C
Group D
1 2
CE culture –
SE-LV –
SE-LV CE culture
– –
Day of age
Group A
Group B
Group C
Group D
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
3 4 7 8 9 10 11 14
– – – – – – – –
– – – – – – – –
2.5 3.3 3.0 2.8 2.5 1.6 1.6 1.3
8.5 8.7 7.9 8.4 7.9 6.7 6.6 7.3
2.7 3.0 2.8 2.8 2.7 1.8 1.7 1.3
8.9 8.4 7.6 6.9b 6.1b 4.7b 4.9b 5.7b
– – – – – – – –
– – – – – – – –
Standard error: liver: 0.199; caeca: 0.357. b Significantly lower than group B. c Significantly lower than C.
liquid nitrogen or stored in RNAlater (Qiagen) until use for immunohistochemistry or studying mRNA expression of selected cytokines, respectively. In experiment 2 the protective effect induced by the CE culture and the SE-LV alone or after combination of both (SE-LV on day 1 followed by the CE culture on day 2 of age) against challenge with SE 147R administered on day 3 of life was compared with an untreated control group (Table 2). The challenge strain was enumerated in caecal contents and liver from 4 birds/group at days 4, 5, 8, 9, 10, 11, 12, and 15 of age using a standard method described above.
2.4. Immunohistochemistry Using immunohistochemistry, the invasion of the SE-LV into lower regions of the caecal mucosa as well as the influx of granulocytes and macrophages into the caecum was examined in experiment 1. Cryostat sections of 7 μm thickness of every chicken caecum were prepared and Table 2 Number (mean log10 cfu/g of 4 birds) of Salmonella Enteritidis 147R (SE 147R; 2 × 105 cfu/ bird PO at 3 days of age) in liver and caecal contents of chickens after pre-treatment with a competitive exclusion (CE) culture or a Salmonella Enteritidis live vaccine (SE-LV) (2 × 108 cfu/bird PO at 1 day of age) without or with subsequent application of a CE culture at day 2 of age (experiment 2). Day of age
Group A
Group B
Group C
Group D
1 2 3
CE culture
SE-LV
SE 147 R
SE 147 R
SE-LV CE culture SE 147 R
– – SE 147 R
Day of age
Group A
Group B
Group C
Group D
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
4 5 8 9 10 11 12 15
0 0 0.3d 0.5d 0d 0.6d 0.7d 0.3d
2.9b,d 3.8d 4.3d 4.2b,d 3.6b,d 4.1d 3.8b,d 4.0d
0 0 0d 0d 0d 0.5d 0.4d 0.3d
4.7d 4.2d 6.4 7.0 6.5 5.3 6.4 5.7
0 0 0d 0d 0d 0d 0d 0a,d
3.5d 2.8d 4.6 4.1b,d 4.6d 3.8d 3.6b,d 3.3d
0.3 0.3 1.8 1.9 1.6 2.2 1.9 1.5
7.7 6.5 6.9 7.0 7.0 6.9 7.4 6.6
a b c d
Significantly lower than group A. Significantly lower than group B. Significantly lower than group C. Significantly lower than group D.
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stained as described previously (Berndt and Methner, 2001). Briefly, the primary antibodies against Salmonella enterica lipopolysaccharide (LPS) (Clone 5D12A, Bio-Rad AbD Serotec), avian granulocytes (kindly provided by F. Van Immerseel, University Gent, Belgium) and monocytes/macrophages (CVI 68.1, Institute for Animal Science and Health, Lelystad, The Netherlands) were used. As negative control, the primary antibodies were replaced with PBS. 2.5. Image analysis Analyses of the number of granulocytes and proportions of immunohistochemically stained areas of macrophages and Salmonella organisms in caecal mucosa were conducted by means of the image analysis system CELL (Olympus) as described previously (Berndt et al., 2007). Briefly, macrophages and Salmonella organisms were monitored by determining the percentage of positive stained areas in whole caecum mucosa (epithelium and lamina propria) and numbers of granulocytes were counted in epithelial und sub-epithelial areas. 2.5.1. Quantitative real-time reverse transcription (RT)-PCR To study gene transcription, total RNA was extracted from chicken caecum samples in experiment 1. RNA extraction, digestion of residual DNA and purity criteria were described previously (Berndt et al., 2007). Avian-specific primers for glycerinaldehyde-3-phosphate (GAPDH), interleukin (IL)-12β (p40), lipopolysaccharide-induced tumour necrosis factor alpha factor (LITAF), inducible nitric oxide synthase (iNOS), IL-1β and IL-10 were developed formerly (Berndt et al., 2007; Braukmann et al., 2012). To determine mRNA expression rates by the QuantiTect SYBR Green real-time one-step RT-PCR Kit (Qiagen), the thermocycler Mx3000P (Agilent) was used with the temperature-time profile including the optimised annealing temperatures as described by Berndt et al. (2007). Each primer pair was tested by a serial dilution of template RNA to ensure optimized amplification and comparable efficiencies of the RT-PCR. For every primer, notemplate controls were performed. The threshold method was used for relative quantification of mRNA level. Normalisation of target genes was performed using the GAPDH as endogenous standard. Results were expressed as fold change (2[−ΔΔCt]) (Pfaffl, 2001) relative to the average of transcripts detected in the control group at each time point. 2.6. Statistical analysis Viable bacterial counts were converted into logarithmic form. For statistical purposes a viable count of log10 b 1.47 from a sample detected positive only after enrichment was rated as log10 = 1.0. A sample which yielded no Salmonella growth after enrichment was rated as log10 = 0. Data were evaluated by analysis of variance (multifactor ANOVA). The factors considered were group and time. P values b 0.05 were regarded as statistically significant (software: statgraphics plus, Inc. Rockville, MD). Statistical analysis of data from immunohistochemistry was conducted using the Mann-Whitney U test (SPSS 19.0, IBM). The normally distributed data of quantitative real-time RT-PCR were statistically evaluated using the Student t-test. Both tests were used for comparison of two independent samples to evaluate differences between the groups at each time point. P values of b 0.05 were considered significant. 3. Results 3.1. Colonisation and invasion of the Salmonella Enteritidis live vaccine with or without combined use of a CE culture The sole oral administration of the SE-LV resulted in a high level of caecal colonisation and systemic invasion but did not induce signs of morbidity (Table 1). The application of the CE culture after the SE-LV did not result in a significant reduction of the vaccine strain in liver
compared to the single use of SE-LV. However, the caecal colonisation of the SE-LV in this group was significantly reduced from day 8 of life onwards in comparison to birds given the live vaccine only. The differences ranged from 1.5 to 2.0 log10 units. In all animals from groups A and D the vaccine strain SE-LV was not detected.
