Institute for Animal Health, Compton, Berkshire RG20 7NN, UK1
Biochemistry Section, Glasgow Veterinary School, Bearsden Rd, Glasgow G61 1QH, UK2
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK3
Author for correspondence: Bernardo Villarreal-Ramos. Tel: +44 1635 578 411. Fax: +44 1635 577 263. e-mail: Bernardo.Villarreal{at}bbsrc.ac.uk
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ABSTRACT |
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Keywords: Salmonella, cattle, htrA, purine
Abbreviations: STSE, Salmonella typhimurium soluble extract; PBS-T, phosphate buffered saline/Tween 20; IFN, interferon gamma
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INTRODUCTION |
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The murine model of salmonellosis has been exploited to identify genes required for survival and replication in this host (Chiang et al., 1999 ). Salmonella strains harbouring such mutations are attenuated and therefore are potential live vaccine candidates against salmonellosis in other species (Mastroeni et al., 1999
). Thus, the murine salmonellosis model has been used to identify candidate mutations to generate attenuated S. typhi for live typhoid vaccine development. Mutations exploited in this way include those associated with aromatic compound dependence (aro) (Hosieth & Stocker, 1981
; Tacket et al., 2000
), purine dependence (pur) (Levine et al., 1987
; OCallaghan et al., 1988
; Everest et al., 1997
) and stress resistance (htrA) (Chatfield et al., 1992
; Strahan et al., 1992
; Tacket et al., 2000
). However, not all mutations that attenuate S. typhimurium in the murine model are fully attenuating for S. typhi in man. For example, although galE mutants of S. typhimurium are attenuated in mice, S. typhi galE strains can cause typhoid-like disease in humans (Hone et al., 1988
).
In addition to their ability to confer protection against virulent challenge, live attenuated Salmonella vaccines can be used as vectors to deliver antigens from other pathogens, thus bringing the ideal single dose multivalent vaccine closer to reality (Levine & Dougan, 1998 ). Few studies have been conducted using Salmonella as a vector for heterologous antigen delivery in species other than the mouse. Studies in cattle have used Salmonella strains harbouring aro mutations (Villarreal-Ramos et al., 1998
). However, in cattle the use of recombinant Salmonella aro mutants as vectors for foreign antigen delivery has been less successful than would have been expected from the results obtained in mice (dOlivera et al., 1997
; Gentschev et al., 1998
; Villarreal-Ramos et al., 2000
). If attenuated salmonellae are to fulfil their potential as live vaccines against salmonellosis in different animal species it is necessary to evaluate different candidate Salmonella vaccines in the target animal species. Further, it will be essential to explore different delivery regimes and understand the mechanisms of immunity induced by Salmonella vectors in different species. In this work we examined the ability of different mutations that are known to attenuate salmonellae in mice to attenuate salmonellae in cattle. We have also examined the influence of the route of administration on the virulence of Salmonella and the immune response induced.
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METHODS |
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Calves.
Four to five week old Friesian bull calves were used throughout. Calves were reared conventionally from birth and tested for the presence of Salmonella as described below. Blood samples were collected by venepuncture from the jugular vein.
Preparation of inocula.
For oral challenge, bacteria were grown overnight in LuriaBertani broth at 37 °C. Appropriate dilutions were made to obtain the number of bacteria required as indicated in the text and in Table 1 for each experiment. The concentration of the inocula was confirmed by plating dilutions on brilliant green agar (Oxoid). Bacteria were resuspended in 20 ml PBS and mixed with an equal volume of antacid solution (5% sodium bicarbonate, 5% magnesium carbonate and 5% magnesium trisilicate; all from BDH) and delivered per os with a syringe (Jones et al., 1988
). Calves were inoculated subcutaneously in the prescapular area with bacteria suspended in a final volume of 1·5 ml PBS, preceded by intravenous injection of 2 ml flunixin meglumine at 50 mg ml-1 (Schering-Plough Animal Health) to reduce the risk of hypersensitivity reactions.
