Transmission patterns of Salmonella in a swine production unit were investigated by statistical analysis of the spatial distribution of isolates and the clustering of genotypes over a 12-week period. The study unit was a breeding-gestation building in a single-site farrow-to-finish swine production system. During the summer of 2003, 1746 pen floor samples were collected during 6 visits conducted at 2-week intervals. Genotyping was performed on the 107 Salmonella isolates obtained using repetitive sequence PCR (3 primers: REP, BOX, ERIC). Genetic similarity was evaluated by DNA fragment matching and hierarchical cluster analysis based on genetic similarities. For each visit, the distance at which spatial clustering of Salmonella isolates occurred was estimated using second order analyses. Significant spatial clustering of Salmonella up to a distance of 15.2m was identified for 4 of the 6 farm visits, those for which the prevalence of Salmonella was the highest. Cluster analysis of genetic similarities identified 4 groups of Salmonella isolates at the level of at least 85% similarity in rep-PCR fragment matching patterns. Genetic clusters were relatively homogeneous for time of visit, with each genetic cluster consisting of isolates from the same or temporally adjacent visits. The correlation between genetic similarity and spatial proximity between pairs of Salmonella isolates, and the correlation between these measures and differences in time of sampling, were evaluated using Mantel's r. There was a strong positive correlation (r=0.62, p<0.0001) between genetic distance and time of sampling, indicating evolution of the Salmonella population over time. For 4 of the 6 visits, a pattern of decreasing genetic similarity between isolates with increasing distance between sampling locations was apparent. However, when viewed over all 6 visits, Salmonella was concentrated in one area of the building and did not move in any specific direction. These results suggest that a particular genotype of Salmonella, if introduced in the breeding-gestation unit of a swine farm would evolve slowly over short time intervals; its spatial distribution would be limited primarily to adjacent or nearby pens. In this study spatial analysis (e.g., Ripley's K-function) and matrix correlation methods (e.g., Mantel's r) expanded upon cluster analysis of genotypic similarities to provide additional interpretable information regarding the spatial distances to which Salmonella is transmitted over time in swine production facilities.

Objective: To investigate whether purchased gilts introduce new Salmonella genotypes into a swine production system and acquire Salmonella infection from resident pigs. Materials and methods: One multi-site and one single-site swine production system from Illinois were recruited on prior evidence of a high prevalence of Salmonella infection. Cohorts of 102 and 120 incoming gilts on the multi-site and single-site farms, respectively, were sampled longitudinally for five to eight visits until the gilts were introduced into the breeding herd. On each visit, fecal and floor samples were obtained from cohort gilts and an approximately equal number of resident pigs in the same or adjacent room or building. All samples were cultured to detect Salmonella. The 123 Salmonella isolates were genotyped using repetitive sequence polymerase chain reaction with REP, BOX, and ERIC primers. Cluster analysis classified the isolates by degree of genetic relatedness. The 45 isolates from the multi-site farm were serotyped. Results: For both farms, the most closely genetically related Salmonella isolates were obtained from the same room on the same visit. For the multi-site farm, there was no evidence of transmission between gilts and resident pigs. For the single-site farm, there was evidence of transmission between gilts and resident pigs in the breeding barns. However, conclusive evidence that incoming gilts introduce new Salmonella genotypes into a swine production system was lacking. Serotyping information supported conclusions drawn from genotyping data. Implications: Spatial separation of purchased gilts from other pigs by all-in, all-out pig flow reduces Salmonella transmission in swine production systems.

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.

Salmonella harborage at slaughter can be viewed as a nsk for human health through contammation of the pork food cham. Better understanding of herd level factors associated with this harborage would be useful to prioritize further study of epidemiology and control of Salmonella 1n pork production. Ileocolic lymph node samples collected at slaughter from 115 Midwest US sw1ne herds were assayed for Salmonella entenca. A subset of these herds was collected sequentially one or two additional times . Herd characteristics and management factors were assessed by a written survey. Risk factors were screened at the univariate level (p < 0.3), then offered for Inclusion by stepw1se analysis including herd I sample as a random statistical effect. Pigs at increased risk of Salmonella harborage at slaughter included those placed in finisher barns at heaver weights (OR 1.2 per 10 kg Increased we1ght), those from larger herds (OR 2 0 comparing upper quintile to lower quintile of herd size), those from herds that allowed VISitors w1th recent (<8 h) contact with other herds (OR 2.2), or those fed pelleted feeds (OR 2.1 ). Further invest1gat1on of these risk factors and potential biological mechanisms will requ1re further study.

