Peroxidase-antiperoxidase and immunogold labeling of Salmonella typhimurium and Salmonella choleraesuis var kunzendorf in tissues of experimentally infected swine

Peroxidase-antiperoxidase immunoenzymatic labeling and immunogold labeling techniques were evaluated for microscopic detection and location of Salmonella organisms in tissues of experimentally infected swine. Salmonella typhimurium and Salmonella choleraesuis var kunzendorf were labeled specifically by the peroxidase-antiperoxidase technique in paraffin-embedded tissues of infected swine for conventional light microscopy and by postembedding immunogold labeling on ultrathin sections for electron microscopy. Salmonella typhimurium had a low tendency to invade the enteric mucosa and did not reveal any predilection for a specific intestinal location. Salmonella choleraesuis var kunzendorf, however, was located preferentially in colon and on the luminal surface of ileal M cells of Peyer patches and had a tendency to invade the enteric mucosa there.     

Safety and efficacy of an attenuated strain of Salmonella choleraesuis for vaccination of swine.

The purpose of this study was to determine the safety and efficacy of a live Salmonella choleraesuis immunizing strain, obtained by repeated ingestion and recovery through porcine neutrophils. The strain was tested in mice and in pigs. The vaccine was safe and effective in controlled experimental trials, using clinical, pathologic, and microbiologic criteria. Vaccinated pigs were able to maintain normal weight gains during the 4-week observation period following challenge inoculation with a high dose of a virulent strain.     

Synergism between porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis in swine

Porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis are two leading causes of economic loss in the swine industry. While respiratory disease is common in both S. choleraesuis and PRRSV infections, the factors that contribute to its development remain largely undefined. We investigated the interaction of PRRSV, S. choleraesuis, and stress in 5-week-old swine. All combinations of three factors (inoculation with S. choleraesuis on Day 0, PRRSV on Day 3, and treatment with dexamethasone on Days 3-7) were used to produce eight treatment groups in two independent trials. Fecal samples, tonsil and nasal swabs, serum samples and postmortem tissues were collected for bacteriologic and virologic examinations. No clinical signs were observed in pigs inoculated with only PRRSV or only S. choleraesuis. In contrast, pigs which were dually infected with S. choleraesuis and PRRSV exhibited unthriftiness, rough hair coats, dyspnea, and diarrhea. The pigs which received all three treatment factors were the most severely affected and 43% (three of seven) of the animals in this group died. Individuals in this group shed significantly higher quantities of S. choleraesuis in feces and had significantly higher serum PRRSV titers compared to other treatments (p < or = 0.05). In addition, S. choleraesuis and PRRSV were shed longer and by more pigs in this group than other groups and S. choleraesuis was recovered from more tissues in this group on Day 21 post inoculation. These results suggested that PRRSV, S. choleraesuis, and dexamethasone acted synergistically to produce a syndrome similar to that observed in the field.     

猪霍乱沙门氏菌的快速分离鉴定

猪霍乱沙门氏菌是引起仔猪副伤寒的主要病原菌,给养猪业造成重大危害。本文旨在建立一套细菌快速分离鉴定方法,为该病流行病学调查提供有效手段。从临床初诊为仔猪副伤寒的病猪采集粪便,接种亚硒酸盐亮绿增菌液增菌后,划线于SS琼脂培养基,挑无色透明菌落纯化。纯化菌落用美国B IOLOG自动细菌鉴定仪GN2肠杆菌鉴定板鉴定,同时用常规生化鉴定管鉴定,结果符合猪霍乱沙门氏菌的生化特征。PCR扩增invA基因,测序鉴定PCR产物,结果与猪霍乱沙门氏菌参考序列同源性达99%以上。分离菌株接种小白鼠,证明其对小白鼠有致病性。用沙门氏菌属标准血清检测证实其抗原式为6,7∶c∶1,5,符合猪霍乱沙门氏菌的抗原特征。应用以上方法从全国各地病料中分离鉴定了40多株猪霍乱沙门氏菌,为猪霍乱沙门氏菌病的流行病学调查提供了依据,同时证明该方法切实可行。     

Assessment of the long-term shedding pattern of Salmonella serovar choleraesuis following experimental infection of neonatal piglets.

In the United States, swine salmonellosis is most often attributed to infections by Salmonella serovar choleraesuis. As a host-adapted pathogen rarely found in nonswine sources, S. choleraesuis is thought to be spread primarily via horizontal transmission, with carrier animals playing an important role. Little has been reported regarding infection of neonatal piglets, particularly regarding their potential to become carriers. Evidence reported herein demonstrates that piglets experimentally infected by S. choleraesuis at 2 days of age were capable of shedding the pathogen for up to 85 days postinfection, at which time the study was concluded. This study also presents findings supporting the use of GN-Hajna as a preenrichment medium for the isolation of S. choleraesuis.     

