samples collected from calves suffering from diarrhoea revealed the isolation
of Salmonella in 17.5% of the cases, while in the contact apparently
healthy calves the incidence was 3.4%. Four salmonella serovares were
elucidated namely, S. typhimurium (7.3% and 1.7%),
Salmonella infections in calves continue to be a major problem worldwide. Substantial economic losses were manifested through mortality and poor growth of infected animals as well as the hazard of transmitting food poisoning to humans. Many outbreaks of salmonella infections has been reported world wide which encountered the most frequently isolated serovars as S. typhimurium, S. enteritidis, S. anatum S. newport, S. cerro, S. montevideo, S. agona and S. dublin which was considered the major host-adapted salmonella for cattle (Mitz et al. 1981, Konrad et al. 1994; Ritchie et al. 2001 and Veling et al. 2002).
The infection was always aggravated by poor hygienic conditions and inadequate nutrition and young calves were most susceptible to infection due to their immature immune responses, undeveloped microflora in their gastrointestinal echo-system and the permanent exposure to the source of infection from the environment and their dams. Calves suffering from acute clinical salmonellosis may shed 108-1010 Salmonella/gram faeces and animals recovering from the infection may continue to be shedders of the organism for 2-12 weeks post infection. Shedding Salmonella by animals without clinical signs may be associated with chronic Salmonella infection (carrier), convalescence after acute infection or a recent starting colonization (Smith et al. 1989, Galland et al. 2000, Roy et al. 2001 and Radke et al. 2002).
Unlike the intermittent shedding of Salmonella in the faeces, antibody levels do not fluctuate greatly on daily basis. Sero-conversion was used in many countries as a tool for Salmonella surveillance and control in cattle, pig and poultry industry at slaughter to identify infected flocks as a regulatory procedures for food safety and security program. Application of ELISA can provide information about the infection status of the animal and the flock, moreover the repeated testing of animals differentiates those recently infected calves (increasing antibody titre) and those convalescent ones (decreasing titres) from salmonella carriers, which would have relatively constant titres (Smith et al. 1989; Nielsen et al. 1995 and Veling et al. 2002).
In order to control and stop the losses incurred by Salmonella in calves formalin-killed Salmonella bacterins has been used commercially in the field in many areas of the world (Wray and Sojka 1984; Roden et al. 1992), Yet, further investigations has to be conducted on the outer membrane protein (OMP) and their immunogenic potentials as a first step for potent vaccine production.
The objective of this study was to investigate the infection of calves with salmonella in certain localities, as well as to evaluate the antigenicity of the outer membrane proteins (OMP) by Western blot and ELISA as a preliminary phase for vaccine production.
A total of 253 faecal samples were collected from 137 diarrheic calves and 116 contact apparently healthy calves aged 1month-1year. At the same time 253 blood samples were collected from the previously mentioned animals in order to correlate the incidence of salmonella to the sero-conversion of each individual animal.
Samples were collected during the period of one year from June 2001 to June 2002 from calves aged (1month - 1year) at governmental and private farms in Giza, Kafr El-Sheikh and Dakahlea governorates.(buffaloe calves were not included in this study).
Specimens were transferred to the laboratory in ice box with minimum delay, where faecal samples were examined bacteriologically for Salmonella isolation and identification. Blood samples were allowed to clott, then centrifuged at 700xg for 15min. Sera were collected and stored in frozen aliquot’s until used in the serological tests.