3.2. Quantification of the Salmonella Enteritidis live vaccine in caecal mucosa After single administration of SE-LV and after combination of the SELV with the CE culture, comparable numbers of small accumulations and single bacteria of the SE-LV strain were observed in the caecal mucosa (Fig. 1). The capability of SE-LV to invade the epithelium and the whole lamina propria was similar in both modes of administration. The highest amounts of Salmonella-positively stained areas were found between days 3 and 7 of life after single administration of SE-LV and between days 4 and 7 of life after application of SE-LV and the CE culture. The administration of the CE culture after the SE-LV did not reveal significant differences in the ability of SE-LV to invade the caecal tissue. In the CE-treated group as well as the control group no Salmonella-LPS was detected.
3.3. Quantification of granulocytes and macrophages in chicken caecum Compared to the controls, the application of the SE-LV induced significantly increased numbers of granulocytes between days 2 and 10 of life (Fig. 2). The combined pre-treatment of chicks with SE-LV and CE culture resulted in significantly elevated numbers of granulocytes at day 3 of life and continued until day 11 of age compared to the control group. Apart from day 7 of life, no significant differences in the occurrence of granulocytes were observed between the group pre-treated with SE-LV alone and the group with combined administration of SELV and CE culture. Furthermore, the percentage of macrophages in the caecum mucosa of SE-LV-treated chicks as well as chicks administered the SE-LV and the CE culture was significantly increased and peaked at day 8 of life (Fig. 3). Compared to the single administration of SELV, no significant differences in the percentage of macrophages were detected after the combined use of SE-LV and CE culture (apart from day 11 of life). Untreated birds as well as CE culture-treated animals showed no or very low numbers of granulocytes and macrophages in caecum without significant differences apart from macrophages on day 10.
3.4. Transcription of immune-related genes in chicken caecum Examination of caecum samples by quantitative real-time RT-PCR revealed an induction of the mRNA expression of selected immune mediators after the administration of SE-LV as well as after combined use of SE-LV and CE culture (Fig. 4). Expression of the pro-inflammatory mediators iNOS, IL-1β, and LITAF was significantly up-regulated in caecum mucosa after single administration of SE-LV. The highest transcription levels were found for iNOS from days 4 to 9 of life. Additionally, the mRNA levels of IL-1β were significantly increased from day 2 of life onwards. Furthermore, the administration of SE-LV resulted in a moderate transcription of IL-12 starting at day 2 of life. The mRNA expression of LITAF and IL-10 was also up-regulated but showed lower mRNA fold changes (under 5-fold increased). In all cases, both the single application of SE-LV and the combination of SE-LV and CE culture showed similar mRNA expression patterns without significant differences. After CE administration, no up-regulation of the iNOS transcription was observed. However, the CE pre-treatment resulted in a low expression of IL-1β and IL-12 and a low mRNA level of LITAF at days 11 and 14 of life.
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Fig. 1. (1) Percentage of Salmonella-positive stained area in caecal mucosa after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Mann-Whitney U test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE. (2 to 5) Representative images of immunohistochemical staining of Salmonella (brown) and cell nuclei (blue) in chicken caecum mucosa at day 4 of life. 2) CE culture, 3) SE-LV, 4) SE-LV + CE culture, and 5) control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.5. Colonisation-inhibition and invasion-inhibition of SE 147R by the CE culture and the SE-LV alone or after combined administration Pre-treatment of chicks with the CE culture resulted in a strong protective effect compared to controls. The number of challenge organisms was significantly reduced in caecal contents by about 3– 4 log10 units and in liver by about 1–2 log10 units (Table 2). The SELV administered on day of hatch also diminished significantly the systemic invasion of the challenge strain compared to controls and even slightly stronger than the CE culture. However, the caecal
colonisation of the challenge strain SE 147R was only initially significantly inhibited (until day 5 of life), later on it was only slightly (not significant) reduced by about 0.5–1.0 log10 units in comparison to control birds. The combined administration of SE-LV and CE culture prevented the systemic invasion of SE 147R completely. The difference to control chickens amounted to nearly 2.0 log10 units. The application of the SE-LV one day prior the CE culture resulted in a significantly reduced caecal colonisation by the challenge strain compared to the untreated control group and to the group administered the SE-LV only.
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Fig. 2. (1) Numbers of granulocytes per square millimeter caecal mucosa after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Mann-Whitney U test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE. (2 to 5) Representative images of immunohistochemical staining of granulocytes (brown) and cell nuclei (blue) in chicken caecum mucosa at day 4 of life. 2) CE culture, 3) SE-LV, 4) SE-LV + CE culture, and 5) control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion This study on combination of exclusion effects by a CE culture and inhibition effects by the attenuated SE-LV has confirmed the considerable exclusion effect of a CE culture against Salmonella exposure in very young chicks (La Ragione and Mead, 2013). Both the caecal colonisation and the systemic invasion of the challenge strain were significantly reduced by the CE culture. To gain information on the mechanisms of this effect it was examined, to the knowledge of the authors for the first time, whether early immunological effects might be induced by the CE culture. The occurrence of granulocytes and macrophages as well as the transcription of immune-related proteins were studied to characterise the
potential of the CE culture to trigger a cellular immune response in the chicken caecum. Heterophils and macrophages contribute to early protecting effects against intestinal infections (Kogut et al., 1994, 1998; Methner et al., 2010; Vazquez-Torres and Fang, 2001). Macrophages especially are essential effector cells concerning the production of different cytokines (IL-1β, IL-12, IL-18) and chemokines (IL-8) (Arango Duque and Descoteaux, 2014). However, compared to untreated control birds, chickens administered the CE culture did not reveal significant differences in any of the parameters examined. It is therefore considered that the protective mechanisms induced by undefined CE cultures are unlikely to be due to the rapid stimulation of the immune system but to different microbiological effects (La Ragione and Mead, 2013).
