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Faecal contamination of carcasses in the slaughterhouse is generally considered to be the source of Salmonella on pork. In this study the hygiene indicator Escherichia coli is used to quantify faecal contamination of carcasses and it is hypothesized that it can be used to predict the quantitative carcass contamination with Salmonella, when the distribution of Salmonella concentrations in faeces is known. Paired pig sample data (faecal samples and carcass swabs) were obtained from five slaughterhouses and analysed for prevalence and concentrations of E. coli and Salmonella. A simple model was developed to describe the faecal contamination of carcasses using the E. coli data. The E. coli results suggested different hygiene performances in different slaughterhouses, and showed that a model assuming that carcasses are predominantly contaminated by their own faeces was not appropriate. Observed Salmonella prevalences were low (on average 1.9% on carcasses) and between slaughterhouses the prevalences ranked differently than the hygiene performance based on the E. coli data suggested. Also, the Salmonella concentrations predicted using E. coli as a faecal indicator were lower than the observed Salmonella concentrations. It is concluded that the faecal carriage of Salmonella together with the faecal contamination of carcasses, as predicted from E. coli data in the animal faeces and hygiene performance of the slaughterhouse, is not sufficient to explain carcass contamination with Salmonella. Our extensive data set showed that other factors than the observed faecal carriage of Salmonella by the individual animals brought to slaughter, play a more important role in the Salmonella carcass contamination of pork.

Impacts • For large slaughterhouses, post-harvest interventions might be cost- effective to achieve a low Salmonella prevalence in pork. However, for small-to-medium sized slaughterhouses, pre-harvest measures might be more feasible. • We describe an example of a Salmonella surveillance-and-control programme using data from the Danish island of Bornholm. • Our analysis shows that it is possible to obtain a low prevalence of Salmonella in pork produced within the programme. Summary Salmonella in pork can be combated during pre- or post-harvest. For large slaughterhouses, post-harvest measures like decontamination might be cost- effective while this is less likely with small-to-medium sized slaughterhouses. In this study, pre-harvest measures might be more relevant. We describe an extended surveillance-and-control programme for Salmonella in finisher pigs, which, to establish equivalence to the Swedish control programme, is intended for implementation on the Danish island, Bornholm. The effect of the pro- gramme on food safety was estimated by analysing Salmonella data from pig carcasses originating from herds that would have qualified for the programme during 2006-2008. Food safety was interpreted as prevalence of Salmonella on carcasses as well as the estimated number of human cases of salmonellosis related to pork produced within the programme. Data from the Danish Salmo- nella programme were obtained from Bornholm. We used a simulation model developed to estimate the number of human cases based on the prevalence of Salmonella on carcass swabs. Herds are only accepted in the programme if they have one or less seropositive sample within the previous 6 months. In this way, the Salmonella load is kept to a minimum. The programme is not yet in opera- tion and pigs that qualify for the programme are currently mixed at slaughter with those that do not qualify. Therefore, we had to assess the impact on the carcass prevalence indirectly. The prevalence of Salmonella in carcass swabs among qualifying herds was 0.46% for the 3 years as a whole, with 2006 as the year with highest prevalence. According to the simulation the expected number of human cases relating to pork produced within the programme was below 10. When the programme is in operation, an extra effect of separating pigs within the programme from those outside is expected to lower the prevalence of Salmonella even further.

This study evaluated the preparation of Shewhart's control charts using the concept of rational subgroups for monitoring the Salmonella antibody ELISA used for surveillance of Danish pig herds. Control charts were prepared for a buffer control sample, a negative serum sample and a positive serum sample. The quality control variables were the natural logarithm (In) of the uncalibrated optical density (OD) for the buffer control sample and the negative serum sample, and the calibrated OD (OD%) for the positive serum sample. Testing round (run) within laboratory robot was chosen as the subgroup, and separate control charts were prepared for five robots. The control limits were set at six times the standard deviation for In(OD) and three times the standard deviation for OD%. Evaluation based on a number of sensitising rules for control charts produced from historical data showed that use of the control charts could reveal systematic analytical errors.

