Abstract
Clinical salmonellosis associated with Salmonella is increasingly reported in finishing swine. Since S. Typhimurium is often associated with these episodes and given that this serotype is among the most often reported in humans, we were interested to determine if various tissues and carcasses from animals coming from herds that were clinically affected were more likely to be contaminated by Salmonella compared to carcasses from animals raised in herds without any history of salmonellosis. Carcasses from animals from affected herds were significantly more contaminated by Salmonella while showing increased titers in antibodies directed against this bacterium. At the opposite, caecal contents and mesenteric lymph nodes from both groups of animals were similarly contaminated by Salmonella. In the second part of the study, we studied the persistence of the bacterium in various tissues after an experimental infection with S. Typhimurium. We found that, after the infection, Salmonella persisted for as many as 7 d in many extraintestinal tissues, while it was present in the feces of infected animals for all 14 d of the experiment. These findings indicated that carcasses from animals that experienced salmonellosis during their growth phase are more likely to be contaminated by this bacterium and that precautions must be taken in order to ensure that clinically affected animals should be kept on the farm for at least 7 d before being shipped for slaughter.
Résumé
Les salmonelloses cliniques associées à Salmonella sont rapportées de plus en plus fréquemment chez les porcs en finition. Puisque S. Typhimurium est souvent relié à ces épisodes et étant donné que ce sérotype est parmi les sérotypes les plus rapportés chez l’humain, nous avons voulu déterminer si les carcasses et les différents tissus des animaux issus d’élevages affectés étaient plus susceptibles d’être contaminés par Salmonella en comparaison de ceux d’animaux provenant d’élevages non affectés par cette condition. Les carcasses provenant d’élevages affectés ont été significativement plus contaminées par la bactérie en plus de démontrer des titres d’anticorps dirigés contre Salmonella plus élevés. Par contre, les contenus caecaux et les nœuds lymphatiques mésentériques des deux groupes ont montré des taux de contamination semblables. Dans la deuxième portion de l’étude, nous avons étudié la persistance de S. Typhimurium dans différents tissus suite à une infection expérimentale. Nous avons trouvé que suite à l’infection, Salmonella a persisté dans plusieurs tissus extraintestinaux jusqu’à 7 jours post-infection alors que la bactérie a été retrouvée dans les fèces pendant les 14 jours de l’expérimentation. Ces résultats indiquent que les carcasses provenant des animaux qui ont manifesté des signes de salmonellose pendant leur croissance sont plus susceptibles d’être contaminées par cette bactérie et que des mesures devraient être prises afin de garder à la ferme les animaux affectés pour une période minimale de 7 jours lors de signes cliniques.
(Traduit par les auteurs)
Introduction
Salmonella enterica has emerged during the last decades as an important public health problem in most developed countries. The main source of infection is consumption of animal products (1). There are over 2500 different serotypes of Salmonella (2). Most serotypes are potential human pathogens even though few serotypes are regularly associated with disease (3). Human infection with multi-resistant S. Typhimurium DT104 has been associated with consumption of beef, chicken, unpasteurized dairy products, and to a lesser extent, with infected animal contacts (4–6). The most common symptoms in humans infected by S. Typhimurium DT104 include diarrhea (100%), fever (80%), abdominal pain (65%), vomiting (45%), and blood in the stool (27%) (7).
In pigs, clinical salmonellosis associated with S. Typhimurium DT104 is reported with increasing frequency (8). While most animals colonized by this bacterium will remain healthy carriers, clinical signs associated with salmonellosis in pigs are yellowish diarrhea with fever, prostration, and/or mortaliy. However, information regarding the distribution and the persistence of S. Typhimurium DT104 in tissues of the pig following infection is limited. Since the disease may occur at the end of the fattening period, from a public health point of view it is critical to better understand the survival of the bacteria in feces and organs following the infection.
Important aspects in the control of Salmonella in the finished product are the detection and preharvest management of affected herds (9). Since a significant proportion of animals from infected herds might be carriers of Salmonella without any clinical signs, it is not clear if the presence of clinical salmonellosis represents an additional threat in terms of food safety. The objectives of the study were 1) to compare at slaughter the bacteriological and serological prevalences of various Salmonella serotypes and phage types in different tissues and feces of animals from herds with and without clinical signs of salmonellosis and 2) to investigate the distribution and the persistence of a multiresistant S. Typhimurium DT104 in organs of experimentally infected piglets.