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Fig. 3. (1) Percentage of macrophage-positive stained area in caecal mucosa after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Mann-Whitney U test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE. (2 to 5) Representative images of immunohistochemical staining of macrophages (brown) and cell nuclei (blue) in chicken caecum mucosa at day 8 of life. 2) CE culture, 3) SE-LV, 4) SE-LV + CE culture, and 5) control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The single oral administration of SE-LV resulted in a high level of intestinal colonisation and systemic invasion. High counts of the Salmonella pretreatment strain in the gut are essential for producing colonisation-inhibition effects (Barrow et al., 1987; Methner et al., 1999). However, SE-LV used alone produced a significant intestinal colonisation-inhibition effect against the challenge strain only for a short time following initial administration but not thereafter. The SELV was produced by chemical mutagenesis (Martin et al., 1996a, 1996b), therefore, the site(s) of these mutations have not been clarified and they may have affected genes which are essential for the expression of the colonisation-inhibition effect (Methner et al., 1997, 2001). In contrast, the SE-LV exhibited a strong invasion-inhibition effect which was also studied for the first time in a registered live Salmonella
vaccine. The SE-LV alone induced an invasion-inhibition effect against the challenge strain which was slightly stronger than the invasioninhibition effect after administration of the CE culture. The mechanisms which might be responsible for the strongly reduced invasion of the challenge strain are however completely different between both preparations. Administration of the CE culture considerably diminished intestinal colonisation of the SE challenge strain and it is considered highly likely that this directly resulted in the reduced number of challenge organisms in the liver. In contrast, the induced influx of heterophils, monocytes and lymphocytes from the blood into the caecal mucosa after administration of SE-LV seems to be the reason for the low number of systemic SE challenge organisms. The special role of granulocytes in preventing the systemic dissemination of Salmonella serovars after
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Fig. 4. Relative quantification of mRNA expression of immune-related proteins in chicken caecum after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Student t-test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE.
pretreatment with Salmonella organisms was shown previously (Methner et al., 2010). The trigger for the induction of the influx of granulocytes and macrophages is the ability of the SE-LV to invade to deeper regions of the intestine (Kogut et al., 1994, 1998) which was shown by the high percentage of Salmonella-positive stained areas in the caecal mucosa. Besides the influx of heterophils and macrophages the highly invasive SE-LV induced also a strong transcription of the pro-inflammatory mediators iNOS, IL-1β, and LITAF as well as the cytokine IL-12 in the chicken caecum. Avian macrophages produce high concentrations of nitric oxide by iNOS (Braukmann et al., 2015; Crippen et al., 2003; Lin et al., 1996) which is involved in the bactericidal activity against Salmonella infections (Chakravortty and Hensel, 2003). It is assumed that in addition to the bactericidal activity of phagocytes, further immune cells are activated after the administration of SELV (Dinarello, 2009; Mastroeni et al., 1998). Therefore, it can be concluded that the early innate immune response induced by the SE-LV
was effective enough to strongly inhibit the systemic invasion and dissemination of the Salmonella challenge strain. 5. Conclusions The postulated potential of inducing additive protective effects by the combined administration of live Salmonella vaccines and a CE culture was partly confirmed in this study. The combined use of a commercial SE-LV with a CE culture did not induce an additive intestinal colonisation-inhibition effect, however, it did result in an additive protective effect that prevented completely the systemic dissemination of the Salmonella challenge strain. To further exploit the efficacy potential of the combined use of CE and vaccination, live Salmonella vaccines are needed that are not only sufficiently attenuated but also still capable to induce both colonisation- and invasion-inhibition effects.
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Conflict of interest statement None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper. Acknowledgements This study was supported by grant no. 0315872A ‘Multifocal strategies to improve gut health and reduce enteritis in poultry and pigs:’ of the European Initiative EMIDA ERA-Net. References Arango Duque, G., Descoteaux, A., 2014. Macrophage cytokines: involvement in immunity and infectious diseases. Front. Immunol. 5, 491. Barrow, P.A., Tucker, J.F., Simpson, J.M., 1987. Inhibition of colonization of the chicken alimentary tract with Salmonella typhimurium gram-negative facultatively anaerobic bacteria. Epidemiol. Infect. 98, 311–322. Berndt, A., Methner, U., 2001. Gamma/delta T cell response of chickens after oral administration of attenuated and non-attenuated Salmonella typhimurium strains. Vet. Immunol. Immunopathol. 78, 143–161. Berndt, A., Wilhelm, A., Jugert, C., Pieper, J., Sachse, K., Methner, U., 2007. Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness. Infect. Immun. 75, 5993–6007. Braukmann, M., Sachse, K., Jacobsen, I.D., Westermann, M., Menge, C., Saluz, H.P., Berndt, A., 2012. Distinct intensity of host-pathogen interactions in Chlamydia psittaci- and Chlamydia abortus-infected chicken embryos. Infect. Immun. 80, 2976–2988. Braukmann, M., Methner, U., Berndt, A., 2015. Immune reaction and survivability of salmonella typhimurium and salmonella infantis after infection of primary avian macrophages. PLoS One 10. http://dx.doi.org/10.1371/journal.pone.0122540. Chakravortty, D., Hensel, M., 2003. Inducible nitric oxide synthase and control of intracellular bacterial pathogens. Microbes Infect. 5, 621–627. Crippen, T.L., Sheffield, C.L., He, H., Lowry, V.K., Kogut, M.H., 2003. Differential nitric oxide production by chicken immune cells. Dev. Comp. Immunol. 27, 603–610. Dinarello, C.A., 2009. Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 27, 519–550. EFSA, 2004. The use of vaccines for the control of Salmonella in poultry. EFSA J. 114, 1–74. EFSA, 2013. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA J. 11, 19–73. Kogut, M.H., Tellez, G.I., McGruder, E.D., Hargis, B.M., Williams, J.D., Corrier, D.E., DeLoach, J.R., 1994. Heterophils are decisive components in the early responses of chickens to Salmonella enteritidis infections. Microb. Pathog. 16, 141–151. Kogut, M.H., Lowry, V.K., Moyes, R.B., Bowden, L.L., Bowden, R., Genovese, K., Deloach, J.R., 1998. Lymphokine-augmented activation of avian heterophils. Poult. Sci. 77, 964–971.
