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A national screening in 2016 identified 88% of Danish pig herds positive for livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA). This highlights the importance of evaluating potential control measures that could reduce the prevalence of LA-MRSA among Danish pig herds. In addition to describing the effects of (1) reduced within-herd transmission, (2) increased biosecurity, and (3) movement restrictions, the eradication of LA-MRSA as a potential control measure was investigated using a simulation model mimicking the spread of LA-MRSA among pig herds between 2006 and 2015. The latter strategy was simulated either as eradication of a random selection of herds for surveillance or as a risk-based selection of herds based on their potential to spread LA-MRSA via pig movements in four different scenarios: low- vs. high-prevalence scenarios with control measures starting in 2007 and in 2010. Almost all control measures showed the potential to reduce the spread of LA-MRSA among pig herds, especially when implemented intensively and when control measures were combined. Risk-based selection of herds for eradication led to a greater relative reduction compared to random selection. In the high-prevalence scenario in particular, combinations including risk-based eradication led to the greatest relative reduction.

Mastitis, often caused by intramammary infection (IMI), is a significant problem in dairy farming globally. Somatic cell count (SCC) is widely used as a parameter for screening IMI in cows that are then treated or culled. We investigated the potential of a new parameter, differential SCC (DSCC), to detect IMI at cow level when SCC is already known. We achieved this using bacterial culture (BC) and PCR to detect 4 categories of pathogens (major, minor, other, and any) in 2 Danish dairy herds. Quarter milk samples were collected from monthly dairy herd improvement samplings over 1 yr and analyzed with BC, whereas cow-level dairy herd improvement samples were analyzed using PCR. Days in milk, parity, and IMI status had a significant effect on DSCC. Using DSCC in addition to SCC significantly improved the indication of IMI compared with using only SCC in the any pathogen category in both herds as well as the minor pathogens category in herd 2 when BC was used for detection. When PCR was used to detect IMI, the use of DSCC in addition to SCC was significant for the other pathogens category in herd 1 and the minor pathogens category in herd 2. Thus, our data revealed that DSCC can add significant information describing IMI status even when SCC is already known; however, this depends on the causative pathogen. Future studies may address how to use DSCC in practice as well as consider the availability of temporal data to potentially gain insight into the course of infection.

The possible spillover from pigs into other production animals incites concern for unresolved reservoirs of human exposure. The present investigation was therefore initiated, to elucidate if Danish veal and dairy farms constitute a reservoir of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) CC398 and to potentially identify the source of introduction. We collected nasal swab samples from 17 Danish veal farms, 2 slaughterhouses, and received bulk tank milk samples from 286 dairy farms. All samples were analyzed by culturing and screening on MRSA selective plates and presumed MRSA was verified by MALDI-TOF and PCR. MRSA isolates were subjected to spa typing and whole-genome sequencing. LA-MRSA was found on two veal farms in one and three calves, respectively, with subsequent follow-up samples found negative. Eight of 286 dairy farms (2.8%) were found LA-MRSA positive and follow-up samples, from five farms showed intermittent detection of LA-MRSA. The spa types, t034 and t011, were the most common while a single isolate from a dairy farm belonged to spa type t843 associated to mecC-MRSA CC130 and is the first report of mecC-MRSA in the Danish dairy production. A phylogenetic analysis showed that some of the isolates grouped within or close to the dominant Danish pig clusters, suggesting spillover into cattle farms. Other isolates clustered outside the dominant pig clusters suggesting that other routes of introduction cannot be excluded. Results of the investigation indicated a contamination of veal farms while some dairy farms seemed to be a permanent reservoir. Thus, Danish cattle represent a low prevalence reservoir of LA-MRSA CC398, which at present, is not of major human health concern.

Bacterial antimicrobial resistance (AMR) in pigs is an important public health concern due to its possible transfer to humans. We aimed at quantifying the relationship between the lifetime exposure of antimicrobials and seven antimicrobial resistance genes in Danish slaughter pig farms. AMR gene levels were quantified by qPCR of total-community DNA in faecal samples obtained from 681 batches of slaughter pigs. The lifetime exposure to antimicrobials was estimated at batch level for the piglet, weaner, and finisher periods individually for the sampled batches. We showed that the effect of antimicrobial exposure on the levels of AMR genes was complex and unique for each individual gene. Several antimicrobial classes had both negative and positive correlations with the AMR genes. From 10–42% of the variation in AMR gene levels could be explained in the final regression models, indicating that antimicrobial exposure is not the only important determinant of the AMR gene levels.

