Identification of patterns of transmission of Salmonella within swine production systems using pulsed field gel electrophoresis (PFGE) and repetitive sequence polymerase chain reaction (REP-PCR): a quantitative analysis

Pulsed field gel electrophoresis (PFGE) using 3 enzymes (Spe I, Xba I, Avr II) and repetitive sequence polymerase chain reaction (REP-PCR) with 3 primers (BOX, ERIC, REP) were compared with respect to their validity as a method for identifying transmission of Salmonella on swine farms. Sixty-eight isolates of Salmonella were obtained from feces of swine, cats, mice, and birds, insect body parts, water and floor samples, and boot scrapings collected on 9 swine farms in Illinois USA. Genetic distances between isolates were calculated using the Dice matching coefficient. Cluster analysis of distance matrices was conducted using the UPG-MA algorithm. There was no significant difference between PFGE and REP-PCR in the genetic diversity detected; however, REP-PCR differentiated between 14 pairs of isolates which PFGE identified as identical. There were no significant differences between PFGE and REP-PCR in identifying all or most close genetic links as isolates from the same farm, the same building, and from the same sampling visit, suggesting ecological validity for both methods. Thus, REP-PCR should be considered as an acceptable and perhaps preferable alternative to PFGE as a genotyping method for studies of Salmonella transmission.     

Testing an ecological model for transmission of Salmonella enterica in swine production ecosystems using genotyping data

An ecological model for transmission of Salmonella enterica in swine production ecosystems was developed, identifying host species, environmental reservoirs, and temporal, spatial, and functional (i.e., stage of production) dimensions. It was hypothesized that transmission was most likely within spatial and functional compartments, between hosts of the same species and abiotic compartments of the same type. Eighteen swine production systems in Illinois, USA, were sampled in four collection cycles (1998, 1999, 2000, 2003). There were 11,873 samples collected, including feces from swine and other mammals and birds, and samples from insects, pen floors, boots, feed, and water. The 460 Salmonella isolates obtained were genotyped using repetitive sequence PCR with three primers—REP, BOX, and ERIC. All isolates from 2000 and 2003 were serotyped, as well as a subsample from 1998 and 1998. Genetic relatedness was estimated from the similarity of fragmentation patterns after gel electrophoresis of PCR products. Cluster analysis identified genetically related isolates. Linking of isolates in tight clusters (similarity ≥85%) was viewed as evidence for transmission. Five farms had a sufficient number of tight clusters for hypothesis testing. The factors most differentiating isolates genetically were farm of origin and time of sampling. Isolates were also differentiated genetically by site, building, room, and pen. There was no consistent association of genotype with stage of production or host/environment reservoir. Serotype analysis confirmed that Salmonella lineages were differentiated by visit and site. Thus, Salmonella transmission was primarily over short distances, i.e., within the same pen or room, with some transmission between rooms and buildings on the same site, but with limited transmission between sites. Transmission was observed across a variety of ecological niches represented by different host species and environmental reservoirs. Genetic differences over time reflected multiple introductions into the ecosystem of different Salmonella genotypes, as well as evolutionary changes within lineages. Intervention strategies to reduce Salmonella prevalence within swine production ecosystems would be best targeted at maintaining spatial barriers to transmission, whereas intervention targeted at specific biological hosts or environmental reservoirs is less likely to be effective.     

Variation in Salmonella enteritidis RAPD-PCR patterns may not be due to genetic differences