The movements of animals were analysed under the conceptual framework of graph theory in mathematics. The swine production related premises of Denmark were considered to constitute the nodes of a network and the links were the animal movements. In this framework, each farm will have a network of other premises to which it will be linked. A premise was a farm (breeding, rearing or slaughter pig), an abattoir or a trade market. The overall network was divided in premise specific subnets that linked the other premises from and to which animals were moved. This approach allowed us to visualise and analyse the three levels of organization related to animal movements that existed in the Danish swine production registers: the movement of animals between two premises, the premise specific networks, and the industry network. The analyses of animal movements were done using these three levels of organisation. The movements of swine were studied for the period September 30, 2002 to May 22, 2003. For daily movements of swine between two slaughter pig premises, the median number of pigs moved was 130 pigs with a maximum of 3306. For movements between a slaughter pig premise and an abattoir, the median number of pigs was 24. The largest percentage of movements was from farm to abattoir (82.5%); the median number of pigs per movement was 24 and the maximum number was 2018. For the whole period the median and maximum Euclidean distances observed in farm-to-farm movements were 22 km and 289 km respectively, while in the farm-to-abattoir movements, they were 36.2 km and 285 km. The network related to one specific premise showed that the median number of premises was mainly away from slaughter pig farms (3) or breeder farms (26) and mainly to an abattoir (1535). The assumption that animal movements can be randomly generated on the basis of farm density of the surrounding area of any farm is not correct since the patterns of animal movements have the topology of a scale-free network with a large degree of heterogeneity. This supported the opinion that the disease spread software assuming homogeneity in farm-to-farm relationship should only be used for large-scale interpretation and for epidemic preparedness. The network approach, based on graph theory, can be used efficiently to express more precisely, on a local scale (premise), the heterogeneity of animal movements. This approach, by providing network knowledge to the local veterinarian in charge of controlling disease spread, should also be evaluated as a potential tool to manage epidemics during the crisis. Geographic information systems could also be linked in the approach to produce knowledge about local transmission of disease.

A method for quantitative evaluation of surveillance for disease freedom has been presented in the accompanying paper (Martin et al., 2007). This paper presents an application of the methods, using as an example surveillance for classical swine fever (CSF) in Denmark in 2005. A scenario tree model is presented for the abattoir-based serology component of the Danish CSF surveillance system, in which blood samples are collected in an ad hoc abattoir sampling process, from adult pigs originating in breeding herds in Denmark. The model incorporates effects of targeting (differential risk of seropositivity) associated with age and location (county), and disease clustering within herds. A surveillance time period of one month was used in the analysis. Records for the year 2005 were analysed, representing 25,332 samples from 3528 herds; all were negative for CSF-specific antibodies. Design prevalences of 0.1-1% of herds and 5% of animals within an infected herd were used. The estimated mean surveillance system component (SSC) sensitivities (probability that the SSC would give a positive outcome given the animals processed and that the country is infected at the design prevalences) per month were 0.18, 0.63 and 0.86, for among-herd design prevalences of 0.001, 0.005 and 0.01. The probabilities that the population was free from CSF at each of these design prevalences, after a year of accumulated negative surveillance data, were 0.91, 1.00 and 1.00. Targeting adults and herds from South Jutland was estimated to give approximately 1.9, 1.6 and 1.4 times the surveillance sensitivity of a proportionally representative sampling program for these three among-herd design prevalences.

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of antibodies against Actinobacillus pleuropneumoniae (Ap) serotypes 2, 6 and 12. The assay was designated MIX-ELISA. Lipopolysaccharide (LPS) from Ap serotypes 2, 6 and 12 was purified using hot phenol-water extraction followed by fractionation by size-exclusion chromatography. A mixture of fractions containing molecules with molecular weight above 50 kDa from all three serotypes was used as antigen. The MIX-ELISA was evaluated with sera from pigs experimentally infected with the serotypes 1, 2, 5b, 6, 7, 8, 10 and 12 of Ap biotype 1. In addition to reaction with sera from pigs inoculated with Ap serotypes 2, 6 and 12, reaction was observed with sera from pigs inoculated with serotype 8. Furthermore, the sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6 or 12 and with sera from herds free of infection with any Ap serotype of biotype 1. The ELISA showed a high herd sensitivity (0.98; 95% confidence interval: 0.89-1.00) and specificity (0.95; 0.88-0.99). The high diagnostic sensitivity and specificity of the assay indicate that screening of herds for Ap infection can be performed using this ELISA. Efficient serological surveillance can be achieved by using such mixed antigen ELISAs coated with size-selected LPS-antigens from the most prevalent serotypes.