The aim of this study was to assess the potential of using multiple data sources currently available in Denmark for monitoring swine diseases. The study included farms that, based on serology, changed from "negative" to "positive" status for Porcine Reproductive and Respiratory Syndrome (PRRS), enzootic pneumonia (Mycoplasma hyopneumonia), and porcine pleuropneumonia (Actinobacillus pluropneumoniae) between January 2014 and September 2017. These corresponded to 45 swine farms working as individual operation units (i.e., their disease status is independent from other farms) and 81 farms that were part of joint operation units (i.e., 2 or more farms considered to be an epidemiological unit, having swine and personnel are transferred among them, that have the same disease status). Additionally, a total of 95 farms with a negative status for these three diseases during the study period were also included in the study. Changes in mortality data, antimicrobial consumption, and vaccine use at herd level were monitored using Shewhart control charts prior to, during, and after these farms were found positive for the three diseases. The analysis was run separately for the different age groups-weaners (up to 30 kg), sows and finishers herds-within each farm. Briefly, the highest percentage of herds generating alarms was generated up to 3 months before they changed their disease status based on mortality (30%) and 1 month after based on antimicrobial use for respiratory diseases (100%). Porcine pleuropneumonia showed to be the disease with the highest impact on these data at herd level; alarms based on the three data streams were generated in the same month that herds changed their status to porcine pleuropneumonia-positive, as well as the following months. Alarms based on vaccine use generally occurred within the same month or after changes in disease status. False alarms were found in 2% (median value) of the herds for the different age groups based on mortality and antimicrobial use for respiratory diseases in healthy farms. Monitoring changes in mortality data, antimicrobial consumption, and vaccine use showed changes (i.e., warnings) at herd level prior to confirmation from diagnostic tests.

Vancomycin-resistant Enterococcus spp. is a major health problem worldwide and livestock have been implicated in constituting a reservoir for the transmission of vancomycin resistance to zoonotic pathogens. Vancomycin resistance determinants can be situated on mobile genetic elements and transferred between bacterial species The livestock reservoir must therefore be included in a risk assessment of the vancomycin resistance burden. Avoparcin, a vancomycin analogue, has not been used in Danish pig production for over 20 years and vancomycin has never been used. The objective of this study was to screen faecal samples from Danish pig farms for nine selected vancomycin resistance determinants. We found at least four different vancomycin resistance determinants in all screened Danish pig farms (665 finisher farms and 78 sow farms). The vancomycin resistance determinants present in vanB or vanG clusters were found at significantly different levels in sow and finisher farms. However, vanA was not detected in any of the farms. In conclusion, vancomycin resistance determinants are still present in Danish pig production 25 years after the ban on avoparcin use.

Background The modern dairy industry routinely generates data on production and disease. Therefore, the use of these cheap and at times even “free” data to predict a given state of welfare in a cost-effective manner is evaluated in the present study. Such register data could potentially be used in the identification of herds at risk of having animal welfare problems. The present study evaluated the diagnostic performance of four routinely registered indicators for identifying herds with high lameness prevalence among 40 Danish dairy herds. Indicators were extracted as within-herd annual means for a one-year period for cow mortality, bulk milk somatic cell count, proportion of lean cows at slaughter and the standard deviation (SD) of age at first calving. The target condition “high lameness prevalence” was defined as a within-herd prevalence of lame cows of ≥ 16% (third quartile). Diagnostic performance was evaluated by constructing and analysing Receiver Operating Characteristic curves and their area under the curve (AUC) for single indicators and indicator combinations. Sensitivity (Se) and specificity (Sp) of the indicators were assessed at the optimal cut-off based on data and compared to a set of predefined cut-off levels (national annual means or 90-percentile). Results Cow mortality had the highest AUC (0.76), while adding the three other indicators to the model did not yield significant increase in AUC. Cow mortality and SD of age at first calving had highest Se (100%, 95% confidence interval (CI): 72–100%), while highest Sp was found for the proportion of lean cows at slaughter (83%, 95% CI: 66–93%). The highest differential positive rate (DPR = 0.53) optimizing both Se and Sp was found for cow mortality. Optimal cut-off points were lower than the presently used pre-defined cut-offs. Conclusions The selected register-based indicators proved to be able to identify herds with high lameness prevalences. Optimized cut-offs improved the predictive ability and should therefore be preferred in official control schemes.