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La Ragione, R., Mead, G.C., 2013. Competetive exclusion. Salmonella in Domestic Animals, second ed. CABI International, Wallingford, UK, pp. 426–454. Lillehoj, E.P., Yun, C.H., Lillehoj, H.S., 2000. Vaccines against the avian enteropathogens Eimeria, Cryptosporidium and Salmonella. Anim. Health Res. Rev. 1, 47–65. Lin, A.W., Chang, C.C., McCormick, C.C., 1996. Molecular cloning and expression of an avian macrophage nitric-oxide synthase cDNA and the analysis of the genomic 5′flanking region. J. Biol. Chem. 271, 11911–11919. Martin, G., Hanel, I., Helmuth, R., Schroeter, A., Erler, W., Meyer, H., 1996a. Immunization with potential Salmonella enteritidis mutants - 1. Production and in vitro characterization. Berl. Munch. Tierarztl. Wochenschr. 109, 325–329. Martin, G., Methner, U., Steinbach, G., Meyer, H., 1996b. Immunization with potential Salmonella enteritidis mutants - 2. Investigations on the attenuation and immunogenicity for mice and young hens. Berl. Munch. Tierarztl. Wochenschr. 109, 369–374. Mastroeni, P., Harrison, J.A., Robinson, J.H., Clare, S., Khan, S., Maskell, D.J., Dougan, G., Hormaeche, C.E., 1998. Interleukin-12 is required for control of the growth of attenuated aromatic-compound-dependent salmonellae in BALB/c mice: role of gamma interferon and macrophage activation. Infect. Immun. 66, 4767–4776. Mead, G.C., 2000. Prospects for ‘Competitive Exclusion’ treatment to control salmonellas and other foodborne pathogens in poultry. Vet. J. 159, 111–123. Methner, U., Barrow, P.A., 1997. Importance of motility of Salmonella Enteritidis and Salmonella Typhimurium on virulence and on the expression of the inhibition phenomenon in vitro and in vivo in SPF chickens. Berl. Munch. Tierarztl. Wochenschr. 110, 391–396. Methner, U., Barrow, P.A., Martin, G., Meyer, H., 1997. Comparative study of the protective effect against Salmonella colonisation in newly hatched SPF chickens using live, attenuated Salmonella vaccine strains, wild-type Salmonella strains or a competitive exclusion product. Int. J. Food Microbiol. 35, 223–230. Methner, U., Barrow, P.A., Berndt, A., Steinbach, G., 1999. Combination of vaccination and competitive exclusion to prevent Salmonella colonization in chickens: experimental studies. Int. J. Food Microbiol. 49, 35–42. Methner, U., Berndt, A., Steinbach, G., 2001. Combination of competitive exclusion and immunization with an attenuated live Salmonella vaccine strain in chickens. Avian Dis. 45, 631–638. Methner, U., Barrow, P.A., Berndt, A., 2010. Induction of a homologous and heterologous invasion-inhibition effect after administration of Salmonella strains to newly hatched chicks. Vaccine 28, 6958–6963. Methner, U., Haase, A., Berndt, A., Martin, G., Nagy, B., Barrow, P.A., 2011. Exploitation of intestinal colonization-inhibition between Salmonella organisms for live vaccines in poultry: potential and limitations. Zoonoses Public Health 58, 540–548. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29, e45. Smith, H.W., Tucker, J.F., 1980. The virulence of salmonella strains for chickens: their excretion by infected chickens. J. Hyg. 84, 479–488. Springer, S., Lehmann, J., Lindner, T., Thielebein, J., Alber, G., Selbitz, H.J., 2000. A new live Salmonella enteritidis vaccine for chickens – experimental evidence of its safety and efficacy. Berl. Munch. Tierarztl. Wochenschr. 113, 246–252. Vazquez-Torres, A., Fang, F.C., 2001. Oxygen-dependent anti-Salmonella activity of macrophages. Trends Microbiol. 9, 29–33.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Combination of competitive exclusion and immunisation with a live Salmonella vaccine in newly hatched chickens: Immunological and microbiological effects M. Braukmann a, P.A. Barrow c, A. Berndt b, U. Methner a,⁎ a b c
Institute of Bacterial Infections and Zoonoses at the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Naumburger Str. 96a, D-07743 Jena, Germany Institute of Molecular Pathogenesis at the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Naumburger Str. 96a, D-07743 Jena, Germany School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, United Kingdom
a r t i c l e
i n f o
Article history: Received 28 January 2016 Received in revised form 25 April 2016 Accepted 7 May 2016 Available online xxxx Keywords: Chicken Competitive exclusion Immune response Inhibition Salmonella live vaccine