Material and methods
Collection of samples at slaughter
Sampling was conducted on finishing pigs in 1 mid-size slaughterhouse, federally inspected, with a capacity of slaughtering 240 pigs per hour in Quebec, between 1999 and 2000. Three criteria were used to select herds with clinical signs. The 1st was diagnosis, based on clinical signs, of salmonellosis by an experienced veterinarian. In addition, 1 or 2 of the following criteria was used to select positive herds. The 2nd criterium was isolation of Salmonella spp. from intestinal or internal organs of affected animals. Finally, the 3rd criterium was isolation of Salmonella spp. from feces collected in many pens containing affected animals without detection of other enteric pathogens. None of these criteria were present in selected herds without clinical signs. In addition to the absence of clinical signs of salmonellosis, herds without clinical signs were selected on the basis of a previous negative serological analysis.
On arrival at the abattoir, animals were kept in pens for a lairage period of 8 to 12 h prior to slaughter. All animals from herds that experienced clinical salmonellosis were kept in separate pens. The holding pens were washed and disinfected every day. The herd of origin was identified by the tattoo number on the animal. In total, 178 pigs from 25 herds without clinical signs and 264 pigs from 19 herds with clinical signs were sampled.
Enzyme-linked immunosorbent assay (ELISA)
Antibodies against Salmonella were evaluated by an indirect ELISA using 12 different antigens from the 4 principal serotypes of groups B, C, N, and E isolated in North America. Antigens were coated on microwell plates (Sarstedt, Montreal, Quebec). The washing buffer used in these tests was phosphate buffered saline solution (PBSS) (Fisher, Nepean, Ontario) containing 500 μL Tween 20 (Fisher). The test and control sera were diluted 1/25 and 1/100 in PBSS-Tween 20 and applied in duplicate for 30 min at 35°C, followed by 3 cycles of washing. Horseradish peroxidase labelled goat antiserum to pig immunoglobulin was diluted 1:1500 in PBSS-Tween 20 and 100 μL was added to each well. The conjugate was incubated for 1 h at 35°C and the wells were washed, as previously described. Finally, 100 μL of substrate was added (50 μL of a mix of 62.5 μL H2O2 3% with 937.5 μL deionized water, 12.5 mL citrate, 125 μL 2,2-azino-di[3-ethylbenzthiazoline] sulfonic acid [ABTS]). After incubation for 15 to 40 min, plates were read at optical density (OD) of 415 nm with positive-negative cut-off of OD% = 40. Bacteriological cultures were performed on 1 gram samples of liver, spleen, mesenteric lymph nodes (MLN), and caecum content collected aseptically (10). Sixteen to 24 h after slaughter, carcass swab samples were collected by the 3 sites sampling scheme, as previously described (11). One gram of diaphragm muscles were taken from these carcasses (10,12).
Bacteriological culture
Primary enrichment of caecal contents, MLN, lung, spleen, diaphragm, and carcasses samples was done in nutrient broth (NB) (1 g in 9 mL/18 to 24 h at 35°C) (Difco Laboratories, Detroit, Michigan, USA). One millilitre was transferred to 9 mL of tetrathionate brilliant green broth (TBG) (BBL Microbiology Systems, Cockeysville, Maryland, USA) and incubated at 42°C for 18 to 24 h. A loopful was inoculated on brilliant green sulfa agar (BGS) (Difco Laboratories) plates containing 20 μg/mL of novobiocin (Sigma Chemical, Oakville, Ontario), and incubated at 37°C for 18 to 24 h. Suspected colonies were striked to triple sugar iron (TSI) (Difco Laboratories) and Christensen’s urea agars (Difco Laboratories). Isolates presumptively identified as Salmonella spp. were further tested by agglutination against polyvalent O-antisera (Poly A-I & Vi; Difco Laboratories), and Salmonella isolates were serotyped at the Health Canada Laboratory in Guelph, under the supervision of Dr. Anne Muckle.