Faecal samples were cultured into tetrathionate broth and Rappaport Vassiliadis broth, incubated at 37°C and 42°C respectively for 24hr. Loopful from these broth cultures were then streaked onto Mac Conkey and S.S agar plates, incubated at 37°C for 24-48hrs and Salmonella suspected colonies were identified morphologically, then biochemically by the API 20E Kit system (BioMereaux, France) and serologically according to the Kauffman-White Scheem by slide agglutination test using polyvalent and monovalent (O) and (H) antisera (Wellcome Research Laboratories, UK.) (Edwards and Ewing, 1972)
Preparation of the Salmonella outer membrane protein (OMP)
The four Salmonella enterica serovars were isolated (Typhimurium, Dublin, Enteritidis and Anatum) were cultured on tryptic soy agar in Roux flasks and incubated at 37°C for 24hr. Bacterial cells were harvested into 10mM HEPES buffer pH 7.4 and washed three times with normal saline. The bacterial cells were sonicated for 15 min. then centrifuged at 1700xg for 20min. The sediment was discarded and the supernatant was centrifuged at 60,000xg and the pellet was dissolved in 2% sarkosyl in 10mM HEPES buffer and was left overnight at 4°C. The preparation was then centrifuged at 60,000xg for 1hr. and the pellet was dissolved in 10mM Tris HCl pH 7.2 and finally dialysed in 0.15MNaCl, 50mMPBS pH 7.4for 24hr. at 4°C.(Filip, 1973; Kudrna 1985; Brusnigina 1988) The protein content of these OMP preparations were then estimated according to (Lowry 1951).
Sodium Dodecyl sulphate-Polyacryamide Gel Electrophoresis (SDS-PAGE) of Salmonella OMP
The four previously prepared Salmonella OMP preparations (Typhimurium, Dublin, Enteritidis and Anatum) were screened for their OMP protein pattern by SDS-PAGE electrophoresis. The SDS-PAGE was performed in a vertical gel slap by the Laemmli discontinuous buffer system (Laemmli 1970). The gel slabs were 140 x 80 x 1.5mm in a vertical electrophoresis unit (Pharmacia). The stacking gel was 4% and the resolving gel was 12%. Equal volumes (25µl) of the different Salmonella OMP preparations and sampling buffer were vortexed for 15 seconds and heated in thermomixer for 5 min then loaded in the gel along with a molecular protein marker range from 14.4kDa to 94kDa. The electophoresis unit was adjusted at 60V for about 4hr. till the marker blue stain (bromothimol blue) in the sampling buffer reaches the end of the gel. The protein bands were fixed in 50% methanol, 7% acetic acid and 43% dist. Water, then visualised by Coommassie blue stain (Giovannaferro,1974 and Seleim 1999).
Western blot analysis
Nitrocellulose membrane of 0.2µm porosity was used to transfer the electrophoresed proteins by the Western blot technique using Bio-Rad transfer unit containing transfer buffer pH8.3 (25mM Tris; 192mM glycine and 20% methanol. The transfer process was run under 60 V for 6hr. at 8°C. At the end of transfer process protein bands were checked on the nitrocellulose membranes by stainig with 1% amido black stain. On unstained nitrocellulose membranes, the unoccupied sites were blocked by blocking buffer (5% gelatin in phosphate buffered saline containing 0.01% Tween-20) (PBST) for 1hr at room temperature with agitation. Membranes were then washed three times for 10min in PBST to wash away the excess of blocking buffer and finally left to dry at room temperature, then cut into strips. The blotted-OMP-protein strips for each Salmonella serovar were incubated with 1:100 diluted sera from calves that were positive isolation of the corresponding serovar. The strips were then washed three times with PBST and incubated with rabbit anti-bovine IgG conjugated with the horse radish peroxidase for 1h at room temperature in dilution 1:2000 in PBS pH 7.4. The conjugate was then washed and the colour was developed by adding the substrate (30mg 4-chloro-1-naphtholdissolved, 10ml cold methanol, 30µl hydrogen peroxide in 50ml PBS pH7.4) (Zamora et al. 1999).
Elution of Immunogenic proteins
All protein bands that reacted specifically in the Western blot reaction were eluted from each of the four salmonella serovar OMP preparations using Bio-Rad protein elution unit to be used in ELISA as coating antigens. Protein content was measured according to Bradford (1976).