a b s t r a c t In addition to evaluating the efficacy potential of a combined use of vaccination and competitive exclusion (CE) against Salmonella exposure in chicks at 3-days of age, a live Salmonella Enteritidis vaccine (SE-LV) and a CE culture were tested for their ability to induce parameters of the innate immunity. Whereas the invasive SE-LV induced an influx of granulocytes and macrophages as well as an increased transcription of several cytokines in the caecal mucosa, the CE culture did not demonstrate any differences in these parameters compared to controls. It is therefore highly probable that the effects observed with CE cultures are not due to the rapid stimulation of the immune system. The combined use of both preparations did not result in an additive intestinal exclusion effect of the challenge strain more pronounced than that after single administration of the CE culture. The combined use of the Salmonella live vaccine and the CE culture resulted in an additive protective effect and prevented completely the systemic dissemination of the Salmonella challenge strain. To exploit the potential of combined use of CE and vaccination further and most effectively, live Salmonella vaccines are needed that are despite their attenuation in virulence still capable to induce both intestinal colonisation- and invasioninhibition effects against Salmonella exposure. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Poultry meat and eggs are considered to be the major source of Salmonella infection for humans (EFSA, 2013). In addition to efficient hygiene regimes at all stages of production, immunisation with both live and inactivated Salmonella vaccines and competitive exclusion (CE) represent the most important methods to increase the resistance of both very young and adult chickens (EFSA, 2004). CE cultures are effective against different serovars (Mead, 2000); however, the mechanisms of protection are not fully understood. Effects like the creation of a restrictive physiological environment, the competition for enteric receptor sites or with other microbes for nutrients, the production of antimicrobial compounds and the stimulation of the immune system are discussed (La Ragione and Mead, 2013). Protective effects induced by vaccination of birds include the reduced intestinal colonisation and the diminished systemic invasion of
⁎ Corresponding author. E-mail address: ulrich.methner@fli.bund.de (U. Methner).
http://dx.doi.org/10.1016/j.rvsc.2016.05.001 0034-5288/© 2016 Elsevier Ltd. All rights reserved.
Salmonella wild-type organisms by mechanisms of the adaptive immunity (EFSA, 2004). In general it is accepted that live Salmonella vaccines are more effective against intestinal and systemic infection than are inactivated vaccine preparations (Lillehoj et al., 2000). Moreover, live Salmonella vaccines are also capable of inducing protective mechanisms effective during the ‘immunity gap’, the time between administration of the vaccine and development of the adaptive immune response. The i) intestinal colonisation-inhibition effect (Barrow et al., 1987; Methner et al., 2011) and the ii) invasion-inhibition effect (Methner et al., 2010) have not been considered until now in the development of live Salmonella vaccines for chickens. The combined use of a CE culture and a live Salmonella vaccine which is able to induce both a colonisation-inhibition and an invasion-inhibition effect, may produce an additive protective impact more pronounced than the single use of either of these methods. A registered live Salmonella Enteritidis vaccine is capable to stimulate a protective adaptive immune response in juvenile chickens (Springer et al., 2000); however, the vaccine has not yet been tested for its potential to induce colonisation- or invasion-inhibition effects. Apart from evaluating the possible protective effects after single and
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combined use of this live Salmonella Enteritidis vaccine and a commercial CE culture against Salmonella Enteritidis exposure in newly hatched chicks, the study also aims to identify parameters of the innate immune response which might bring about the effects observed. 2. Materials and methods 2.1. Chickens Specific pathogen-free White Leghorn chickens were hatched at the facilities of the Friedrich-Loeffler-Institute from eggs obtained from Charles River Deutschland GmbH. Experimental and control groups were kept in cages in separate negative pressure rooms. Commercial feed (coarse meal without antibacterial additives) and public drinking water were both available ad libitum. The single groups were managed separately (including cleaning and feeding regimes) to prevent crosscontamination between the groups effectively throughout the trials. Animal experiments were performed in accordance with the German Animal Protection Act and approved by an ethical committee (Animal Ethics approval number: 04-005/11- 01 December 2011). 