Salmonella Enteritidis is a leading cause of gastroenteritis associated with consumption of contaminated poultry meat and eggs. Because pulsed-field gel electrophoresis (PFGE) has limited utility in distinguishing between clonal Salmonella Enteritidis isolates, random amplified polymorphic DNA (RAPD) PCR has been recommended as an alternative molecular fingerprinting tool. This study's objective was to determine whether increasing PCR stringency would improve the repeatability of RAPD DNA patterns based on assessment of target sites within the genome. An in silico PCR was performed to predict amplification products from an Salmonella Enteritidis genome sequence for three different RAPD primers (1247, 1283, and OPA4) and to determine whether any primer would be more likely to amplify variable regions within the genome. A comparison of within- and between-isolate similarities in RAPD patterns was performed using primer 1247, which was predicted by in silico analysis to yield a variable size range of amplicons. In order to reduce artifactual variability associated with the method, three different methods for template preparation were evaluated. All were found to provide comparable results with respect to the similarities observed with repeated analyses of the same Salmonella Enteritidis isolates (n = 18, P = 0.91). Although the median within-isolate similarity (76.0%) was significantly greater than the median between-isolate similarity (66.7%; P = 0.001), duplicate RAPD-PCR runs of the same Salmonella Enteritidis isolates produced DNA patterns that ranged in similarity between 61.5 and 100%. These results indicate that the repeatability of RAPD-PCR is insufficient to distinguish genetic differences among related and unrelated Salmonella Enteritidis isolates.     

Antimicrobial resistance in generic Escherichia coli isolated from swine fecal samples in 90 Alberta finishing farms

The objective of this study was to determine the prevalence of antimicrobial resistance in generic Escherichia coli isolates obtained from 90 Alberta finisher swine farms. Up to 5 isolates were obtained from each of 269 pooled fecal samples and were classified as susceptible or resistant according to Clinical and Laboratory Standards Institute guidelines. Of the 1322 isolates, 166 (12.6%) were susceptible to all 15 antimicrobials. No resistance to amikacin, ceftiofur, ceftriaxone, or ciprofloxacin, antimicrobials of importance in human medicine, was observed. Relatively low frequencies of resistance were observed to gentamicin (1.1%), amoxicillin/clavulanic acid (0.7%), and cefoxitin (0.7%). Higher frequencies of resistance were observed for tetracycline (78.9%), sulfisoxazole (49.9%), streptomycin (49.6%), ampicillin (30.6%), chloramphenicol (17.6%), kanamycin (10%), and trimethoprim/ sulfamethoxazole (6.4%). Among the isolates resistant to > or = 2 antimicrobial classes, 20.8%, 20.6%, 18.2%, 7.0%, 1.8%, 0.2%, and 0.2% were resistant to 2, 3, 4, 5, 6, 7, and 8 antimicrobials, respectively. The most common multidrug-resistance patterns (resistance to > or = 2 antimicrobial classes) were streptomycin-tetracycline (9.4%), streptomycin-sulfisoxazole-tetracycline (6.2%), and ampicillin-streptomycin-sulfisoxazole-tetracycline (6.1%). More clustering (higher intra-class correlation coefficients) in antimicrobial resistance was observed for isolates at the same visit than for isolates from different visits in the same farm, indicating that sampling more farms, testing fewer isolates per visits, and taking longer periods between visits may be appropriate and more efficient for a better understanding of potential shifts in resistance over time.     

Association between genetic sequence homology of Porcine reproductive and respiratory syndrome virus and geographic distance between pig sites

The objective of this study was to investigate whether geographic distance was correlated with genetic homology among isolates of Porcine reproductive and respiratory syndrome virus (PRRSV) from a single pork-producing company. We analyzed geographic distance, temporal distance, and percentage similarity in the PRRSV nucleotide sequence among 62 farms, applying the Mantel test for correlation between distance matrices and PRRSV sequencing. Genomic similarity had a significant (P < 0.01) negative (rM = -0.217) correlation with geographic distance. These findings indicate that, under the conditions of this study, the greater the distance between farms, the less the genetic homology among PRRSV isolates.     

Comparison of antimicrobial resistance in generic Escherichia coli and Salmonella spp. cultured from identical fecal samples in finishing swine