Statistical analysis
Linear logistical regressions were performed to evaluate the association between groups of animals from each category (clinically affected herd or not) as dependent variables and the various status of bacteriological culture (or serology) for each tissue sample, using the Wald test to calculate P-value of each association.
Animals
For experimental infection, 44 early weaned (8 d) crossbred piglets from 1 herd, without history of clinical salmonellosis, were used. They were housed in isolation facilities of the Canadian Food Inspection Agency at St-Hyacinthe, Quebec. They were given corn-based pelleted feed supplemented with milk products without any growth promoters or antibiotics products. Feed samples were cultured to detect Salmonella spp. by taking a composite sample of 100 g from many feed bags. Potable water was supplied ad libitum.
Bacteria
Salmonella Typhimurium DT104 # 4393 strain, originating from a clinical case of salmonellosis, was used for inoculation. This strain was resistant to chloramphenicol, sulfamethoxazole, trimethoprim, streptomycin, amoxicillin, clavulanic acid, tetracycline, ampicillin, spectinomycin, sulfisoxazole, and neomycin. It was found sensitive to enrofloxacin, gentamicin, and ceftiofur. The frozen stock culture was subcultured in NB overnight at 35°C. From this culture, 5% was inoculated into fresh broth and incubated, as described before, for 18 h (OD was 0.6 at 600 nm). The culture was centrifuged at 7650 × g for 20 min at 4°C. The supernatant was removed, and the pellet resuspended in a 1/10 volume of NB and kept on ice (13). The culture’s OD (2.5 at 600 nm) was measured to reach a final concentration of approximately 2.7 × 1010 colony-forming units per mL (cfu/mL). The bacterial concentration was determined by plating serial dilutions of initial inoculum onto blood agar (14,15).
Experimental procedures
Piglets were divided into 2 groups. The 1st group (n = 41) was exposed and the 2nd group (n = 3) served as the uninoculated control. They were kept in isolated rooms, between 10 to 11 piglets per room for exposed animals, with solid concrete floors. Unexposed controls were kept in a separate room. The piglets were monitored to detect Salmonella spp. on pre-exposure days 34, 27, 20, 6, and 0 (day of exposure) by culturing rectal and tonsil swabs and feces. Rectal temperatures were taken pre-exposure days 27, 20, 18, and 15. At 6 wk of age animals were infected by squirting an inoculum of approximately 2.7 × 1010 cfu/mL directly down the throat. They were challenged orally with a single dose of 5 mL of undiluted culture. Control animals received 5 mL of sterile culture medium. Feces were allowed to accumulate for 3 d after exposure. Floors were washed daily except for the first 3 d post exposure. After challenge, rectal temperatures and rectal swabs were taken on days 1, 2, 3, 5, 7, 10, and 14. Clinical signs, including diarrhea, were monitored daily following challenge. Piglets were necropsied at random on post exposure hours 12 and days 1, 2, 3, 5, 7, 10, and 14. One control was necropsied on post exposure days 5, 7, and 10. Rectal swabs and samples from selected body organs were collected at necropsy for bacteriology. Material from tonsillar surfaces was obtained by vigorously rubbing the surfaces with a sterile swab. Rectal specimens were obtained by insertion of a sterile swab 8 to 10 cm.
Necropsy procedures
All animals were euthanatized by intravenous injection (T-61; Hoechst Canada, Regina, Saskatchewan). Skin was washed by vigorously scrubbing the surfaces with a hibitane solution. Body cavities were carefully opened to prevent contamination of internal organs by intestinal content. Samples were taken aseptically, using sterile instruments, and bags. Samples of 2 to 5 g from the quadriceps muscle, superior inguinal lymph nodes, liver, spleen, lung, diaphragm, kidney, palatine tonsil, cervical lymph nodes, MLN, ileum, Peyer’s Patches, caecum, and colon, and rectal swabs were collected for qualitative bacteriology. Tissues from the gut area were removed last to avoid cross-contamination between areas. Tissue samples were collected twice for bacteriologic analysis and polymerase chain reaction (PCR) assay. The PCR samples were stored at –20°C and processed later. Bacteriology was done as described previously (bacteriological culture).