The objectives of this study were to elucidate at which age tonsillar colonisation by Actinobacillus pleuropneumoniae occurs in pigs and relate this occurrence to the presence of colostral antibodies to A. pleuropneumoniae. The infection patterns were studied in an isolated cohort of pigs, which consisted of the offspring from five sows originating from a conventional pig herd. The sows were transferred to isolated research facilities before farrowing. A. pleuropneumoniae was detected on the tonsils of all sows. After a nursing period of 3 weeks, the pigs were weaned and reared isolated from other pigs until slaughter. The pigs were examined repeatedly for the presence of A. pleuropneumoniae on the tonsils and for antibodies to A. pleuropneumoniae using bacteriological and serological techniques, respectively.A. pleuropneumoniae was detected in the tonsils of one pig as early as 11 days after birth, showing that A. pleuropneumoniae can be transmitted from sow to offspring during a 3-week nursing period. The cumulative proportion of pigs carrying A. pleuropneumoniae in their tonsils increased significantly between the age of 4-12 weeks. This age period corresponded to the age at which the proportion of pigs with detectable levels of colostral antibodies to the different serotypes of A. pleuropneumoniae was declining. Since these two events take place in the same age period, we expect a possible biological association between the level of the passive immunity and the degree of tonsillar colonisation. The median duration of tonsillar colonisation was estimated to approximately 7-8 weeks.

The objective was to develop a blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Actinobacillus pleuropneumoniae (Ap) serotype 12 in pig serum. Lipopolysaccharide (LPS) from Ap serotype 12 was purified and used as antigen in the assay. Antibodies to the LPS antigen in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotype 12 infection. The blocking ELISA showed no cross-reaction when tested with sera from pigs experimentally infected with 12 other serotypes of Ap biotype 1. The sensitivity and specificity of the blocking ELISA on the herd level was evaluated by testing sera from pig herds naturally infected with Ap serotypes 2 and/or 12 and from herds declared free of infection with Ap. The Ap serotype 12 blocking ELISA showed a herd sensitivity of 0.77 (95% confidence interval, 0.62-0.88) and a herd specificity of 1.00 (0.95-1.00) with a cut-off value at 40% relative absorbance or 60% inhibition. The assay may be used advantageously as a confirmatory test in serological surveillance programmes for Ap infections in SPF systems for pig production.

Surveillance programmes based on laboratory screening tests are increasingly used to document freedom from disease in order to facilitate trade. The following aspects must be considered when designing such programmes: diseases to be selected; epidemiology of the diseases; unit of analysis (animal or herd); target age group (or target farm type); test characteristics and sample size. Issues related to these aspects are discussed and illustrated using the example of serological surveillance for exotic viral diseases in the pig population of Denmark. Sampling designs based on individual animal samples are compared with herd-based sampling (two-stage sampling). While the latter is likely to require a larger sample size, the increased level of information and the reliability of the results obtained are considered to be worth the expense. Issues related to the development of international standards for declaring freedom from disease are discussed. The authors conclude that international standards are desirable, providing that these standards represent scientifically valid principles.

Background We know from previous studies that increasing age, abnormal vital signs and abnormal acid-base status are strongly associated with in-hospital mortality in unselected patients admitted acutely to hospital. A model including this information will make us able to explore associations and predict the risk for future patients. Our aim was to describe a Bayesian model for prediction of adverse outcome in the acute ill adult patient admitted to hospital, based on already existing data from the ‘Acute Admission Database’. Methods The model is as tatic Bayesian network, i.e. as tochastic model where all interdependence is described by conditional probabilities. The net consists of nodes representing variables and pointed arrows of influence. The probabilities connected to the nodes and arrows are conditional probabilities showing how the state of a variable influences the probability distribution for the states of another variable. We based the model on already existing data from the ‘Acute Admission Database’ and imported data from 6279 patients consecutively admitted to Hillerod Hospital through the Emergency Department into the Bayesian net program, Netica “3.7” © Norsys Software Corp. We included the risk factors identified in this cohort in previous studies as nodes, and represented the known associations with directed arrows. Results We tested the use of the model by simulating the path of an acutely ill patient: a male patient, 70 years old and presenting with vomiting blood. By using this evidence in the nodes of relevance, we could assess the most probable distribution of the other nodes, including the outcome of interest. We simulated that more data became available for instance vitals signs and triage categories. This new evidence changed the nodes and finally we entered information about a venous blood gas, which changed the probability distribution of the outcome measures as more evidence was gained. Conclusion By using already existing data, we were able to build a Bayesian network, which can be used to estimate the risk of adverse outcome and serve as a decision support system in assessing future patients admitted acutely to hospital.