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Bacteriological analysis.
The presence of Salmonella in faeces before challenge was determined by enrichment in selenite brilliant green broth (SBG) (Difco) and Rappaport medium [0·45% tryptone (Difco), 124 mM NaCl (BDH), 10 mM KH2PO4 (BDH), 180 mM MgCl2.6H2O (BDH), 0·012% malachite green (BDH)] (Rappaport et al., 1956 ) and by direct inoculation onto brilliant green agar (Oxoid) for colony counts. The presence of Salmonella in faeces after challenge was determined daily both by enrichment and by direct plating of dilutions of faecal homogenates. Briefly, 1 g faeces was placed in 9 ml PBS, pH 7·5 and mixed to obtain a homogeneous suspension. Five drops of 20 µl each were plated on brilliant green agar plates. The results were expressed as geometric mean counts (g sample)-1. Bacteraemia was monitored daily by enrichment of 1 ml blood in SBG and Rappaport medium and subsequent inoculation onto brilliant green agar. After killing the following samples were removed from calves: ileum wall, ileum contents, ileal lymph node (the distal comma shaped node at the end of the chain of jejuno-ileal lymph nodes), caecum wall, caecum contents, caecal lymph node, colon wall, colon contents, colonic lymph node, liver, hepatic lymph node, spleen, lung, bronchial lymph node, blood and bile. From each sample, 1 g was placed in 9 ml H2O and homogenized, where necessary, using mortar, pestle and sand. Dilutions of the homogenates were plated on brilliant green agar plates as described above. The presence of Salmonella was also determined by enrichment of 1 ml homogenate in SBG and Rappaport medium and subsequent inoculation onto brilliant green agar. Using this technique, the limit of detection by direct plating of bacterial homogenates is 500 bacteria g-1 (i.e. 1 bacterium in a 20 µl droplet=50 bacteria ml-1 from the original 10 ml homogenate). Therefore, if bacteria were not detected by direct plating but were detected by enrichment in broth, the counts were assumed to be less than 500 bacteria (g sample)-1.
ELISA.
ELISA to detect antibodies to LPS was carried out as described by Demarco de Hormaeche et al. (1988 ). Each well of Microtest III 96-well plates (Falcon) was coated with 2·5 µg smooth Westphal LPS from S. typhimurium (Sigma) in 10 mM glycine, 20 mM NaCl, 0·2 mM EDTA, 0·01 mM NaF and 24 mM sodium deoxycholate. After overnight incubation at 37 °C, the plates were incubated with blocking buffer, consisting of 3% BSA Fraction V (Sigma) in PBS and 0·05% Tween 20 (Sigma) (PBS-T). After 1 h at 37 °C, the plates were washed with PBS-T. Fifty microlitres of cattle serum diluted in 0·3% BSA in PBS-T was added to individual wells in duplicate. To detect IgG1, IgG2 and IgM, sera were diluted 1:1000, 1:100 and 1:100, respectively. After incubation for 1 h at 37 °C the plates were washed and 50 µl B37 (monoclonal antibody to bovine IgG1), B192 (monoclonal antibody to IgG2) or B67 (monoclonal antibody to IgM) diluted in 0·3% BSA in PBS-T was added to each well. Monoclonal antibodies were diluted 1:250, a previously determined optimum concentration. After 1 h incubation at 37 °C, plates were washed with PBS-T and 50 µl goat anti-mouse horseradish peroxidase conjugate (Dako) diluted 1:1000 in 0·3% BSA in PBS-T was added to each well according to the manufacturers instructions. After 1 h at 37 °C, 100 µl 3,3',5,5'-tetramethylbenzidine (ICN), prepared according to the manufacturers instructions, was added to each well. After 10 min, 25 µl 4 M H2SO4 was added to each well and plates were read at 450 nm in a Titretek Multiskan MCC/340 ELISA reader.