Selective enrichment of tissues for PCR procedures
The MLN and ileum from pigs experimentally infected with strain 4393 were investigated. Selective enrichment was done, as previously described. Briefly, 1 gram of each tissue was aseptically cut with a scalpel and incubated with 9 mL of NB for 18 to 24 h at 35°C. Then, 1 mL was inoculated in 9 mL of TBG and incubated for 18 to 24 h at 42°C, followed by plating on BGS and XLTD4 (Difco Laboratories) and incubated at 37°C for 18 to 24 h. The TBG were kept at room temperature for 5 d for a delayed plating on BGS and XLTD4. Up to 5 suspect Salmonella colonies on each BGS and XLTD4 were inoculated on TSI and urea and incubated at 37°C for 18 to 24 h.
DNA isolation
Cell lysates for each isolate were prepared by suspending a loopful of bacteria grown on TSI in 1 mL of sterile distilled water. Cells were centrifuged (5000 × g for 5 min) and the pellets were washed 1 more time in distilled water. The pellets were resuspended in lysis buffer containing 200 μL of chelex 5% (Biorad, Mississauga, Ontario) and 20 μL of proteinase K 10 mg/mL (Sigma). This solution was vortexed for 10 to 15 s and incubated for 1 h at 56°C. All samples were then vortexed for 10 s and heated for 8 min at 98°C. Lysates were vortexed for 10 s and chilled for 5 min on ice. Bacterial cell debris were removed by centrifuging at 6000 × g for 3 min. Supernatants were kept at –20°C until use.
Polymerase chain reaction and electrophoresis
Three microliters of the template DNA were added to a mixture (22.8 μL) containing 15 μL Dnase free water (Invitrogen, Burlington, Ontario), 2.5 μL 10 × PCR-reaction-buffer, 0.75 μL MgCl2 (50 mM) (Invitrogen), 0.3 μL deoxyribonucleotide triphosphates (dNTP) (200 μM) (Roche, Laval, Quebec), 0.5 μL of each primer (10 pmol/μL) (Invitrogen), and 0.25 μL Taq DNA polymerase (5 U/μL) (Invitrogen). Primers of oligonucleotide sequences to detect genes coding for florfenicol resistance (flost), integron (int), invasion (inv), and virulence (spvC) were used as previously described (16). The amplification was performed in a UnoII DNA thermocycler (Biometra, Göttingen, Germany). The conditions for the PCR were an initial denaturation at 94°C for 90 s followed by 30 cycles each of 45 s denaturation at 94°C, 45 s annealing at 60°C, and 90 s extension at 72°C, and a final extension step of 3 min at 72°C. The PCR products were cooled to 4°C (16). Ten microliters of each PCR product were electrophoresed on a 1.5% agarose gel for 45 min (Sigma) at a constant voltage of 100 V in TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM ethylene diamine tetra acetate [EDTA]). Amplicons were stained with ethidium bromide (1 μg/mL) for 30 min, destained 10 min in distilled water, visualized on a ultra violet (UV) light induced fluorescence (Bio/Can Scientific, Mississauga, Ontario), and photographed on a polaroid film type 667 (Fisher). As negative control, all components of the reaction mixture except template DNA were used. The experimental strain # 4393 was used as positive control. Culture from TSI tubes with positive result were transferred on blood agar plate and incubated at 35°C for 18 to 24 h. Isolates were stored at –20°C in glycerol.
Results
Salmonella was isolated from at least 1 tissue sample in 226 out of the 442 sampled animals. Salmonella was recovered in significantly higher rates in carcasses from animals belonging to herds that showed clinical signs of salmonellosis compared to unaffected herds (Table I). Percentages of animals that were positive by serology were also significantly higher in animals from clinically affected herds. No statistically significant difference was observed in the percentages of positive caecal content or culture of mesenteric lymph nodes between both groups of animals. Overall, the percentage of extraintestinal tissues colonized by the bacteria was the same for animals from affected herds and animals from regular herds. However, no diaphragm was found to be positive to Salmonella. Furthermore, the serotypes observed in both groups of animals were similar with the exception of S. Anatum that was observed more frequently in tissues of animals from affected herds (Tables II and III).
Table I.