Objectives: To describe the prevalence and serovars of Salmonella enterica in ileocolic lymph nodes of slaughtered swine in a sample of Midwest US herds and to assess as methods of study pooling and freezing of lymph-node samples prior to bacterial culture. Materials and methods: Ileocolic lymph nodes from 30 pigs from each of 146 herds were sampled at slaughter. Tissue from five pigs was pooled for one bacterial culture. Retained frozen tissues from the same pigs were cultured individually (n = 82 herds) from a subset of those with Salmonella-positive pools (n = 100 herds). A mathematical relationship was described to predict approximate individual prevalence based on number of positive pools. Isolates were serotyped. To test for effects of freezing on test sensitivity, lymph nodes from 100 pigs were cultured both fresh and after freezing. Results: Salmonellae were detected in 100 of 146 herds (68.5%). The mean number of positive pools per herd was 1.75, and the mean within-herd, individual-pig prevalence was 6.98% (95% CI, 4.88% – 9.07%). Freezing of samples did not result in decreased detected prevalence. Individual prevalence could be approximately predicted by pool results, although with low precision. Implications: Salmonellae were found in two-thirds of the herds studied. Culture of pooled samples with subsequent culture of retained frozen tissues from positive pools may be an effective way to test a larger number of herds on a given budget through laboratory-cost savings. However, pooling without culture of individuals from positive pools results in imprecise prevalence estimation.

ABSTRACT API 20E and invA PCR were evaluated for the identification of Salmonella enterica isolates from swine farms. API 20E had the highest agreement with other tests at the 99.9% likelihood level. Both tests had 100% sensitivity and 96% specificity compared to 16S rRNA sequencing. Compared to serotyping, both tests had 96% sensitivity; specificity was 86% for API 20E and 79% for invA PCR.

API 20E and invA PCR were compared for diagnostic accuracy for Salmonella for 310 bacterial isolates from 3 Illinois swine farms.Reactions based on Triple Sugar Iron agar, Lysine Iron Agar, and Salmonella O (poly A/B) antisera tests were also considered.Repetitive sequence PCR (rep-PCR) using REP, BOX, and ERIC primers, identified the genetic basis for diagnostic classification.Cluster analysis and multidimensional scaling grouped isolates based on diagnostic and genetic characteristics.The invA PCR had higher agreement with other tests (particularly poly A/B antisera) than API 20E in Salmonella classification.Cluster analysis identified several clusters of isolates that were API 20E positive but negative by other tests, suggesting lower specificity for API 20E than invA PCR.Rep-PCR genotyping supported a genetic basis for diagnostic test result differences.This suggests that invA PCR should be considered as a cost and time saving alternative to API 20E in the diagnosis of Salmonella.

Thirty swine production units in the midwestern United States were studied to assess the relationship of herd-level prevalence of Salmonella on the farm prior to slaughter versus at slaughter. Fecal samples were collected from 30 pigs on each farm within 48 h of slaughter, and 30 ileocecal lymph node samples were collected in the same shipment cohort at slaughter. Samples were cultured by conventional methods, and Salmonella identity was confirmed by serotyping. Overall, 11.7% (n = 105) of the fecal samples and 14.9% (n = 133) of the ileocecal lymph node samples were positive for Salmonella. Seventeen of the farms (56.7%) had one or more positive fecal samples, and 24 (80.0%) had one or more positive ileocecal lymph node samples. Twenty-four recognized serotypes and three additional distinct antigenic types were identified. Among all isolates, 56.5% had serotypes that were duplicated both on the farm and at slaughter for a particular cohort, whereas the remaining samples lacked a duplicate serotype in the other sample type. There was a positive correlation in the prevalence of Salmonella between fecal samples and ileocecal lymph node samples (Spearman's p = 0.75; 95% confidence interval [CI], 0.62 to 0.89). Linear regression analysis was used to identify two farms that biased the regression estimates. Excluding these farms, 62% of the variance in farm slaughter Salmonella prevalence was accounted for by on-farm prevalence. The analyses suggest that the prevalence of Salmonella spp. at slaughter can be predicted from preslaughter on-farm sampling and vice versa.