The objectives of this study were to investigate the associations between antimicrobial resistance patterns in generic Escherichia coli and Salmonella spp. isolates recovered from identical pen pooled fecal samples, and to evaluate potential clustering of multiple isolates of these organisms within identical fecal samples. Up to 5 generic E. coli (n = 922 isolates) and Salmonella spp. (n = 922 isolates) isolates were obtained from each of 188 pen pooled fecal samples that had been collected from 45 finishing swine farms in Alberta in 2000, and tested for susceptibility to 15 antimicrobials. No isolates of either organism were resistant to 3rd generation cephalosporins or fluoroquinolones, which in Canada are considered antimicrobials of very high importance to human health. Approximately twice as many generic E. coli isolates as Salmonella spp. isolates were resistant to at least 1 antimicrobial. In addition, E. coli isolates showed more multidrug-resistance patterns. No significant association was observed between the resistance phenotypes of Salmonella spp. and E. coli at the fecal sample level. More clustering at the sample level was observed for proportions of antimicrobial resistance (AMR) in Salmonella spp. isolates than E. coli indicating that in future studies it might be sufficient to test fewer than 5 Salmonella spp. isolates per sample.     

Epidemiological study of Yersinia enterocolitica in swine herds in Québec

The objectives of this study were the identification of the different contamination sources of Yersinia enterocolitica, as well as the determination of the prevalence and the distribution of the different genotypes in swine herds. The owners of 20 farms, located in the Richelieu-Yamaska region, agreed to participate in the study. Each farm was visited a minimum of 5 times between May and October 1997, and, at each visit, 20 environmental and 10 fecal samples were collected. Yersinia enterocolitica isolates were identified, serotyped, and submitted to a genetic characterization by pulsed-field gel electrophoresis. The correlation coefficient (0.61) between prevalence in environment and in feces was significant (P = 0.004). Among the 153 positive samples, 93.5% belonged to serotype 0:3. The comparison of PFGE profiles revealed that all environmental Y. enterocolitica isolates had a profile identical to that of isolates recovered in feces from the corresponding farms. Also, when the genetic profiles of isolates recovered from feces collected at the first visit were compared with the profiles of isolates obtained from the subsequent visits, the same profile was observed on every farm. We concluded that environment does not represent the main source of contamination of swine by Y. enterocolitica and that, in most instances, the same strain persists in a barn from one production lot to another.     

Longitudinal study of Salmonella species in 90 Alberta swine finishing farms

The aim of this study was to determine the farm prevalence of Salmonella in 90 Alberta finishing swine farms over a 5-month period, to evaluate Salmonella distribution in the farm environment and to describe Salmonella serovar diversity on these farms. Ten veterinary practitioners selected 90 Alberta swine farms based on an annual production of > or =2000 market pigs per farm and the willingness of the producers to participate in the study. Between May and September 2000, twenty samples were collected from finishing swine and the environment of each farm. The annual production of selected farms represented approximately 25% of the market swine production in Alberta. Participating farms were geographically representative of major swine production areas in Alberta. Sixty (66.7%) farms had at least one Salmonella-positive sample, with confidence interval (CI) of 57.1-77.2%. Salmonella were detected in 14.3% of fecal and 20.1% of environmental samples. The number of Salmonella-positive samples per farm ranged from 1 to 19. Among environmental samples, Salmonella were most frequently recovered from boots (38.6%) and the main drain (31.8%). Twenty-two serovars were detected on the 60 Salmonella-positive farms. Serovars Typhimurium (78 isolates), Derby (71 isolates) and Infantis (47 isolates) were the most common. A single serovar was detected on 58 farms, while 2, 3 and >3 serovars were detected on 15, 10 and 7 farms, respectively. The Salmonella farm status changed frequently over the 5-month period indicating the dynamic nature of Salmonella infections on these farms.     

New insights into the genetics of EBLV-1 from Germany

Previous epidemiological studies on EBLVs indicated a distinct geographical distribution of EBLV-1 in Germany. In this study, 48 isolates were selected to further investigate the spatial and temporal distribution of EBLV-1 variants in Germany. The nucleoprotein-gene (N), the nucleoprotein-phosphoprotein spanning untranslated region (NP-UTR) and the UTR between G- and L-gene of each isolate were sequenced using direct cycle sequencing. Results of the subsequent phylogenetic analysis of the N-gene confirmed previous studies on EBLVs, showing a high sequence identity among German EBLV-1a isolates, and a correlation between genetic and temporal and spatial distance, respectively, was shown. Our results indicate that the GL-UTR is not suitable for phylogenetic analyses. Interestingly, 6 nt insertions in two isolates as well as a single nucleotide insertion in a different isolate were detected in the N-P UTR. Within the UTR between G- and L-gene one isolate showed a 35 nt deletion. The effect of those changes on viral properties remains elusive as such mutations have not been described for lyssaviruses before.     