To detect antibodies to a soluble extract of S. typhimurium (STSE), 96-well plates were coated with 50 µl STSE, prepared as described previously (Villarreal et al., 1992 ), diluted to 2 µg ml-1 in 0·1 M carbonate buffer [0·1 M Na2CO3, 0·1 M NaHCO3; both from BDH), pH 9·6. After overnight incubation at 4 °C, the plates were incubated for 1 h at 37 °C before adding 100 µl blocking buffer [2% casein (Hammarsten, BDH) in PBS, 0·05% Tween 20 (Sigma), pH 7·0] to each well. Thereafter the protocol followed was essentially the same as for the LPS ELISA, except that 0·2% casein in PBS-T was used as the diluent.
Measurement of interferon (IFN
) and haptoglobin in sera of cattle inoculated with S. typhimurium 4/74.
IFN in sera was measured by ELISA (CSL, Bovigamm) according to the manufacturers instructions.
The concentration of haptoglobin in sera was assessed using a method based on the preservation of the haemoglobin peroxidase activity of haptoglobin in mild acidic conditions (Makimura & Suzuki 1982 ). The method was modified for use on an automated biochemical analyser (MIRA, Roche Diagnostics) to reduce interference from serum albumin (Eckersall et al., 1999
). In this method, using the automated biochemical analyser, 2·5 µl serum was mixed with 200 µl of met-haemoglobin solution (0·06 mg ml-1 in 0·9% NaCl), prepared as described by Makimura & Suzuki (1982
). After 50 s, 90 µl SB-7 chromogen solution (Tridelta Development) was added. After a further 25 s, 50 µl 0·12% H2O2 substrate (diluted in H2O) was added and the increase in absorbance at 600 nm over the following 50 s was monitored. The assay was quantified by comparison with dilutions of a bovine serum sample with a known concentration of haptoglobin, originally being calibrated with purified bovine haptoglobin (Horadagoda et al., 1994
).
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RESULTS |
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To assess the ability of S. typhimurium 4/74 to infect cattle via the parenteral route, six calves were inoculated subcutaneously with 1·5x109 (calves 5, 6 and 7) or 1·8x109 (calves 3, 4 and 8) S. typhimurium 4/74 (Table 1). Calves 3, 4 and 5 succumbed to infection while calves 6, 7 and 8 survived this high challenge dose. All the subcutaneously challenged cattle developed pyrexia and bacteraemia and secreted S. typhimurium in their faeces (Table 1
). Calves 3, 4 and 5 succumbed to infection 3, 3 and 7 d after inoculation, respectively. Post-mortem bacteriological analysis of killed calves indicated that the salmonellae had spread systemically (Table 2
). In calves 4 and 5, the number of S. typhimurium 4/74 cells associated with the digestive tract was greater than the number of salmonellae associated with systemic sites such as the spleen, lung and liver. In calf 3, the number of S. typhimurium 4/74 cells isolated from the spleen, lungs and hepatic nodes was greater than the number of bacteria isolated from the digestive tract tissues. Four to five weeks after inoculation, the calves that survived (6, 7 and 8), were killed and a post-mortem bacteriological analysis performed as indicated in Methods (Table 2
). S. typhimurium 4/74 was only detected in one animal after enrichment of organ homogenates.
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Measurement of IFN and antibodies to LPS in the sera of calves challenged with S. typhimurium 4/74
In a preliminary attempt to relate immunological parameters to susceptibility to infection, the concentrations of IFN were measured in the sera of infected calves (Table 4
). There was no obvious correlation between the concentration of IFN
, the route of inoculation or susceptibility to infection. Calves 1 and 2, inoculated orally, had the highest serum IFN
concentrations on days 2 and 1 respectively. The IFN
concentrations remained higher than they were before inoculation until both calves died. Calves 3, 4, 6 and 7, which were inoculated subcutaneously, had the highest serum IFN
concentrations 1 d after inoculation, whereas calf 5 had detectable serum IFN
on day one with the highest concentration on day two after inoculation. Calf 8 had detectable serum IFN
on day one after infection with the highest concentration at 67 d after inoculation.