Serological and bacterial culture of Salmonella spp. in selected tissues and carcasses from herds with or without a history of clinical salmonellosis
| Clinical signs | No clinical signs | |||
|---|---|---|---|---|
| Samples | Prevalence (%) | Number of herds | Prevalence (%) | Number of herds |
| Serum samples | 34.81a ± 9.55b | 23 | 14.84a ± 9.0 | 27 |
| Caecal contents | 53.11 ± 11.05 | 24 | 50.13 ± 14.8 | 19 |
| Mesenteric lymph nodes | 15.02 ± 6.84 | 24 | 16.45 ± 10.84 | 19 |
| Carcass swabs | 9.21c ± 5.96 | 25 | 3.77c ± 3.62 | 25 |
| Liver | 5.11 ± 13.65 | 10 | 11.04 ± 17.73 | 12 |
| Spleen | 4.00 ± 12.15 | 10 | 2.71 ± 9.18 | 12 |
Statistically significant (P < 0.01) difference
95% confidence interval
Table II.
Distribution at slaughter of serotypes and phage types isolated from different tissues of pigs from herds without a history of salmonellosis
| Liver | Spleen | MLN | Feces | Diaphragm | Carcass | Total | |
|---|---|---|---|---|---|---|---|
| S. Anatum | — | — | — | 3 | — | — | 3 |
| S. Brandenburg | — | — | — | — | — | 1 | 1 |
| S. Derby | 1 | 2 | 6 | 20 | — | 2 | 31 |
| S. Heidelberg DT9 | 1 | 1 | — | — | — | — | 2 |
| S. Heidelberg DT25 | — | — | — | 1 | — | — | 1 |
| S. Heidelberg DT29 | — | — | 1 | — | — | — | 1 |
| S. Heidelberg DT32 | — | — | — | 2 | — | — | 2 |
| S. Heidelberg DT39 | 1 | — | — | — | — | — | 1 |
| S. Heidelberg DT47 | 1 | — | — | — | — | — | 1 |
| S. Ohio | — | — | — | 2 | — | — | 2 |
| S. Ohio var. 14 + | — | — | — | 5 | — | — | 5 |
| S. Senftenberg | — | — | — | 5 | — | — | 5 |
| S. Typhimurium DT12 | 3 | — | 1 | — | — | 1 | 5 |
| S. Typhimurium DT104 | — | — | 1 | — | — | — | 1 |
| S. Typhim. var. Copenhagen DT104 | — | — | — | 5 | — | — | 5 |
| S. Typhimurium DT104a | 1 | — | — | 1 | — | — | 2 |
| S. Typhim. var. Copenhagen DT104a | — | — | — | 2 | — | — | 2 |
| S. Typhim. var. Copenhagen DT104b | — | — | — | 1 | — | — | 1 |
| S. Typhimurium DT193 | — | — | 3 | 2 | — | — | 5 |
| S. Typhimurium DT208 | — | — | — | 1 | — | — | 1 |
| I rough-O:b:l:w | — | — | — | 2 | — | — | 2 |
| I;4,12:-:- | — | — | — | 1 | — | — | 1 |
| Total | 8 | 3 | 12 | 53 | 0 | 4 | 80 |
MLN — mesenteric lymph nodes; DT — definitive type
Table III.