Pneumonia associated with Salmonella choleraesuis infection in swine: 99 cases (1987-1990).

Salmonella choleraesuis was isolated in pure or mixed bacterial cultures from 153 swine necropsied between Jan 1, 1987 and Dec 31, 1990. Pneumonia was seen in 99 of 109 swine from which this bacterium was isolated in the absence of other pathogenic bacteria. Pneumonia was seen more frequently than hepatitis, splenomegaly, or colitis. Pleuropneumonia that was grossly indistinguishable from the pleuropneumonia associated with Actinobacillus pleuropneumoniae was seen in 29 of 99 swine from which S choleraesuis was the only bacterium isolated.     

Antimicrobial susceptibility of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis recovered from Taiwanese swine.

Minimum inhibition concentrations (MICs) were determined for ampicillin, ceftiofur, cephalothin, chloramphenicol, enrofloxacin, gentamicin, lincomycin, lincospectin (lincomycin/spectinomycin), neomycin, premafloxacin, spectinomycin, sulfamethoxazole/trimethoprim, and tetracycline against a total of 180 isolates of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis (60 each) clinically isolated from pigs on farms in Taiwan from 1994 to 1996. No more than 3 isolates per farm were used. Ceftiofur had the highest activity in vitro against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, with MIC90 values of 0.03, 2, and 1 microg/ml, respectively. Premafloxacin was highly active against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, with MIC90 values of 2, 8, and 0.5 microg/ml, respectively, which were lower than those with enrofloxacin (MIC90 8, 32, and 2 microg/ml, respectively). Neomycin was moderately active against A. pleuropneumoniae and E. coli, with MIC90 values of 8 and 64 microg/ml, respectively, but was inactive with S. choleraesuis. Gentamicin showed high activity against A. pleuropneumoniae (MIC90 of 2 microg/ml) but was only moderately active with E. coli and S. choleraesuis (MIC90 of 64 and 32 microg/ml). Cephalothin was highly active against isolates of A. pleuropneumoniae (MIC90 of 1 microg/ml) but was inactive with E. coli (MIC90 of 128 microg/ml). Lincomycin had moderate activity (MIC90 of 32 microg/ml) against A. pleuropneumoniae. Chloramphenicol, lincomycin, and tetracycline were inactive with E. coli and S. choleraesuis (MIC90 > 128 microg/ml). In conclusion, ceftiofur and premafloxacin were highly active against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, enrofloxacin and gentamicin were highly to moderately active; cephalothin was highly active against A. pleuropneumoniae and moderately active against S. cholearesuis; chloramphenicol, lincomycin, and tetracycline were active only with A. pleuropneumoniae; neomycin was moderately active against A. pleuropneumoniae and E. coli. The other antimicrobials tested were inactive.     

Long-term survival and infectivity of Salmonella choleraesuis

It is believed that Salmonella Choleraesuis, the host-adapted serotype of swine, does not survive well outside the host. We examined the survival capability as well as the presence of latent DNA of S. Choleraesuis in swine feces. Pigs were infected with S. Choleraesuis and feces was collected and pooled on days 2, 4, 7, and 10 post inoculation (PI). Feces was stored in a wet and a dry form and survival was measured over 13 months. Salmonella Choleraesuis was recovered from wet feces through 3 months of storage. In a desiccated (dry) form, S. Choleraesuis was recovered from at least 13 months. Direct PCR analysis did not detect S. Choleraesuis subsequent to the final culture recovery for any stored sample. We also examined the infectivity of S. Choleraesuis resident in dry feces. Six or 13 week old pigs were inoculated with dry feces that had been stored either 2 months or 4 months, respectively. Pigs were inoculated either intranasally or by mixing dry feces with the swine ration. Although clinical signs were mild, S. Choleraesuis was widely disseminated among the tissues of all the pigs inoculated. This study demonstrates that S. Choleraesuis remains viable and infective in the environment. Therefore, contaminated fecal matter can serve as a reservoir for S. Choleraesuis as well as other Salmonella spp. Control measures must consider this environmental reservoir as a source of new infections.     