2.2. Bacterial strains and culture Salmovac SE (IDT Biologika), a registered Salmonella Enteritidis live vaccine (SE-LV) strain (phage type PT4) was used for oral immunisation of the chickens on day 1 of life with or without combined use of a commercial CE culture (Aviguard, Microbial Developments Ltd.). To facilitate accurate enumeration of the vaccine strain in caecal contents and liver, a spontaneous nalidixic acid-resistant (N) mutant was produced for immunisation (Smith and Tucker, 1980). The resistance has no perceptible impact on the in vivo results (Barrow et al., 1987; Methner et al., 2010, 2011). To confirm this assumption, the original nonresistant SE-LV and the nalidixic acid-resistant SE-LV were compared using in vitro experiments for both, their adhesion and invasion in a cell-culture model as well as for their ability to inhibit the growth of other Salmonella organisms in nutrient broth (Methner and Barrow, 1997). Both variants of the SE-LV did not differ in these characteristics (data not shown). The viable count of the attenuated SE-LV administered PO via crop instillation was 2 × 108 colony forming units (cfu) per bird. The CE culture was dissolved in accordance with the manufacturer's instruction and administered via crop instillation. Oral infection of the chicken was carried out with a rifampicin (R) resistant variant (Methner et al., 2011) of the comprehensively characterised strain Salmonella enterica subspecies enterica serovar Enteritids 147 (SE 147R, phage type PT4) (Methner et al., 2010, 2011) at a dose of 2 × 105 cfu/bird. All strains had been stored in a Cryobank system (Mast Diagnostica) at − 20 °C. 2.3. Experimental design and bacteriology Caecal colonisation and systemic invasion of the attenuated SE-LV after single administration on day 1 of life as well as in combination with a CE culture (SE-LV on day 1 followed by the CE culture on day 2 of age) without Salmonella challenge was studied in experiment 1 (Table 1). SE-LV was enumerated in caecal contents and in liver at days 3, 4, 7, 8, 9, 10, 11, and 14 of life from 4 birds/group, respectively, by a standard plating method (Methner et al., 2001, 2010). Homogenised organ samples were diluted and plated on brilliantgreen phenol red agar (SIFIN) with sodium nalidixate (50 μg/mL) and incubated at 37 °C for 18–24 h. Caecal contents and liver samples from all birds in groups A–D (Table 1) were also pre-enriched in buffered peptone water (SIFIN), incubated at 37 °C for 18–24 h and streaked onto brilliant-green phenol red agar with sodium nalidixate (SIFIN). Additionally, caeca from each animal of the groups administered the SE-LV alone or in combination with the CE culture, a control group and a group given the CE culture only were taken and frozen in
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Table 1 Number (mean log10 cfu/g of 4 birds) of Salmonella Enteritidis live vaccine (SE-LV) in liver and caecal contents of chickens after oral administration of 2 × 108 cfu/bird at 1 day of age without or with subsequent application of a competitive exclusion (CE) culture at day 2 of age (experiment 1). Day of age
Group A
Group B
Group C
Group D
1 2
CE culture –
SE-LV –
SE-LV CE culture
– –
Day of age
Group A
Group B
Group C
Group D
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
3 4 7 8 9 10 11 14
– – – – – – – –
– – – – – – – –
2.5 3.3 3.0 2.8 2.5 1.6 1.6 1.3
8.5 8.7 7.9 8.4 7.9 6.7 6.6 7.3
2.7 3.0 2.8 2.8 2.7 1.8 1.7 1.3
8.9 8.4 7.6 6.9b 6.1b 4.7b 4.9b 5.7b
– – – – – – – –
– – – – – – – –
Standard error: liver: 0.199; caeca: 0.357. b Significantly lower than group B. c Significantly lower than C.
liquid nitrogen or stored in RNAlater (Qiagen) until use for immunohistochemistry or studying mRNA expression of selected cytokines, respectively. In experiment 2 the protective effect induced by the CE culture and the SE-LV alone or after combination of both (SE-LV on day 1 followed by the CE culture on day 2 of age) against challenge with SE 147R administered on day 3 of life was compared with an untreated control group (Table 2). The challenge strain was enumerated in caecal contents and liver from 4 birds/group at days 4, 5, 8, 9, 10, 11, 12, and 15 of age using a standard method described above.
2.4. Immunohistochemistry Using immunohistochemistry, the invasion of the SE-LV into lower regions of the caecal mucosa as well as the influx of granulocytes and macrophages into the caecum was examined in experiment 1. Cryostat sections of 7 μm thickness of every chicken caecum were prepared and Table 2 Number (mean log10 cfu/g of 4 birds) of Salmonella Enteritidis 147R (SE 147R; 2 × 105 cfu/ bird PO at 3 days of age) in liver and caecal contents of chickens after pre-treatment with a competitive exclusion (CE) culture or a Salmonella Enteritidis live vaccine (SE-LV) (2 × 108 cfu/bird PO at 1 day of age) without or with subsequent application of a CE culture at day 2 of age (experiment 2). Day of age
Group A
Group B
Group C
Group D
1 2 3
CE culture
SE-LV
SE 147 R
SE 147 R
SE-LV CE culture SE 147 R
– – SE 147 R
Day of age
Group A
Group B
Group C
Group D
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
Liver
Caecal contents
4 5 8 9 10 11 12 15
0 0 0.3d 0.5d 0d 0.6d 0.7d 0.3d
2.9b,d 3.8d 4.3d 4.2b,d 3.6b,d 4.1d 3.8b,d 4.0d
0 0 0d 0d 0d 0.5d 0.4d 0.3d
4.7d 4.2d 6.4 7.0 6.5 5.3 6.4 5.7
0 0 0d 0d 0d 0d 0d 0a,d
3.5d 2.8d 4.6 4.1b,d 4.6d 3.8d 3.6b,d 3.3d
0.3 0.3 1.8 1.9 1.6 2.2 1.9 1.5
7.7 6.5 6.9 7.0 7.0 6.9 7.4 6.6
a b c d
Significantly lower than group A. Significantly lower than group B. Significantly lower than group C. Significantly lower than group D.