Objectives: To determine whether purchase and isolation of gilts and boars, purchase of semen for artificial insemination (AI), sharing of boars with other herds, herd size, and confinement housing were risk factors for porcine reproductive and respiratory syndrome (PRRS) virus infection and clinical PRRS in swine herds during the early years of the pandemic.Methods: A herd demographic and management survey of 103 swine herds in Illinois was conducted in 1992 and repeated in 1997 to determine whether PRRS virus infection and clinical disease had been diagnosed during the intervening period. Multivariable logistic regression analysis was conducted to identify risk factor associations.Results: Larger herd size was associated with an increased risk, and isolation of purchased gilts with a decreased risk, of both PRRS virus infection and clinical PRRS. Purchase of semen for AI was associated with an increased risk of PRRS virus infection.Implications: Swine producers should certify that purchased semen is free of PRRS virus, gilts should be isolated prior to introduction to the herd, and biosecurity measures should be implemented to prevent PRRS virus infection, particularly in larger herds.

Pulsed field gel electrophoresis (PFGE) using restriction enzymes AvrII, SpeI, and XbaI, and repetitive sequence polymerase chain reaction (Rep-PCR) using BOX, ERIC, and REP primers, were compared with respect to their ability to detect genetic differences among 68 Salmonella isolates from nine Illinois swine farms. Both genotyping methods had high reproducibility of fragment numbers (reliability>0.9) and sizes (reliability>0.85), and produced approximately the same number of DNA fragments, but Rep-PCR fragment profiles had considerably greater variation. Genetic distances between isolates were calculated from fragment size matching. There was good agreement between the genetic distance matrices for the composite (3-enzyme and 3-primer) methods (Mantel's r=0.83). PFGE detected slightly greater variation in genetic distances among isolates, but failed to differentiate seven pairs of isolates, three of which were sampled at least 1 month apart and therefore unlikely to be truly identical genetically. In contrast, Rep-PCR identified no isolates as genetically identical. In cluster analyses based on genetic distances, there were moderate differences between PFGE and Rep-PCR (about 2/3 agreement in tight cluster membership). Both PFGE and Rep-PCR were able to differentiate isolates of the same serotype. However, some serotypes (Agona, Anatum, Derby, Infantis, Worthington) were distributed across clusters. There was less agreement between individual primer/enzyme and composite results for Rep-PCR than for PFGE. This greater independence of results for individual primers for Rep-PCR accounted in part for the greater discriminative ability of the composite method. Both composite methods indicated that most Salmonella transmission occurred within a farm and that there was no preference for transmission between specific ecological compartments. Given the equally high reliability of both genotyping methods, the greater discriminative ability of Rep-PCR recommends it as the preferred method for precise detection of transmission links.

BACKGROUND:Determination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny. However, there is little information available on how well the genetic relationships inferred from different genotyping methods agree with true genetic relationships. In this report, two genotyping methods - restriction fragment analysis (RFA) and partial genome DNA sequencing - were each compared to complete DNA sequencing as the definitive standard for classification.RESULTS:Using the Genbank database, 16 different types or subtypes of papillomavirus were selected as study samples, because numerous complete genome sequences were available. RFA was achieved by computer-simulated digestion. The genetic similarity of samples, based on RFA, was determined from the proportion of fragments that matched in size. DNA sequences of four specific genes (E1, E6, E7, and L1), representing partial genome sequencing, were also selected for comparison to complete genome sequencing. Laboratory error was not taken into account. Evaluation of the correlation between genetic similarity matrices (Mantel's r) and comparisons of the structure of the derived dendrograms (partition metric) indicated that partial genome sequencing (for single genes) had higher agreement with complete genome sequencing, achieving a maximum Mantel's r = 0.97 and a minimum partition metric = 10. RFA had lower agreement, with a maximum Mantel's r = 0.60 and a minimum partition metric = 18.CONCLUSIONS:This simulation indicated that for smaller genomes, such as papillomavirus, partial genome sequencing is superior to restriction fragment analysis in representing genetic relatedness among isolates. The generalizability of these results to larger genomes, as well as the impact of laboratory error, remains to be demonstrated.