Distribution at slaughter of serotypes and phage types isolated from different tissues of pigs from herds with a history of salmonellosis
| Liver | Spleen | MLN | Feces | Diaphragm | Carcass | Total | |
|---|---|---|---|---|---|---|---|
| S. Anatum | — | — | 6 | 20 | — | 1 | 27 |
| S. Brandenburg | — | — | — | 6 | — | 6 | 12 |
| S. Derby | 2 | — | 9 | 25 | — | 1 | 37 |
| S. Heidelberg DT20 | — | — | 7 | 8 | — | — | 15 |
| S. Infantis | — | — | — | 5 | — | 1 | 6 |
| S. Ohio | 2 | 2 | 3 | 10 | — | — | 17 |
| S. Ohio var. 14 + | — | 1 | 4 | 16 | — | 3 | 24 |
| S. Senftenberg | — | — | — | 14 | — | 3 | 17 |
| S. Senftenberg (1) | — | — | — | 3 | — | — | 3 |
| S. Typhimurium DT12 | — | — | — | 1 | — | — | 1 |
| S. Typhim. var. Copenhagen DT12 | — | — | 1 | — | — | — | 1 |
| S. Typhimurium DT36 | — | — | 2 | — | — | — | 2 |
| S. Typhimurium DT104 | — | — | 1 | — | — | — | 1 |
| S. Typhim. var. Copenhagen DT104 | — | — | 1 | 2 | — | — | 3 |
| S. Typhim. var. Copenhagen DT104a | — | — | — | 2 | — | — | 2 |
| S. Typhimurium DT186 | — | — | — | 1 | — | — | 1 |
| S. Typhimurium DT193 | — | — | 1 | 7 | — | — | 8 |
| S. Typhim. var. Copenhagen DT193 | — | — | 1 | — | — | — | 1 |
| S. Typhim. var. Copenhagen DT194 | — | — | — | — | — | 1 | 1 |
| S. Typhimurium DT208 | — | — | — | 1 | — | — | 1 |
| S. Typhim. var. Copenhagen DT721 | — | — | — | 1 | — | — | 1 |
| S. Typhim. var. Copenhagen U302 | — | — | 1 | 4 | — | 1 | 6 |
| S. Typhimurium untypable | 1 | 1 | 1 | 1 | — | 5 | 9 |
| S. Typhim. var. Copenhagen untypable | — | — | — | 1 | — | — | 1 |
| I:4,12:i:-DT104a | — | — | — | 2 | — | — | 2 |
| I:4,5,12:r:- | — | — | — | 1 | — | — | 1 |
| I:rough-O:fg:- | — | — | — | 1 | — | — | 1 |
| I:rough-O:i:1,2 | — | — | 1 | 1 | — | — | 2 |
| I:rough-O:b:l,w | — | — | — | 1 | — | — | 1 |
| I:rough-O:-:l,w | — | — | 1 | — | — | — | 1 |
| S. unknown | — | — | — | 2 | — | 1 | 3 |
| Total | 5 | 4 | 40 | 136 | 0 | 23 | 208 |
MLN — mesenteric lymph nodes; DT — definitive type
Since we observed S. Typhimurium in a significant proportion of extraintestinal tissues in both groups of animals, we were then interested in the 2nd part of the study to evaluate the persistence in various tissues of bacteria following an experimental infection. Thus, we inoculated groups of animals using a S. Typhimurium DT104 strain in order to reproduce a mild infection clinically characterized by a yellowish diarrhea and fever like those mostly observed in the field. Following the inoculation, we were able to isolate the bacteria for up to 7 d after in almost all samples from extraintestinal tissues (Table IV). This persistence in extraintestinal tissues was accompanied by a yellowish diarrhea and an elevation of body temperature. Bacteria were recovered from the intestinal tissues, MLN, and palatine tonsils for the entire experimentation.
Table IV.
Distribution of Salmonella Typhimurium DT104 # 4393 in tissues of experimentally infected piglets following oral inoculation
| Post exposure days | ||||||||
|---|---|---|---|---|---|---|---|---|
| Samples | + 0.5 d | + 1 d | + 2 d | + 3 d | + 5 d | + 7 d | + 10 d | + 14 d |
| Liver | 4/5 | 5/5 | 5/5 | 5/5 | 5/5 | 2/5 | 0/6 | 0/5 |
| Spleen | 5/5 | 4/5 | 4/5 | 5/5 | 5/5 | 2/5 | 1/6 | 0/5 |
| Lung | 3/5 | 5/5 | 5/5 | 5/5 | 4/5 | 3/5 | 1/6 | 0/5 |