Precise estimates of disease transmission rates are critical for epidemiological simulation models. Most often these rates must be estimated from longitudinal field data, which are costly and time-consuming to conduct. Consequently, measures to reduce cost like increased sampling intervals or subsampling of the population are implemented. To assess the impact of such measures we implement two different SIS models to simulate disease transmission: A simple closed population model and a realistic dairy herd including population dynamics. We analyze the accuracy of different methods for estimating the transmission rate. We use data from the two simulation models and vary the sampling intervals and the size of the population sampled. We devise two new methods to determine transmission rate, and compare these to the frequently used Poisson regression method in both epidemic and endemic situations. For most tested scenarios these new methods perform similar or better than Poisson regression, especially in the case of long sampling intervals. We conclude that transmission rate estimates are easily biased, which is important to take into account when using these rates in simulation models.

Due to an increased incidence of human infections, livestock-associated methicillin-resistant Staphylococcus Aureus (LA-MRSA) in pigs and its spread into the human population has been a major public and political issue in Denmark. Similar concerns are also being raised about LA-MRSA in other Western European countries. At a time when the proportion of LA-MRSA-positive pig farms was low, Norway adopted a ‘trace and destroy’ strategy aimed at keeping LA-MRSA out of the pig population. However, to date, no country with a high proportion of LA-MRSA-positive pig herds has chosen to use an eradication strategy. This study analyses the cost and complexities of conducting an LA-MRSA eradication program in a situation where a large proportion of herds are positive. The total cost of the eradication program was estimated based on the following components: 1) planning, 2) monitoring and testing, 3) cleaning and disinfection, 4) production gains and losses, 5) net reduction in breeding exports, and 6) loss of genetic progress, including the costs of a mitigating caesarean section strategy in breeding herds. Costs were related to the depopulation of 1 million sows, to gilt supply (as this was an important limiting factor during an eradication program in Denmark), and to aggregated losses linked to a temporary halt on breeding progress. Using conservative assumptions, the total eradication costs were estimated at €1.8 billion, broken down into: planning costs (3%), monitoring and testing (6%), cleaning and disinfection (19%), production gains and losses (33%), net loss from breeding exports (19%) and loss of genetic progress (20%). The long-term effects of an LA-MRSA eradication program for Danish pig production were uncertain and were therefore not taken into account in the analysis. Due to an increased incidence of human infections, livestock-associated methicillin-resistant Staphylococcus Aureus (LA-MRSA) in pigs and its spread into the human population has been a major public and political issue in Denmark. Similar concerns are also being raised about LA-MRSA in other Western European countries. At a time when the proportion of LA-MRSA-positive pig farms was low, Norway adopted a ‘trace and destroy’ strategy aimed at keeping LA-MRSA out of the pig population. However, to date, no country with a high proportion of LA-MRSA-positive pig herds has chosen to use an eradication strategy. This study analyses the cost and complexities of conducting an LA-MRSA eradication program in a situation where a large proportion of herds are positive. The total cost of the eradication program was estimated based on the following components: 1) planning, 2) monitoring and testing, 3) cleaning and disinfection, 4) production gains and losses, 5) net reduction in breeding exports, and 6) loss of genetic progress, including the costs of a mitigating caesarean section strategy in breeding herds. Costs were related to the depopulation of 1 million sows, to gilt supply (as this was an important limiting factor during an eradication program in Denmark), and to aggregated losses linked to a temporary halt on breeding progress. Using conservative assumptions, the total eradication costs were estimated at €1.8 billion, broken down into: planning costs (3%), monitoring and testing (6%), cleaning and disinfection (19%), production gains and losses (33%), net loss from breeding exports (19%) and loss of genetic progress (20%). The long-term effects of an LA-MRSA eradication program for Danish pig production were uncertain and were therefore not taken into account in the analysis.

Societal pressure to limit the use of antibiotics in livestock production systems, including dairy cattle systems, is consistently increasing. To motivate farmers to reduce antibiotic usage, it is important to understand the factors that determine whether a cow will be treated with antibiotics or not. If farmers' usual practices regarding antibiotic treatments are taken into account, they may be motivated to adopt control measures that can facilitate prudent use of antibiotics and are at the same time cost-effective. In this study, we analyzed database recordings of milk yield and somatic cell count from the routine milk recording scheme, clinical registrations of mastitis and PCR results, and cow factors such as days in milk and parity in relation to antibiotic treatments for 518 dairy herds in Denmark. Farm-wise logistic regressions were used to predict antimicrobial treatment based on these factors. The resulting regression coefficients of 422 herds were further analyzed by principal component analysis and clustering to determine the driving predictors for treatment in different groups of farms. The results showed that determinants that were most important for predicting antibiotic treatments vary from one farm to another. Health indicators such as PCR or somatic cell count were most indicative for treatment on some farms, whereas other groups seemed to depend more on production factors (milk yield) or later culling of the cows. This shows that farmers behave differently and differences can be identified in register data. This information can be considered when developing cost-effective herd-specific control measures of mastitis to promote prudent use of antibiotics in Danish dairy cattle farms.