裸果木种群遗传多样性特点及与地理气候因子关联研究

本文利用ISSR标记技术,选取15对引物,对分布于西北荒漠区12个裸果木种群的遗传多样性以及与地理气候因子的相关性进行了分析。结果表明：群体内多态性位点百分率（PPL）为88.81%,种群水平为69.93%;Nei’s基因多样性指数（He）变化范围为0.2356~0.2697,Shannom信息指数（I）变化范围为0.3509~0.4057,具较高的遗传多样性水平。基因分化系数（Gst）为0.2790,种群间遗传分化较小,种群内存有丰富的遗传变异。经Mantel检验,种群遗传距离与地理距离有相关性（R²=0.2144）。利用NTSYSpc-2.1软件进行UPGMA法聚类,各种群样品间的遗传相似系数在0.81～0.99之间,按照遗传距离远近,裸果木12个种群可分为4大类群。经相关性分析,种群遗传多样性与纬度、海拔存在正相关（P<0.05）与年均温存在负相关（P<0.05）。     

Spatial clustering of swine influenza in Ontario on the basis of herd-level disease status with different misclassification errors

This approach maximizes sensitivity of serology-based monitoring systems by considering spatial clustering of herds classified as false positive by herd testing, allowing outbreaks to be detected in an early phase. The primary objective of this study was to determine whether swine herds infected with influenza viruses cluster in space, and if so, where they cluster. The secondary objective was to investigate the combining of a multivariate spatial scan statistic with herd test results to maximize the sensitivity of the surveillance system for swine influenza. We tested for spatial clustering of swine influenza using the Cuzick–Edwards test as a global test. The location of the most likely spatial clusters of cases for each subtype and strain in a sample of 65 sow and 72 finisher herds in 2001 (Ontario, Canada), and 76 sow herds in 2003 (Ontario, Canada) was determined by a spatial scan statistic in a purely spatial Bernoulli model based on single and multiple datasets.

A case herd was defined by true herd-disease status for sow or finisher herds tested for H1N1, and by apparent herd-disease status for sow herds tested for two H3N2 strains (A/Swine/Colorado/1/77 (Sw/Col/77) and A/Swine/Texas/4199-2/98 (Sw/Tex/98)). In sow herds, there was no statistically significant clustering of H1N1 influenza after adjustment for pig-farm density. Similarly, spatial clustering was not found in finisher herds. In contrast, clustering of H3N2 Sw/Col/77 (prevalence ratio = 12.5) and H3N2 Sw/Tex/98 (prevalence ratio = 15) was identified in an area close to a region with documented isolation of avian influenza isolates from pigs.

For the H1N1 subtype tested by ELISA, we used an approach that minimized overall misclassification at the herd level. This could be more applicable for detecting clusters of positive farms when herd prevalence is moderate to high than when herd prevalence is low. For the H3N2 strains we used an approach that maximized herd-level sensitivity by minimizing the herd cut-off. This is useful in situations where prevalence of the pathogen is low. The results of applying a multivariate spatial scan statistic approach, led us to generate the hypothesis that an unknown variant of influenza of avian origin was circulating in swine herds close to an area where avian strains had previously been isolated from swine. Maximizing herd sensitivity and linking it with the spatial information can be of use for monitoring of pathogens that exhibit the potential for rapid antigenic change, which, consequently, might then lead to diminished cross-reactivity of routinely used assays and lower test sensitivity for the newly emerged variants. Veterinary authorities might incorporate this approach into animal disease surveillance programs that either substantiate freedom from disease, or are aimed at detecting early incursion of a pathogen, such as influenza virus, or both.     