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).

The aim of the present study was to evaluate ribotyping as an epidemiological tool for Actinobacillus pleuropneumoniae and apply the method in studies of A. pleuropneumoniae infections in Danish pig herds. The evaluation of ribotyping was based on the 13 international reference strains and 106 epidemiologically unrelated Danish field strains representing the nine serotypes of biotype 1 (1, 2, 5A/B, 6, 7, 8, 10, 12, and K2:O7) and one serotype 14 of biotype 2. Enzymes Cfo I and Hind III were chosen for generation of ribotype patterns. Ribotyping of the reference strains resulted in 10 Cfo I types and 11 Hind III types. Ribotyping of the Danish strains resulted in 17 different Cfo I ribotypes and 24 different Hind III ribotypes. Combining Hind III- and Cfo I-ribotyping divided the Danish strains into 26 different types. The stability, reproducibility and typability of ribotype patterns were good, and the discriminatory power was between 0.85–0.89. The relatively low discriminatory power was caused by four predominant types, containing 61% of the isolates. The typing system was applied in studies of routes of infection of specific pathogen-free (SPF) pig herds and included 112 strains of A. pleuropneumoniae . Airborne transmission from neighboring conventional pig farms was investigated in 12 cases of infected SPF herds. Transmission via vehicles transporting pigs between SPF herds was investigated in nine cases while transmission by trading of pigs between SPF herds was investigated in two cases. Serotype 2 was isolated from all SPF herds included in this study, except one, emphasizing the high prevalence of this serotype in Denmark. By ribotyping, airborne transmission was indicated in five of 12 cases, transmission via pig transporting vehicle was indicated in six of nine cases, and transmission via trading was indicated in one of two cases. In many cases findings of predominant ribotypes made interpretations of suspected routes of transmission difficult. The relationship of strains based on ribotypes was calculated using Dices coefficient and clustered by UPGMA. Hind III ribotypes of serotype 2 strains were closely related, though only showing 43% similarity to Hind III ribotypes of remaining serotypes.

200 SPF pigs were infected by aerosol with Mycoplasma hyopneumoniae and the development of clinical signs, serological and pathological reactions were studied. Mean time to onset of coughing was 13 days. A mean delay of 9 days was observed from onset of coughing until seroconversion against M. hyopneumoniae as measured by ELISA. At an individual level, the sensitivity for this ELISA was estimated to 98-100% and the specificity to 93-100%. Pasteurella multocida was isolated from the majority of the lungs 4 weeks post inoculation with M. hyopneumoniae and the lung lesions in pigs were significantly larger when P. multocida was present as compared to pigs with M. hyopneumoniae alone. An evaluation of cultivation, immunofluorescence, ELISA and polymerase chain reaction for demonstration of M. hyopneumoniae in lungs showed that all four methods have a high sensitivity in the acute stages of pneumonia. In the later stages the sensitivity of cultivation was superior to the other methods. No differences in specificity were observed between the methods. The antigen-ELISA OD values and the immunofluorescence scores revealed a strong positive correlation. Nasal swabs were additionally used for demonstration of M. hyopneumoniae and the polymerase chain reaction was found superior to the other methods.

A monoclonal antibody-based blocking enzyme-linked immunosorbent assay (ELISA) was used for serological surveillance of Mycoplasma hyopneumoniae infection in pig herds. A follow-up study was conducted on "herd predictive values" previously reported for this ELISA. Of those herds giving positive results by this ELISA, 42% were subsequently found to be infected, while 100% of herds giving negative results were uninfected. Previous reports recorded positive and negative herd predictive values of 39% and 99.8%, respectively. Among naturally-infected animals, reaction in colostrum was more frequent than in serum, and this difference was most pronounced if the colostrum samples were obtained shortly before or after farrowing. Coughing was found to be the most reliable clinical indicator of infection, but surveillance through clinical herd inspections alone failed to detect 30% of infected herds. The time required for seroconversion following natural exposure to M. hyopneumoniae differed in two herds using different management systems: in one herd antibodies were first detected three weeks post-exposure, while in the other herd antibodies were not detected until five weeks after exposure.