Evaluation of stocking density and subtherapeutic chlortetracycline on Salmonella enterica subsp. enterica shedding in growing swine

The objective of this research was to determine the effect of stocking density and inclusion of subtherapeutic chlortetracycline in the diet on Salmonella fecal prevalence and antimicrobial resistance in growing swine. A 2 x 2 factorial design was employed on a privately owned commercial swine farm. Four finisher rooms were included in the study. Two of the rooms received 50 g/tonnes of chlortetracycline in the ration, two rooms received no antimicrobials in the feed. In each room, alternate pens were assigned to either high stocking density (0.60 m2/pig) or low stocking density (0.74 m2/pig). Pigs were placed in the finisher rooms at 10 weeks of age and followed for 6 weeks. Individual fecal samples were collected from the floors of each pen and cultured once weekly. Antimicrobial resistance phenotypes were determined. Data were analyzed using multilevel, multivariable logistic regression. Pigs fed chlortetracycline were at increased odds (OR 6.88, 95% CI 2.77-17.12) to shed Salmonellae. No other associations between treatments (CTC and stocking density) and Salmonella prevalence or reduced susceptibility to antimicrobials were identified. Variance in the odds of a fecal sample to be positive was distributed mostly at the lowest level, the individual fecal sample. The increased risk of shedding associated with inclusion of subtherapeutic chlortetracycline in swine diets is discordant with previous results by our group, suggesting farm or strain specific factors may impact this association. Understanding this risk may provide a potential intervention for controlling Salmonella pre-harvest.     

Studies on the pathogenesis of Salmonella typhimurium and Salmonella choleraesuis var kunzendorf infection in weanling pigs

Twenty-six 4-week-old pigs were randomly allotted to 4 groups: group 1--orally inoculated with Salmonella typhimurium; group 2--orally dosed with S choleraesuis; and groups 3 and 4, with surgically constructed intestinal loops--loops inoculated with either S typhimurium or S choleraesuis. One pig each from groups 1 and 2 was killed at 8, 12, 24, 48, 72, 96, and 120 hours after inoculation. One pig each from groups 3 and 4 was killed at 2, 4, 6, 8, 12, and 24 hours after intestinal loop inoculation. Inoculation of S typhimurium resulted in acute enterocolitis of variable severity, whereas inoculation of S choleraesuis resulted initially in septicemia followed by formation of large necrotic and ulcerative lesions in the colonic mucosa. The most consistent systemic lesion of S choleraesuis infection was interstitial pneumonia and multifocal hepatic necrosis. Salmonella typhimurium and S choleraesuis were ultrastructurally within enterocytes of ligated ileal loops. Intracellular bacteria were morphologically intact, occurred free in the cytoplasm and membrane bound, and caused no detectable cytotoxic effect to the cell. Both S typhimurium and S choleraesuis penetrated the intestinal mucosa and were isolated from mesenteric lymph nodes at 2 hours after inoculation.     

Effect of efrotomycin in feed on the quantity, duration, and prevalence of shedding and antibacterial susceptibility of Salmonella typhimurium in experimentally infected swine

The influence of efrotomycin administered at the rate of 16 mg/kg of feed in 10 Salmonella typhimurium-inoculated pigs was determined by comparing this group with a group of 10 pigs inoculated with S typhimurium that were given nonmedicated feed. Two control groups of 4 noninoculated pigs each, 1 group medicated with efrotomycin at 16 mg/kg of feed, the other nonmedicated, also were evaluated. An inoculum of 1.7 x 10(10) colony-forming-units/pig induced colonization of S typhimurium in all 20 pigs. Evaluation of the quantity of shedding did not reveal a clear or consistent treatment-related increase in S typhimurium counts; mean differences between the nonmedicated and medicated groups never exceeded 1 log unit. On the last day of the study (day 56 of the medication), 8 nonmedicated and 9 medicated pigs were determined to be infected with S typhimurium via enrichment procedures, so there was no difference in duration of shedding, and there were no significant differences in prevalence of shedding between the nonmedicated and medicated groups at any of the sampling times. Of 1,340 S typhimurium colonies isolated from the nonmedicated and medicated groups, 1,330 were susceptible to all 12 antibacterials tested, indicating no treatment-related effect on susceptibility. At necropsy, S typhimurium was not isolated from any liver or spleen specimens, and was isolated from only 2 of 20 lymph nodes. However, S typhimurium was isolated via enrichment from the cecal contents from all 20 pigs. There were no treatment-related differences in feed consumption, weight gain, or feed efficiency. Appreciable differences in the measurements were not found between the efrotomycin-medicated and nonmedicated pigs.     

蓝狐猪霍乱沙门氏菌的分离鉴定

某蓝狐场２只成狐于１９９６年１１月先后发病,临床症状为呕吐、腹泻、眼有脓性分泌物,病程分别为７和１２天,病理变化主要为杆脏、脾脏、淋巴结肿大,有小坏死灶。作者从脾脏和淋巴结中分离出２株猪霍乱沙门氏菌,且证明２株菌有较强的致病性。     

Effect of feeding chlortetracycline or virginiamycin on shedding of salmonellae from experimentally-infected swine