抗蚜苜蓿品种(系)SSR标记的遗传多样性分析

用6对SSR引物对9个抗蚜苜蓿(Medicago spp.)品种(系)进行了遗传多样性检测,共检测到69个位点。多态位点百分率为91.3%(63个位点),平均等位基因数为1.913 0,平均有效等位基因数为1.482 2,平均遗传多样性指数为0.293 4,遗传距离为0.156 6～0.623 2。聚类分析结果表明:抗蚜品种M8和品系HA-3具有较近的亲缘关系,二者的相似系数最大,为0.855 0。而品种S7和X2亲缘关系较远,相似系数最小,为0.536 2。此外,高抗品系HA-3与低抗品种Hunter River的相似系数为0.739 1,其遗传背景差异性相对较大。     

Use of molecular fingerprinting to assist the understanding of the epidemiology of Salmonella contamination within broiler production

1. We analysed Salmonella isolates by conventional sero- and phage-typing, as well as by molecular techniques within the broiler production chain in two integrated companies. The most prevalent serovars were selected for genetic fingerprinting. 2. Isolates were first screened by plasmid profiling; subsequently, the most common plasmid types within the prevalent zoonotic serovars (enteritidis and typhimurium) and S. agama were further characterised by PstI-SphI ribotyping, and XbaI pulsed field gel electrophoresis (PFGE). 3. Salmonella binza, S. kedougou, and S. 4,12:d:- were endemic in the feed mills over long periods of time, and a variety of plasmid types for each of the serovars were found in the premises. 4. A similar situation was found with S. binza and S. senftenberg within the hatchery in company B. The Salmonella serovars which were resident in those locations were also the ones most widely distributed throughout the broiler flocks. 5. Plasmid profiling was useful to subdivide clusters of isolates within serovars, but for each serovar a high percentage (36 to 79%) of the isolates tested fall within a prevalent plasmid type. 6. A more detailed genetic analysis of the isolates by a multiple typing approach allowed for further strain differentiation, and allowed some epidemiological conclusions to be drawn.     

Molecular epidemiologic analysis of Mycobacterium bovis isolates from Mexico

OBJECTIVE: To assess phylogenetic relationships among Mycobacterium bovis isolates by use of random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) fingerprinting and to relate genetic profiles of isolates to epidemiologic characteristics. ANIMALS: 400 cattle with tuberculosis. PROCEDURE: Mycobacterium bovis was isolated from various organs of cattle slaughtered in 6 geographic regions of Mexico. Most cattle were adult Holsteins from large herds that did not participate in a tuberculosis control program. Four random primers and 2 selected primers were used in RAPD-PCR fingerprinting of 88 isolates. Pairwise genetic distance between isolates was obtained and subjected to cluster analysis with bootstrapping to test for levels of support. RESULTS: 98 different fragments were obtained; there was broad genetic diversity among isolates, and each isolate had a unique RAPD-genotype, including those originating from the same herd. Clustering by geographic location, affected organ, or severity of lesion was not detected. Linkage disequilibrium analysis suggested that M. bovis was highly clonal and that mutations develop at a rapid rate among isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Use of RAPD-PCR could not differentiate M. bovis isolates by epidemiologic characteristics or identify common sources of infection.     

冰草属植物种质资源遗传多样性的ISSR分析

用ISSR标记对来自国内外的33份冰草材料的遗传多样性进行了检测。从93条ISSR引物中共筛出11条能扩增出清晰条带并具有多态性的引物,33个样品DNA共获得84个扩增位点,其中多态性位点59个,平均每个引物扩增位点为7.64个。品种间遗传相似系数在0.083～0.706,表现出丰富的遗传多样性。利用UPGMA聚类分析,以遗传相似系数0.52为界限,33份材料划分为4类,聚类基本符合地理来源相近的材料聚为一类,呈现出一定的地域性分布规律。     

Lysine decarboxylase-negative Salmonella enterica serovar enteritidis: antibiotic susceptibility, phage and PFGE typing