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DISCUSSION |
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The difference in pathogenesis of S. typhimurium in cattle and mice is of relevance in the design and development of safe and effective vaccines. This is particularly important as the mouse is now most frequently used as the animal of choice for identifying potentially attenuating gene mutations in Salmonella spp. Indeed, mutations in both the aro and htrA genes were originally identified as attenuating in mice and were subsequently used as components of rationally attenuated S. typhi vaccine candidates. Mutations in aro genes have been shown to significantly attenuate Salmonella strains in several animal species including cattle (Smith et al., 1984 ; Jones et al., 1991
) and humans (Tacket et al., 1992
). Mutations in htrA were also used to prevent the detectable systemic spread of S. typhi in volunteers challenged with S. typhi aro vaccines (Tacket et al., 2000
). The htrA gene encodes a periplasmic protease which increases the resistance of salmonellae to peroxide and which appears to contribute to the ability of salmonellae to resist the bactericidal action of macrophages (Johnson et al., 1991
). The apparent virulence of S. typhimurium 4/74 htrA in calves after oral challenge may reflect differences in the ability of bovine macrophages to kill S. typhimurium. Alternatively, S. typhimurium may grow in different host sites in bovine tissues. However, at this time we cannot rule out the possibility that other Salmonella strains may be attenuated by htrA or purE mutations in cattle.
IFN can be produced by T cells and NK (natural killer) cells in response to exposure to bacterial antigens. In naïve animals IFN
is produced predominantly by NK cells. In mice IFN
can be detected in vivo in the sera of mice inoculated intravenously with salmonellae 3 d after inoculation (Mastroeni et al., 1996
; Ramarathinam et al., 1991
). Depletion of IFN
during the early stages of salmonellosis in mice led to an increase in the rate of growth of virulent salmonellae (Muotiala & Makela, 1990
). Equally, administration of exogenous IFN
in the early stages of salmonellosis in mice leads to a decrease in the rate of growth of salmonellae (Muotiala & Makela, 1990
). We looked for a relationship between the route of infection and production of IFN
in cattle inoculated with S. typhimurium 4/74, but found no correlation. Calves inoculated orally or subcutaneously that either survived or succumbed to the infection produced IFN
. An acute phase reaction has been demonstrated in cattle infected with S. typhimurium. The production of haptoglobin is stimulated during the acute phase reaction by a combination of pro-inflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF
being released from macrophages and other cells at the site of lesions or infection. Potentially, IFN
can be involved in this pathway by activation of macrophages, but in our studies, with the difference between the subcutaneous and oral routes of inoculation it would appear that the IFN
and haptoglobin responses were not correlated. The acute phase response detected in the subcutaneously inoculated cattle was moderate. Recent studies show that the acute phase response to conditions such as clinical mastitis, pulmonary thromboembolism and acute respiratory disease can lead to serum concentrations of 23 mg haptoglobin ml-1 (Horadagoda et al., 1999
) and here we observed a rise to only about 25% of these levels. The acute phase response is not likely to be a useful diagnostic tool in orally infected cases of salmonellosis (e.g. natural infection), but could be a useful tool in experimental infection, especially for monitoring the response to subcutaneous inoculation.
In conclusion, our studies, taken together with those of others, highlight significant differences in the pathogenesis of salmonellosis between mice and cattle. This conclusion is supported by data obtained using different routes of challenge and different mutations that influence the growth of salmonellae in vivo. Further studies using different Salmonella strains deleted in different genes should be of value in identifying the differences in basic pathogenic mechanisms and improve our understanding of host specificity in Salmonella, a poorly understood area at the present time.
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ACKNOWLEDGEMENTS |
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Received 28 March 2000;
revised 21 July 2000;
accepted 10 August 2000.
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