Objectives: To evaluate the impact of endemic infection with porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), transmissible gastroenteritis virus-porcine respiratory coronavirus (TGEV-PRCV), pseudorabies virus (PRV), Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae (APP) on the reproductive performance of sows. Methods: Seventeen groups of 30 to 60 sows and gilts in seven herds were monitored over a 2-year period by serological testing for the pathogens listed above. Litter size, number of stillborn (including mummies), average weaning weight, preweaning deaths, and interfarrowing interval were recorded for the sows that were tested. Multiple linear regression analyses were performed to evaluate the association of the measures of reproductive performance with serological test results. Results: Infection with PRRSV was consistently associated with poorer reproductive performance. Sows that had antibodies to PRRSV had, on average, 0.1 to 0.9 more stillborn piglets per litter than did seronegative animals. The average interfarrowing interval was 3 to 10 days longer for PRRSV-seropositive sows. A consistent correlation between reproductive performance and serological results could not be found for SIV, TGEV-PRCV, PRV, M hyopneu-moniae, or APP. Implications: Even in herds where no clinical signs of the disease are present, reproductive performance may be substantially inferior in sows with high levels of antibody against PRRSV.

In a controlled experiment, 16 wild-trapped raccoons were exposed to 1 of 2 genetically modified live pseudorabies virus (PRV) vaccines used in swine. One vaccine had genes deleted for thymidine kinase (TK(-)) and glycoprotein G (gG(-)); the other had an additional deletion for glycoprotein E (gE(-)). These vaccines were administered orally and intranasally at four dose levels: 10(3), 10(4), 10(5), and 10(6) TCID(50). The 21 days survival rate was 37.5% for the gG(-)TK(-) vaccine; all of the survivors developed antibodies to PRV. All animals receiving the gG(-)gE(-)TK(-) vaccine survived; 75% (all except the lowest dose) developed anti-PRV antibodies. Survivors were challenged intranasally with a 3.2x10(3) TCID(50) dose of the virulent wildtype PRV Shope strain. Two of the remaining three gG(-)TK(-) vaccinated raccoons survived the challenge; for the gG(-)gE(-)TK(-) vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody titers were more likely to survive the challenge with the virulent PRV; there was a 100% mortality rate for raccoons lacking detectable anti-PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent virus.

In today's intensive pork production systems, herd health is a major factor determining the profitability of the business. Among the economically most important diseases in the Midwest are porcine reproductive and respiratory syndrome, pseudorabies, transmissible gastroenteritis, swine influenza and pneumonia caused by Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae. Several studies have evaluated the financial impact of acute outbreaks of these diseases in swine operations (Miller & Kliebenstein, 1985, Mullan et a. 1994, Poulson et al., 1993, Rougoor et al., 1996). However, the predominant problem in many swine operations is a subclinical infection of the herd rather than acute outbreaks of diseases. Infected pigs are not obviously sick or die, but perform poorly. The economic consequences of subclinical infection can be slow growth, decreased feed efficiency, low fertility, increased numbers of abortions and stillbirths, and smaller litter sizes (Baysinger et al., 1997). For farms operating on an 'all in, all out' basis, the difference in weight gain between infected and non-infected pigs can be an additional problem, because finishing pigs do not reach market weight at the same time.