| Kidney | 5/5 | 5/5 | 3/5 | 5/5 | 3/5 | 4/5 | 0/6 | 0/5 |
| Diaphragma | 4/5 | 5/5 | 5/5 | 5/5 | 3/5 | 1/5 | 0/6 | 0/5 |
| Cervical LN | 4/5 | 5/5 | 3/5 | 5/5 | 5/5 | 4/5 | 0/6 | 0/5 |
| Tonsils | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 6/6 | 5/5 |
| Inguinal LN | 3/5 | 4/5 | 4/5 | 5/5 | 5/5 | 3/5 | 1/6 | 0/5 |
| Quadriceps | 2/5 | 5/5 | 4/5 | 5/5 | 3/5 | 3/5 | 0/6 | 0/5 |
| MLN | 5/5 | 5/5 | 4/4 | 5/5 | 5/5 | 5/5 | 6/6 | 5/5 |
| Ileum | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 6/6 | 5/5 |
| P. Patches | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 6/6 | 5/5 |
| Caecum | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 6/6 | 5/5 |
| Colon | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 6/6 | 5/5 |
| Feces | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 6/6 | 5/5 |
| Temperature (°C) ± sχ̄ | 39.8 ± 0.3 | 40.4 ± 0.1 | 40.6 ± 0.1 | 40.1 ± 0.1 | 40.0 ± 0.1 | 38.0 ± 0.4 | 39.9 ± 0.3 | 39.4 ± 0.1 |
| Clinical signs at necropsy | Pleurisis, ileitis, peritonitis | No data | Ileitis, ileocolitis, diarrhea | Ileitis, ileocolitis | Mild ileitis, ileocolitis | Pneumonia, ileitis, ileocolitis | Enlarged MLN, Enteritis | Mild leitis |
Diaphragms were collected with a piece of muscle
Cervical LN — Cervical lymph nodes; Inguinal LN — Inguinal lymph nodes; MLN — Mesenteric lymph nodes; P. Patches — Peyer’s Patches;
Temperature (°C) ± sχ̄ — mean rectal temperature ± standard error of the mean for all piglets infected at this period
In order to confirm the presence of the multiresistant strains in tissues of inoculated animals, we adapted a multiplex PCR assay (16) to detect the multiresistant experimental strain in selected tissues. Using this technique we were able to detect positive tissues in 67 tissues out of 82 where the bacteriological culture was positive (Table V). The multiresistant strain was not found in negative-control piglets.
Table V.
Bacteriology and multiplex polymerase chain reaction (PCR) on selected tissues following experimental infection by Salmonella Typhimurium DT104 in pigs
| Test | |||
|---|---|---|---|
| Days post exposure | Tissues | Bacteriology | PCR |
| + 0.5 d | MLN | 5/5 | 1/5 |
| Ileum | 5/5 | 3/5 | |
| + 1 d | MLN | 5/5 | 4/5 |
| Ileum | 5/5 | 5/5 | |
| + 2 d | MLN | 4/4 | 4/5 |
| Ileum | 5/5 | 5/5 | |
| + 3 d | MLN | 5/5 | 5/5 |
| Ileum | 5/5 | 5/5 | |
| + 5 d | MLN | 5/5 | 5/5 |
| Ileum | 5/5 | 5/5 | |
| + 7 d | MLN | 5/5 | 5/5 |
| Ileum | 5/5 | 4/5 | |
| + 10 d | MLN | 6/6 | 5/6 |
| Ileum | 6/6 | 2/6 | |
| + 14 d | MLN | 5/5 | 5/5 |
| Ileum | 5/5 | 4/5 | |
MLN — mesenteric lymph nodes
Discussion
In order to achieve a better control of Salmonella in pork products, many countries have put in place control programs that include measures to be taken at the herd level. In the past few years in Canada, as well as in many other countries, an increase in the number of clinical cases of salmonellosis associated with S. Typhimurium was noted in animals and humans (8,17,18). Infections with this serotype are often associated with septicemia in animals. Since this serotype was regularly found in meat products and ultimately in humans, we were interested in this study to determine if the occurrence of clinical salmonellosis in swine would have an impact on the prevalence of the bacteria in tissues and carcasses.