Background: The acute phase proteins (APP) serum amyloid A (SAA), haptoglobin, and fibrinogen are valuable blood bi-omarkers in equine inflammatory diseases, but knowledge of factors influencing their concentrations in blood and peritonealfluid (PF) of horses with colic is needed.Objectives: The objective of this study was to investigate the influence of demographics (age, sex, breed), disease process (sim-ple obstruction, strangulating obstruction, inflammatory), disease location, disease duration, hypovolemia, and admission hospi-tal on concentrations of APP, lactate and white blood cell counts (WBC) in horses with colic admitted to 2 referral hospitals.Animals: The study included 367 horses with colic admitted at 2 referral hospitals.Methods: Prospective multicenter observational study of clinical data, as well as blood and PF biomarkers. Associationsbetween biomarker concentrations and clinical variables were analyzed using multivariate linear regression analysis.Results: Increasing pre-admission duration of colic was associated with increased concentrations of APP in blood andPF. Blood concentrations of SAA and fibrinogen were associated with disease process (inflammatory, strangulations, simpleobstructions) in more colic duration groups (5–12 and >24 hours) than any of the other biomarkers. No relevant associationsbetween demographic factors, hospital, or hydration status and the measured biomarkers were found.Conclusions and Clinical Importance: In horses with colic, concentrations of APP are associated mainly with diseaseprocess and duration of colic and may thus be used for assessment of disease independently of demographic or geographicfactors. Serum amyloid A may be a diagnostic marker for use in colic differential diagnosis, but further evaluation is needed.Key words: Fibrinogen; Haptoglobin; Peritoneal fluid; Serum amyloid A.

SUMMARYAntimicrobial resistance (AMR) in pig populations is a public health concern. There is a lack of information of spatial distributions of AMR genes in pig populations at large scales. The objective of the study was to describe the spatial pattern of AMR genes in faecal samples from pig farms and to test if the AMR genes were spatially randomly distributed with respect to the geographic distribution of the pig farm population at risk. Faecal samples from 687 Danish pig farms were collected in February and March 2015. DNA was extracted and the levels of seven AMR genes (ermB, ermF, sulI, sulII, tet(M), tet(O) and tet(W)) were quantified on a high-throughput real-time PCR array. Spatial differences for the levels of the AMR genes measured as relative quantities were evaluated by spatial cluster analysis and creating of risk maps using kriging analysis and kernel density estimation. Significant spatial clusters were identified for ermB, ermF, sulII and tet(W). The broad spatial trends in AMR resistance evident in the risk maps were in agreement with the results of the cluster analysis. However, they also showed that there were only small scale spatial differences in the gene levels. We conclude that the geographical location of a pig farm is not a major determinant of the presence or high levels of AMR genes assessed in this study.