Swine from a herd routinely fed subtherapeutic levels of chlortetracycline (CTC) were fed a diet containing 55 mg of CTC/kg, a diet containing 55 mg of virginiamycin/kg, or a control diet. All animals were inoculated with Salmonella typhimurium that was susceptible to tetracycline. The quantity, duration and prevalence of shedding of S. typhimurium were determined. The infecting organism was first recovered from the animals fed CTC or the control diet on d 2, from animals fed virginiamycin on d 7 and from animals in a second control group on d 10. The infecting organism was recovered in fewer samples obtained during the initial 7 d postinfection than in those obtained during the last 24 d of the study. Little transfer of resistance to the infecting organism seemed to have occurred from the resident microflora because only two isolates (1%) had resistant patterns that differed from that of the infecting organism. Feeding CTC or virginiamycin to swine did not significantly increase or prolong shedding of an experimentally infected tetracycline-susceptible strain of S. typhimurium. Neither antibiotic affected the drug resistance of the infecting organism.     

沙门氏菌对不同品种猪生产性能及免疫功能的影响

研究不同品种猪在沙门氏菌攻毒压力下生产性能及免疫功能的变化。在试验的7、14、21d,测定两品种猪的健康状态与沙门氏菌压力下的平均日增重(ADG)、平均日采食量(ADF)、血清IGF-1(胰岛素样生长因子)及抗体水平。结果显示:在7、14、21d时,长大二元杂交猪的ADG、ADF都显著高于松辽黑猪(0.01);在试验7、21d,松辽黑猪与长大二元杂交猪的血清IGF-1差别不显著,但在14d时,在沙门氏菌的压力下,长大二元杂交猪的IGF-1高于松辽黑猪(0.05);在7、14d松辽黑猪的血清抗体水平显著高于长大二元杂交猪(0.01),但21d时差异不显著。     

Porcine neutrophil function in the presence of virulent and avirulent Salmonella choleraesuis

Porcine polymorphonuclear leukocytes (PMNLs) may be activated by bacteria to begin phagocytosis followed by oxidative and non-oxidative mechanisms of killing. The purpose of this study was to identify differences between virulent and avirulent Salmonella choleraesuis (S. choleraesuis) strains, 38 and 9 respectively, in their interactions with porcine PMNLs using five different assays. (1) Staphylococcus aureus (S. aureus) ingestion was determined by exposure of porcine PMNLs to a mixture of S. choleraesuis and 125I labeled S. aureus. There was a 2.98% and 22.20% decrease in S. aureus ingestion by mouse-avirulent S. choleraesuis 9 and mouse-virulent S. choleraesuis 38 respectively. (2) Iodination of proteins was done by exposing zymosan stimulated porcine PMNLs to S. choleraesuis in the presence of 125I and measuring its incorporation into porcine PMNL proteins. This assay indicated a 73.7% and 74.7% decrease in iodination by S. choleraesuis 9 and S. choleraesuis 38, respectively. (3) Cytochrome c reduction was performed by using porcine PMNLs, zymosan, and S. choleraesuis to determine the bacterial effect on superoxide anion production. S. choleraesuis 9 and S. choleraesuis 38 inhibited superoxide anion production by 78.0% and 92.6%, respectively. (4) Lactoferrin release from porcine PMNLs was measured by an ELISA using the supernatant from the cytochrome c assay. Results indicate a 52.0% and 61.0% increase in lactoferrin release by S. choleraesuis 9 and 38 respectively. (5) The bactericidal assay was performed by counting cfus of S. choleraesuis after preliminary incubation with porcine PMNLs, followed by killing of extracellular S. choleraesuis and lysis of porcine PMNLs. Survival of S. choleraesuis 9 and E. coli (control) were 7.50% and 1.37%, respectively, in contrast to 52.62% survival of the virulent S. choleraesuis 38. These results indicate that both strains inhibited protein iodination and caused a slight increase in lactoferrin release, but the virulent S. choleraesuis 38 inhibited S. aureus ingestion, cytochrome c reduction, and survived porcine PMNL killing more effectively than the avirulent S. choleraesuis 9.     

Effect of 50 kilobase-plasmid, pKDSC50, of Salmonella choleraesuis RF-1 strain on pig septicemia.

Salmonella choleraesuis strains with and without 50-kilobase plasmid (pKDSC50) were intravenously inoculated into Yorkshire pigs. By the inoculation of 7.2 x 10(5) - 3.5 x 10(7) cells, RF-1 strain with pKDSC50, but not 31N-1 strain without the plasmid, caused a septicemia. The inoculation of 8.7 x 10(9) RF-1 cells killed pigs at 2-4 day postinfection with severe hemorrhage on the whole body. Pigs with a similar number of 31N-1 cells (8.3 x 10(9) cells), showed milder hemorrhage, and they died at 6 day postinfection. These results indicated that pKDSC50 is required for RF-1 strain to express the full virulence causing a heavy cutaneous pig septicemia.