The overall prevalence of Salmonella observed in caecal contents samples is much higher than those recently observed in other studies (19–21), including one study performed in part in this abattoir. During this study, arrangements were taken to ensure that herds that had clinical salmonellosis were processed in the slaughterhouse that agreed to participate. Although precautions were taken to put animals from these herds in particular pens that were washed and disinfected after each lot was slaughtered, the animals were otherwise processed as usual. Since many opportunities for cross contamination existed for animals from affected herds to contaminate animals from clinically healthy herds (transportation, unloading devices, and corridors), the findings that fecal material from both groups of animals were similarly contaminated by Salmonella suggest that such cross contamination occurred and resulted in much higher contamination rates than usual in apparently healthy animals. Since it is hard to control the environment during the slaughter period, it would be advisable in current field operations to ensure that animals from highly seropositive herds, contaminated herds, or both be introduced into the plant after regular lots, and slaughtered at the end of the day to minimize cross-contamination. In further experiments it might be advisable to sample different types of lots on different days of operation. Furthermore, regular sampling performed at this packing plant before and after the completion of this study revealed individual carcass contamination rates comparable and even below those of other packing plants (data not shown), indicating that the slaughter of highly contaminated herds in this study might have resulted in higher contamination rates in nonaffected herds. Nevertheless, despite the higher carriage rates observed in animals entering the packing plant, it was possible, after the slaughter process, to obtain an overall final carcasses contamination rate (7.8%) comparable to those obtained in some recent studies in North America (11).
Despite the fact that feces and MLN from both groups of animals were equally positive for Salmonella, we observed that carcasses from animals from clinically affected herds had significantly higher positive isolation rates of Salmonella than carcasses from nonaffected herds, indicating that carcasses from these animals should be considered a higher risk when developing Salmonella control programs. In addition, as expected, higher seropositivity rates were also observed in animals from affected lots. The fact that MLN from nonaffected herds were equally contaminated suggests a very recent infection of naïve animals from these herds during transportation or the lairage period before the slaughter process, since it generally takes 6 to 8 h before colonization of MLN after the initial infection. On the other hand, the results obtained suggest that the bacterial load in feces or on the skin might have been higher in animals from affected herds compared to those from nonaffected herds. It was previously reported that animals infected with higher doses of Salmonella may shed substantial numbers of bacteria for longer periods of time (22), while it is unlikely that such levels of contamination occur in feces from recently infected animals.
Various serotypes were recovered in both groups of animals. Salmonella Anatum was found in much higher percentages than reported in another study (23). However, since this study was not designed to be representative of the current field situation in Quebec, but to follow clinically affected herds, no conclusion can be drawn concerning a possible higher prevalence of this serotype in the field. Given the fact that clinical infections associated with S. Cholerasuis are very rare in Canada, and since S. Typhimurium is the only other serotype that was linked in the past to septicemia in swine, it was rather surprising to observe that the presence of S. Typhimurium was not significantly associated with the presence of clinical signs in animals from affected herds. Nevertheless, S. Typhimurium was also regularly recovered from nonaffected animals. A total of 17 S. Typhimurium DT104 isolates, a S. Typhimurium phage type that is of highest concern for public health, were cultured in this study, while 13 of these isolates were found in caecal content.
In the 2nd part of this study, after an experimental infection, S. Typhimurium was found in extraintestinal tissues for as many as 7 d post exposure and in feces throughout the experiment. Since the persistence in tissues was associated with fever, it would be advisable to avoid the expedition at slaughter of febrile animals when clinical salmonellosis is observed in a herd at the end of the fattening period. Alternatively, animals from affected pens should be kept on farm for at least 7 d at the end of the clinical episode. In this study, the PCR assay was positive for most tissues found positive at bacteriological culture, despite the fact that PCR was done using frozen tissues. The use of this multiplex PCR allowed for the simultaneous detection of the bacterium at the genus level as well as the identification of multiresistant strains in the feces and some tissues. This feature might be of interest in developing control programs focused on multiresistant Salmonella strains. However, the fact that few samples were found positive at the bacteriological culture without being found positive at PCR might indicate that these animals might have been infected by other strains before the infection despite the serial negative sampling that was performed prior to the infection.
Overall, the results obtained in this study indicate that hog carcasses from herds affected by clinical salmonellosis are more likely to be contaminated by Salmonella and that following the infection, the bacterium persists in tissues for at least 1 wk, indicating that special precautions should be taken during the slaughter of these animals.
Acknowledgments
This work was supported by the Conseil de recherches en sciences naturelles et en génie du Canada (CRSNG) and la Fédération des producteurs de porcs du Québec. The authors thank Julie Légaré and Jannick Beaurivage for technical assistance.
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