One hundred twenty Salmonella Enteritidis isolates collected from 1992 to 2005 in Nagasaki prefecture (65 isolates from 40 outbreak cases, 44 from sporadic diarrhea patients, and 11 from chicken-related products) were investigated by their antibiotic susceptibility profiles, phage typing, and pulsed-field gel electrophoresis (PFGE) typing. Out of them, 18 were identified as lysine decarboxylase (LDC)-negative isolates, and 15 showed resistance toward streptomycin. Based on the PFGE typing, the isolates were classified into five clusters by UPGMA clustering method. Three LDC-negative isolates belonged to cluster A and were of phage type (PT) 4 and isolated between 2000 and 2004. Other 15 LDC-negative isolates belonged to cluster E. They were PT1, reacted but did not conform (RDNC), or untypable and were isolated between 2001 and 2004. LDC-negative isolates of the cluster A differed from LDC-negative isolates of the cluster E in antibiotic susceptibility profiles, phage typing, and PFGE typing. LDC-negative isolates of the cluster E were isolated after 2001 in Nagasaki prefecture.     

大雁、斑头雁和天鹅源沙门氏菌血清及脉冲场凝胶电泳分型研究

为了解大雁、斑头雁及天鹅源沙门氏菌流行特征和脉冲场凝胶电泳（PFGE）分型,本研究对33株沙门氏菌进行血清型鉴定,采用PFGE对其进行基因分型,并用BioNumerics 6.6软件进行聚类分析。鉴定出丙型副伤寒沙门氏菌6株、汤卜逊沙门氏菌19株、乙型副伤寒沙门氏菌1株、伊鲁木沙门氏菌1株、奥斯陆沙门氏菌1株、5株未定型。PFGE聚类分型共分为16个型别,其相似度为53.7%~100.0%,且大多数菌株与人源菌株相似度超过80%。结果表明,大雁、斑头雁及天鹅源沙门氏菌相同血清型PFGE带型高度相似,可能来自同一克隆株,不同血清型PFGE带型差别较大。     

PFGE Genotyping and Serotype Analysis of Salmonella Isolated from Goose,Anserindicus and Cygnus

To understand the Salmonella epidemic characteristics and pulsed field gel electrophoresis（PFGE）genotyping of goose,Anserindicus and Cygnus,33 strains of Salmonella were serotype typed by PFGE and genotyped with BioNumerics 6.6 cluster analysis software.The results showed that there were 16 PFGE patterns,which displayed 6 strains Salmonella paratyphi C,19 strains Salmonella thompson,1 strains Salmonella paratyphi B,1 strain Salmonella irumu,1 strain Salmonella oslo,5 undifferentiated.PFGE similarity is about 53.7% to 100.0%,and most of the strains and anthropogenic strains similarity was more than 80%.The results showed that goose,Anserindicus and Cygnus Salmonella serotypes same PFGE type with a high degree of similarity,and possibly from the same clone,quite different serotypes PFGE band pattern difference.     

Drag swab efficiency factors when sampling chicken manure

This study examines drag swabbing distance, media for moistening the drag swabs, and site selection when sampling a laying facility by drag swabbing manure piles. Manure piles at a laying facility in California's San Joaquin Valley were sampled with drag swabs over various distances. Samples were cultured for Salmonella spp. with standard laboratory methods, and most probable number calculations. Salmonella spp. counts were expected to be highly variable because of reported clustering. Therefore, total bacteria and Escherichia coli, which were assumed to have a more uniform distribution on the surface of the manure, were additionally used as proxies for Salmonella. Media for moistening the swabs were compared by seeding postswabbing samples with Salmonella typhimurium, and culturing at different delay times. Total bacterial counts were compared between samples that were obtained from either wet or dry surfaces. Numbers of Salmonella spp. and total bacteria peaked within 120 feet of swabbing distance. Higher total bacteria counts were obtained by swabbing wet areas rather than dry areas, but the distance that could be swabbed effectively was shorter in wet areas. Moistening media selected for the swab resulted in statistically different culture counts, but did not show any important difference in maintaining Salmonella viability over a 48-hr period when the samples were kept at refrigerated temperatures. Once swabs became fully loaded with fecal material, bacterial numbers failed to increase with further use. Overuse of a swab may result in failure to detect Salmonella enteritidis on chicken manure if the distribution of this organism is clustered.