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stained as described previously (Berndt and Methner, 2001). Briefly, the primary antibodies against Salmonella enterica lipopolysaccharide (LPS) (Clone 5D12A, Bio-Rad AbD Serotec), avian granulocytes (kindly provided by F. Van Immerseel, University Gent, Belgium) and monocytes/macrophages (CVI 68.1, Institute for Animal Science and Health, Lelystad, The Netherlands) were used. As negative control, the primary antibodies were replaced with PBS. 2.5. Image analysis Analyses of the number of granulocytes and proportions of immunohistochemically stained areas of macrophages and Salmonella organisms in caecal mucosa were conducted by means of the image analysis system CELL (Olympus) as described previously (Berndt et al., 2007). Briefly, macrophages and Salmonella organisms were monitored by determining the percentage of positive stained areas in whole caecum mucosa (epithelium and lamina propria) and numbers of granulocytes were counted in epithelial und sub-epithelial areas. 2.5.1. Quantitative real-time reverse transcription (RT)-PCR To study gene transcription, total RNA was extracted from chicken caecum samples in experiment 1. RNA extraction, digestion of residual DNA and purity criteria were described previously (Berndt et al., 2007). Avian-specific primers for glycerinaldehyde-3-phosphate (GAPDH), interleukin (IL)-12β (p40), lipopolysaccharide-induced tumour necrosis factor alpha factor (LITAF), inducible nitric oxide synthase (iNOS), IL-1β and IL-10 were developed formerly (Berndt et al., 2007; Braukmann et al., 2012). To determine mRNA expression rates by the QuantiTect SYBR Green real-time one-step RT-PCR Kit (Qiagen), the thermocycler Mx3000P (Agilent) was used with the temperature-time profile including the optimised annealing temperatures as described by Berndt et al. (2007). Each primer pair was tested by a serial dilution of template RNA to ensure optimized amplification and comparable efficiencies of the RT-PCR. For every primer, notemplate controls were performed. The threshold method was used for relative quantification of mRNA level. Normalisation of target genes was performed using the GAPDH as endogenous standard. Results were expressed as fold change (2[−ΔΔCt]) (Pfaffl, 2001) relative to the average of transcripts detected in the control group at each time point. 2.6. Statistical analysis Viable bacterial counts were converted into logarithmic form. For statistical purposes a viable count of log10 b 1.47 from a sample detected positive only after enrichment was rated as log10 = 1.0. A sample which yielded no Salmonella growth after enrichment was rated as log10 = 0. Data were evaluated by analysis of variance (multifactor ANOVA). The factors considered were group and time. P values b 0.05 were regarded as statistically significant (software: statgraphics plus, Inc. Rockville, MD). Statistical analysis of data from immunohistochemistry was conducted using the Mann-Whitney U test (SPSS 19.0, IBM). The normally distributed data of quantitative real-time RT-PCR were statistically evaluated using the Student t-test. Both tests were used for comparison of two independent samples to evaluate differences between the groups at each time point. P values of b 0.05 were considered significant. 3. Results 3.1. Colonisation and invasion of the Salmonella Enteritidis live vaccine with or without combined use of a CE culture The sole oral administration of the SE-LV resulted in a high level of caecal colonisation and systemic invasion but did not induce signs of morbidity (Table 1). The application of the CE culture after the SE-LV did not result in a significant reduction of the vaccine strain in liver
compared to the single use of SE-LV. However, the caecal colonisation of the SE-LV in this group was significantly reduced from day 8 of life onwards in comparison to birds given the live vaccine only. The differences ranged from 1.5 to 2.0 log10 units. In all animals from groups A and D the vaccine strain SE-LV was not detected.
3.2. Quantification of the Salmonella Enteritidis live vaccine in caecal mucosa After single administration of SE-LV and after combination of the SELV with the CE culture, comparable numbers of small accumulations and single bacteria of the SE-LV strain were observed in the caecal mucosa (Fig. 1). The capability of SE-LV to invade the epithelium and the whole lamina propria was similar in both modes of administration. The highest amounts of Salmonella-positively stained areas were found between days 3 and 7 of life after single administration of SE-LV and between days 4 and 7 of life after application of SE-LV and the CE culture. The administration of the CE culture after the SE-LV did not reveal significant differences in the ability of SE-LV to invade the caecal tissue. In the CE-treated group as well as the control group no Salmonella-LPS was detected.
3.3. Quantification of granulocytes and macrophages in chicken caecum Compared to the controls, the application of the SE-LV induced significantly increased numbers of granulocytes between days 2 and 10 of life (Fig. 2). The combined pre-treatment of chicks with SE-LV and CE culture resulted in significantly elevated numbers of granulocytes at day 3 of life and continued until day 11 of age compared to the control group. Apart from day 7 of life, no significant differences in the occurrence of granulocytes were observed between the group pre-treated with SE-LV alone and the group with combined administration of SELV and CE culture. Furthermore, the percentage of macrophages in the caecum mucosa of SE-LV-treated chicks as well as chicks administered the SE-LV and the CE culture was significantly increased and peaked at day 8 of life (Fig. 3). Compared to the single administration of SELV, no significant differences in the percentage of macrophages were detected after the combined use of SE-LV and CE culture (apart from day 11 of life). Untreated birds as well as CE culture-treated animals showed no or very low numbers of granulocytes and macrophages in caecum without significant differences apart from macrophages on day 10.
3.4. Transcription of immune-related genes in chicken caecum Examination of caecum samples by quantitative real-time RT-PCR revealed an induction of the mRNA expression of selected immune mediators after the administration of SE-LV as well as after combined use of SE-LV and CE culture (Fig. 4). Expression of the pro-inflammatory mediators iNOS, IL-1β, and LITAF was significantly up-regulated in caecum mucosa after single administration of SE-LV. The highest transcription levels were found for iNOS from days 4 to 9 of life. Additionally, the mRNA levels of IL-1β were significantly increased from day 2 of life onwards. Furthermore, the administration of SE-LV resulted in a moderate transcription of IL-12 starting at day 2 of life. The mRNA expression of LITAF and IL-10 was also up-regulated but showed lower mRNA fold changes (under 5-fold increased). In all cases, both the single application of SE-LV and the combination of SE-LV and CE culture showed similar mRNA expression patterns without significant differences. After CE administration, no up-regulation of the iNOS transcription was observed. However, the CE pre-treatment resulted in a low expression of IL-1β and IL-12 and a low mRNA level of LITAF at days 11 and 14 of life.
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Fig. 1. (1) Percentage of Salmonella-positive stained area in caecal mucosa after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Mann-Whitney U test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE. (2 to 5) Representative images of immunohistochemical staining of Salmonella (brown) and cell nuclei (blue) in chicken caecum mucosa at day 4 of life. 2) CE culture, 3) SE-LV, 4) SE-LV + CE culture, and 5) control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.5. Colonisation-inhibition and invasion-inhibition of SE 147R by the CE culture and the SE-LV alone or after combined administration Pre-treatment of chicks with the CE culture resulted in a strong protective effect compared to controls. The number of challenge organisms was significantly reduced in caecal contents by about 3– 4 log10 units and in liver by about 1–2 log10 units (Table 2). The SELV administered on day of hatch also diminished significantly the systemic invasion of the challenge strain compared to controls and even slightly stronger than the CE culture. However, the caecal
colonisation of the challenge strain SE 147R was only initially significantly inhibited (until day 5 of life), later on it was only slightly (not significant) reduced by about 0.5–1.0 log10 units in comparison to control birds. The combined administration of SE-LV and CE culture prevented the systemic invasion of SE 147R completely. The difference to control chickens amounted to nearly 2.0 log10 units. The application of the SE-LV one day prior the CE culture resulted in a significantly reduced caecal colonisation by the challenge strain compared to the untreated control group and to the group administered the SE-LV only.