ELISA ProcedureThe ELISA was performed in polystyrene 96 well microtiter plates (Dyatech). A checkerboard titration was performed to determine the optimum antigen concentration and conjugate dilution. The plates were coated separately with 5ug/ml of each Salmonella eluted-OMP preparations in 100µl carbonate bicarbonate buffer pH9.6 and kept for 2hrs at 37°C followed by overnight incubation at 4°C for antigen adsorption. Antigens which did not bind to the polystyrene plates were removed by washing three times with PBS pH 7.2-Tween 20 (0.05%) (PBST). Sites on the plates which were not occupied by the coating antigens were blocked by adding 100µl of 0.1% gelatine solution (blocking buffer) and allowed to incubate for 1hr. at 37°C. The plates were washed three times with the previously mentioned washing buffer to wash away the excess of the blocking buffer. 100µl calf sera diluted 1:100 in PBST were then pipetted in the ELISA plates and incubated at 37°C for 1hr. Plates were then washed again three times with PBST. Then 100µl rabbit anti-bovine IgG conjugated to horse radish peroxidase (Kirkegaard and Perry Laboratories) was added in dilution 1:3000 (in PBS containing 0.05%Tween-20). The colour was developed by adding 3,3,5,5-tetramethyl-benzidine (TMB) as substrate and the blue developed colour was then converted to yellow by addition of 100µl of 1M H2SO4 and the plates were read at 450nm. The positive control sera was used from animals that were identified as salmonella positive infected, while the negative control sera were collected from animals that had no history of infection and were proven negative for salmonella isolation. The OD values of serum samples greater than 0.32 for S. typhimurium ELISA, greater than 0.3 for S. dublin or S. enteritidis ELISA and 0.27 for S. anatum were considered positive, which is the mean value of the negative sera plus two standard deviations (SD) (John et al.1993; Galland et al. 2000).
Episodes of diarrhoea in calves were reported in some governmental and private farms in Giza, Kafr El-Sheikh and Dakahlea governorates. Faecal samples collected from these calves as well as the contact apparently showed higher incidence of Salmonella 17.5% and 3.4% respectively. Four different Salmonella serovares were elucidated namely, Typhimurium 7.3% and 1.7%, S. dublin 5.1% and 0.8%, Enteritidis 2.9% and 0.8% and Anatum 2.2% and 0% from diarrhoic and apparently healthy calves respectively (Table 1 and Figure 1). The antigenic structure of all the 28 Salmonella isolates were confirmed by the Kauffman-White Scheem (slide agglutination test) using polyvalent and monovalent (O) and (H) antisera (Table 1).
The OMP electrophoretic analysia of the four Salmonella serovars was displayed in Figure 3 (a) The four serovars shared many protein bands as intense protein bands in the range from 20K and 45K was a general character of the four OMP preparations (common Salmonella antigen), while fainter bands were revealed at the higher molecular weight end (higher than 67K) aswell as at the lower molecular weight end (lower than 20K) (Figure3-a). In the Western blot analysis, serum antibodies from calves infected with S. typhimurium (serogroup B) reacted with protein bands at 17K, 24K, and 31K.
The OMP of S. dublin and S. enteritidis reacted relatively similar in the Western blot reaction. Two protein bands were demonstrated,14.4K and 24K. as both serovars belong to serogroup D1 and the greater similarity in their somatic antigen structure, O:1, O:9 and O:12.
Only one protein band (24K) was reacting in the Western blot reaction of S. anatum blotted OMP (serogroup E1) and reacted specifically with serum from infected calves.(Figure 3-b).
ELISA results detected the presence of Salmonella antibodies in the serum of diarrheic calves in 24.1% of the animals (33 out of 137), while the rate of isolation of salmonella from these diarrheic calves was 17.5% (24 out of 137). In apparently healthy calves ELISA detected the presence of Salmonella antibodies in their serum in 9.5% of the animals (11 out of 116), while the rate of isolation of salmonella from these apparently healthy calves was 3.4% (4 out of 116).(Table 1 and 2). There was some discrepancies in ELISA results with isolation results. Yet ELISA recorded higher incidence of salmonella infection with the four serovars , both in diarrhoic and apparently healthy calves.(Table 2 and Figure 2).