The objective of this 2-year field survey was to sample multiple ecological compartments within swine production systems to identify potential sources of Salmonella infection for swine. Twelve single-site production systems within Illinois were identified by slaughter sampling to have detectable Salmonella in swine and therefore selected for study. There were four visits to each farm during a 5-month period. Fecal samples were obtained from swine and other wild and domestic mammals. Arthropods and environmental samples of feed, water, pen floors, boots, and bird feces were also collected. All 8,066 samples obtained were cultured to detect Salmonella. Salmonella was detected on 11 of the 12 farms. There were 206 positive cultures, including samples from swine (83), pen floors (54), boots (32), flies (16), mice (9), cats (3), and birds (3). Swine shedding Salmonella in feces were detected on 9 of the 12 farms. The more Salmonella-abundant ecological compartments were cats (12% of samples positive), boots (11%), bird feces (8%), flies (6%), and mice (5%); 2.1% of 4,024 swine samples were positive. All 221 feed samples were negative for Salmonella. There was a correlation between a farm having a high prevalence of shedding Salmonella in pigs and a high abundance on pen floors, flies, and boots. The most common serotypes detected were Derby, Agona, Worthington, and Uganda, which were distributed throughout the ecosystem, suggesting widespread transmission across ecological compartments. The ubiquitous distribution of Salmonella suggests that an effective control strategy must target multiple compartments of the swine production ecosystem.

Transmission of Toxoplasma gondii on swine farms was investigated using a deterministic dynamic computer simulation model. A primary focus was to evaluate a feline T. gondii vaccine. Animal populations (swine and cats) were compartmentalized based on the stage of T. gondii infection. Simulations were run under conditions of closed and equilibrium population size. Model parameters were varied in a factorial experimental design to test the following hypotheses: T. gondii infection in finishing pigs decreases with (1) vaccination of susceptible cats, (2) an increase in the proportion of cats captured for vaccination, (3) a decrease in the initial number of cats, (4) a decrease in the initial T. gondii prevalence in cats and (5) a decrease in oocyst-survival time. Seeding conditions included a total of 10, 20, 30, 40 or 50 cats, initial T. gondii prevalences in cats of 30, 60 or 90%, vaccination of 0, 50 or 75% of the cats and two vaccination schedules (the field schedule from a prior trial and a weaning-vaccination schedule). Simulations were run at oocyst-survival times of 52, 39 and 26 weeks. T. gondii prevalence in finishing pigs was recorded every week for 10 years. The probability of elimination of T. gondii from finishing pigs increased with a decrease in the number of cats and a decrease in oocyst-survival time. The last-year average prevalence was used as the outcome in a multiple linear regression analysis. Decreased T. gondii prevalence in finishing pigs was the result of a decrease in the initial number of cats on the farm (squared semipartial correlation coefficient (sr(2))=47%), decreased oocyst survival (sr(2)=35%), using the weaning-vaccination schedule (sr(2)=7%) and vaccination versus non-vaccination (sr(2)=5%). Unexpectedly, the initial T. gondii prevalence in cats had no effect on T. gondii prevalence in finishing pigs. The simulation supports the field trial indicating vaccine effectiveness. However, vaccination had less impact on decreasing T. gondii infection in finishing pigs than a decrease in the number of farm cats.

Restriction fragment length polymorphism (RFLP) analysis and partial-genome DNA sequencing are commonly used to infer genetic relationships among pathogens. This study compares the application of both techniques to the analysis of 16 pseudorabies virus isolates collected during a 1989 outbreak. Genetic distances derived from RFLP and DNA sequence data were not significantly correlated with geographic distances between farms from which isolates were collected. RFLP-based genetic distance was, however, strongly correlated with temporal distance between isolates (days separating time of isolation). Sequence-based genetic distance was significantly correlated with temporal distance only when synonymous changes (nucleotide changes not leading to amino acid changes) were considered separately. Conversely, non-synonymous changes were correlated with the host species of origin of the viral isolate. These results indicate that selectively-neutral genetic changes most accurately reflect historical relationships, but that non-neutral changes most accurately reflect the biological environment of the viral isolate (e.g. host immune system).

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.