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Fig. 2. (1) Numbers of granulocytes per square millimeter caecal mucosa after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Mann-Whitney U test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE. (2 to 5) Representative images of immunohistochemical staining of granulocytes (brown) and cell nuclei (blue) in chicken caecum mucosa at day 4 of life. 2) CE culture, 3) SE-LV, 4) SE-LV + CE culture, and 5) control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion This study on combination of exclusion effects by a CE culture and inhibition effects by the attenuated SE-LV has confirmed the considerable exclusion effect of a CE culture against Salmonella exposure in very young chicks (La Ragione and Mead, 2013). Both the caecal colonisation and the systemic invasion of the challenge strain were significantly reduced by the CE culture. To gain information on the mechanisms of this effect it was examined, to the knowledge of the authors for the first time, whether early immunological effects might be induced by the CE culture. The occurrence of granulocytes and macrophages as well as the transcription of immune-related proteins were studied to characterise the
potential of the CE culture to trigger a cellular immune response in the chicken caecum. Heterophils and macrophages contribute to early protecting effects against intestinal infections (Kogut et al., 1994, 1998; Methner et al., 2010; Vazquez-Torres and Fang, 2001). Macrophages especially are essential effector cells concerning the production of different cytokines (IL-1β, IL-12, IL-18) and chemokines (IL-8) (Arango Duque and Descoteaux, 2014). However, compared to untreated control birds, chickens administered the CE culture did not reveal significant differences in any of the parameters examined. It is therefore considered that the protective mechanisms induced by undefined CE cultures are unlikely to be due to the rapid stimulation of the immune system but to different microbiological effects (La Ragione and Mead, 2013).
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Fig. 3. (1) Percentage of macrophage-positive stained area in caecal mucosa after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Mann-Whitney U test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE. (2 to 5) Representative images of immunohistochemical staining of macrophages (brown) and cell nuclei (blue) in chicken caecum mucosa at day 8 of life. 2) CE culture, 3) SE-LV, 4) SE-LV + CE culture, and 5) control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The single oral administration of SE-LV resulted in a high level of intestinal colonisation and systemic invasion. High counts of the Salmonella pretreatment strain in the gut are essential for producing colonisation-inhibition effects (Barrow et al., 1987; Methner et al., 1999). However, SE-LV used alone produced a significant intestinal colonisation-inhibition effect against the challenge strain only for a short time following initial administration but not thereafter. The SELV was produced by chemical mutagenesis (Martin et al., 1996a, 1996b), therefore, the site(s) of these mutations have not been clarified and they may have affected genes which are essential for the expression of the colonisation-inhibition effect (Methner et al., 1997, 2001). In contrast, the SE-LV exhibited a strong invasion-inhibition effect which was also studied for the first time in a registered live Salmonella
vaccine. The SE-LV alone induced an invasion-inhibition effect against the challenge strain which was slightly stronger than the invasioninhibition effect after administration of the CE culture. The mechanisms which might be responsible for the strongly reduced invasion of the challenge strain are however completely different between both preparations. Administration of the CE culture considerably diminished intestinal colonisation of the SE challenge strain and it is considered highly likely that this directly resulted in the reduced number of challenge organisms in the liver. In contrast, the induced influx of heterophils, monocytes and lymphocytes from the blood into the caecal mucosa after administration of SE-LV seems to be the reason for the low number of systemic SE challenge organisms. The special role of granulocytes in preventing the systemic dissemination of Salmonella serovars after
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Fig. 4. Relative quantification of mRNA expression of immune-related proteins in chicken caecum after oral application of a competitive exclusion (CE) culture, a Salmonella Enteritidis live vaccine (SE-LV), the combination of SE-LV and CE culture as well as in the control group (experiment 1). Significant differences (P b 0.05) were calculated using the Student t-test. a: CE vs. control; b: SE-LV vs. control; c: SE-LV + CE vs. control; d: SE-LV vs. SE-LV + CE.
pretreatment with Salmonella organisms was shown previously (Methner et al., 2010). The trigger for the induction of the influx of granulocytes and macrophages is the ability of the SE-LV to invade to deeper regions of the intestine (Kogut et al., 1994, 1998) which was shown by the high percentage of Salmonella-positive stained areas in the caecal mucosa. Besides the influx of heterophils and macrophages the highly invasive SE-LV induced also a strong transcription of the pro-inflammatory mediators iNOS, IL-1β, and LITAF as well as the cytokine IL-12 in the chicken caecum. Avian macrophages produce high concentrations of nitric oxide by iNOS (Braukmann et al., 2015; Crippen et al., 2003; Lin et al., 1996) which is involved in the bactericidal activity against Salmonella infections (Chakravortty and Hensel, 2003). It is assumed that in addition to the bactericidal activity of phagocytes, further immune cells are activated after the administration of SELV (Dinarello, 2009; Mastroeni et al., 1998). Therefore, it can be concluded that the early innate immune response induced by the SE-LV
was effective enough to strongly inhibit the systemic invasion and dissemination of the Salmonella challenge strain. 5. Conclusions The postulated potential of inducing additive protective effects by the combined administration of live Salmonella vaccines and a CE culture was partly confirmed in this study. The combined use of a commercial SE-LV with a CE culture did not induce an additive intestinal colonisation-inhibition effect, however, it did result in an additive protective effect that prevented completely the systemic dissemination of the Salmonella challenge strain. To further exploit the efficacy potential of the combined use of CE and vaccination, live Salmonella vaccines are needed that are not only sufficiently attenuated but also still capable to induce both colonisation- and invasion-inhibition effects.
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