The OMP electrophoretic analysis of the four salmonella serovars (Typhimurium, Dublin Enteritidis and Anatum) (Figure 3-a) showed that the serovars shared many protein bands which constitute the common genus antigen. The intense protein region which occupied the the range from 20K and 45K constituted the majority of the Salmonella specific OMP bands. The fainter bands which were revealed at the higher molecular weight region (higher than 67K) and at the lower molecular weight region (lower than 20K) were bands related or associated to the OMP or residues of flagellar and pilus protein (lower than 20K) or could be residues of Salmonella toxines (higher than 67K) (Brusnigina 1988; Kim et al., 1991; Robin et al. 2000; Santos et al. 2002; Zhang et al. 2002). OMP preparation from S. dublin and S. enteritidis showed greater similarities in their electrophoretic patterns, as both serovars belongd to the sero-group D1 and share many factors in the somatic antigens, O:1, O:9 and O:12. Many other virulence factors were ex pressed by Salmonella which pathologically influence the host cells and specific genes encoding these factors were regulated by many environmental conditions and regulatory pathways which can ba altered by many bacterial, host and environmental factors.(Robin et al.2000 Santos et al. 2002 and Zhang et al. 2002).
Western blot analysis, serum antibodies from calves infected with
S. typhimurium (serogroup B) reacted with protein bands at molecular weight of 17K, 24K, and 31K. This range of protein band has been claimed as it is the major outer membrane protein of Salmonella enterica serovar Typhimurium (Collinson et al., 1993 and Zamora et al., 1999). The OMP of the two serovars Dublin and Enteritidis that belong to serogroup D1 reacted relatively similar with the antisera collected from calves that were infected with their respective serovars. Two protein bands were demonstrated, 14.4K and 24K.
Only one protein band (24K) from the blotted OMP of S. anatum (serogroup E1) reacted with serum from infected calves.(Figure 1-b). Unmarkable cross reactivity was noticed in Western blot analysis between S. typhimurium, S. dublin and S. enteritidis due to the variable sharing of their somatic antigenic structure. Other protein band could be elicited and released in the serum of infected calves, due to the severe inflammatory response and release of antibodies, chemokines, cytokines and interleukines (Bayar et al., 1982 and Zamora et al., 1999 and Santos et al.2002).
OMP-ELISA results detected the presence of salmonella antibodies in the serum of diarrheic calves in 24.1% of the animals (33 out of 137), while the rate of isolation of salmonella from these diarrheic calves was 17.5% (24 out of 137). In apparently healthy calves ELISA detected the presence of salmonella antibodies in their serum in 9.5% of the animals (11 out of 116), while the rate of isolation of salmonella from these apparently healthy calves was 3.4% (4 out of 116).(Table 1 and 2) This could be attributed to the intermittent shedding of Salmonella by animals without clinical signs could be due to chronic Salmonella cases, convalescence after acute infection or a recent colonization (Smith et al. 1989, Seleim 1999 Galland et al. 2000, Roy et al. 2001 and Radke et al. 2002 Veling et al. 2002). Unlike the intermittent shedding of Salmonella in the faeces, antibody levels in the serum which do not fluctuate on daily basis and sero-conversion was used in many countries as a tool to monitor the immune status of large klocks of cattle, sheep, pig and poultry industry at slaughter to identify infected flocks as a regulatory procedures for food safety and security program. Application of ELISA was a key tool to provide information about the infection status of the animal and the flock, moreover the repeated testing of animals differentiates those recently infected calves (increasing antibody titre) and those convalescent ones (decreasing titres) from salmonella carriers, which would have relatively constant titres (Smith et al. 1989; Nielsen et al. 1995 and Veling et al. 2002). A slight discrepancies were recorded in detecting Salmonella infected animals using OMP ELISA. In serum from diarrhoic calves, there was Three serum samples cross reacted, one calf showed reactively positive with S. typhimurium ELISA and S. dublin ELISA . There were also two cases that gave positive results with S.dublin ELISA and S. enteritidis ELISA. In the serum from apparently healthy calves only one serum sample cross reacted between S.dublin ELISA and S. enteritidis ELISA (table2).
In order to control and stop the losses incurred by Salmonella in calves formalin-killed Salmonella bacterins has been used commercially in the field in many areas of the world (Wray and Sojka 1984; Roden et al. 1992). Yet out of these result we could conclude that Salmonella OMP can play an essential role in the induction of immune response in the animals and can be employed as an effective candidate vaccine which can confer solid and active immunity and evade the hefty expenses of treatment of Salmmonella infected calves which lately has aquired multiple potent antibiotic resistence (Donkergoed et al. 1999, Galland et al. 2000 and Carlson et al.2002).
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