Background:Seizures are common in dogs and can be caused by non-epileptic conditions or epilepsy. The clinical course of newly diagnosed epilepsy is sparsely documented.The objective of this study was to prospectively investigate causes for seizures (epileptic and non-epileptic) in a cohort of dogs with new-onset untreated seizures, and for those dogs with newly diagnosed epilepsy to investigate epilepsy type, seizure type and the course of disease over time, including the risk of seizure recurrence. Untreated client-owned dogs experiencing new-onset seizures were prospectively enrolled in a longitudinal observational study including clinical investigations and long-term monitoring at the Copenhagen University Hospital for Companion Animals. A baseline clinical assessment was followed by investigator/owner contact every eight weeks from inclusion to death or end of study. Inclusion of dogs was conducted from November 2010 to September 2012, and the study terminated in June 2014.Results:One hundred and six dogs were included in the study. Seventy-nine dogs (74.5%) were diagnosed with epilepsy: 61 dogs (77.2%) with idiopathic epilepsy, 13 dogs (16.5%) with structural epilepsy and five dogs (6.3%) with suspected structural epilepsy. A non-epileptic cause for seizures was identified in 13 dogs and suspected in 10 dogs. Four dogs in which no cause for seizures was identified experienced only one seizure during the study. In dogs with idiopathic epilepsy 60% had their second epileptic seizure within three months of seizure onset. Twenty-six dogs with idiopathic epilepsy (43%) completed the study without receiving antiepileptic treatment. The natural course of idiopathic epilepsy (uninfluenced by drugs) was illustrated by highly individual and fluctuating seizure patterns, including long periods of remission. Cluster seizures motivated early treatment. In a few dogs with a high seizure frequency owners declined treatment against the investigators advice.Conclusions:Epilepsy is the most likely diagnosis in dogs presenting with new-onset seizures. The course of idiopathic epilepsy is highly individual and might not necessarily require long-term treatment. This must be considered when advising owners about what to expect with regard to treatment and prognosis. BackgroundSeizures are common in dogs and can be caused by non-epileptic conditions or epilepsy. The clinical course of newly diagnosed epilepsy is sparsely documented.The objective of this study was to prospectively investigate causes for seizures (epileptic and non-epileptic) in a cohort of dogs with new-onset untreated seizures, and for those dogs with newly diagnosed epilepsy to investigate epilepsy type, seizure type and the course of disease over time, including the risk of seizure recurrence. Untreated client-owned dogs experiencing new-onset seizures were prospectively enrolled in a longitudinal observational study including clinical investigations and long-term monitoring at the Copenhagen University Hospital for Companion Animals. A baseline clinical assessment was followed by investigator/owner contact every eight weeks from inclusion to death or end of study. Inclusion of dogs was conducted from November 2010 to September 2012, and the study terminated in June 2014.ResultsOne hundred and six dogs were included in the study. Seventy-nine dogs (74.5%) were diagnosed with epilepsy: 61 dogs (77.2%) with idiopathic epilepsy, 13 dogs (16.5%) with structural epilepsy and five dogs (6.3%) with suspected structural epilepsy. A non-epileptic cause for seizures was identified in 13 dogs and suspected in 10 dogs. Four dogs in which no cause for seizures was identified experienced only one seizure during the study. In dogs with idiopathic epilepsy 60% had their second epileptic seizure within three months of seizure onset. Twenty-six dogs with idiopathic epilepsy (43%) completed the study without receiving antiepileptic treatment. The natural course of idiopathic epilepsy (uninfluenced by drugs) was illustrated by highly individual and fluctuating seizure patterns, including long periods of remission. Cluster seizures motivated early treatment. In a few dogs with a high seizure frequency owners declined treatment against the investigators advice.ConclusionsEpilepsy is the most likely diagnosis in dogs presenting with new-onset seizures. The course of idiopathic epilepsy is highly individual and might not necessarily require long-term treatment. This must be considered when advising owners about what to expect with regard to treatment and prognosis.

The aim of this study was to explore spatio-temporal mortality patterns in Danish swine herds from December 2013 to October 2015, and to discuss the use of mortality data for syndromic surveillance in Denmark. Although it has previously been assessed within the context of syndromic surveillance, the value of mortality data generated on a regular and mandatory basis from all swine herds remains unexplored in terms of swine surveillance in Denmark. A total of 5010 farms were included in the analysis, corresponding to 1896 weaner herds, 1490 sow herds and 3839 finisher herds. The spatio-temporal analysis included data description for spatial, temporal, and spatio-temporal cluster analysis for three age groups: weaners (up to 30kg), sows and finishers. Logistic regression models were used to assess the potential factors associated with finisher and weaner herds being included within multiple-herd clusters. The spatio-temporal distribution of mortality changed over time, and suggested a general increase in mortality for the months of January and July for the three age groups. A large number of single-herd clusters (i.e. clusters with only one herd), and fewer multiple-herd clusters (i.e. clusters with at least two herds) were found. The herd size affected whether weaner herds were within multiple-herd clusters, and factors such farm type, SPF status and presence of atrophic rhinitis had an impact on finisher herds being inside vs. outside multiple-herd clusters in the univariable analysis. However, due to a strong correlation between variables, only farm type remained in the multivariable analysis for the finisher herds. The higher mortality observed for the months of January and July could be linked to infrequent updates of the data used to calculate mortality. The presence of single-herd clusters might indicate welfare and disease issues, while multiple-herd clusters could suggest the presence of infectious diseases within the cluster area. The impact of farm type is linked to the fact that larger farms specialize in only one age group, with high biosecurity and more specialized personnel, and subsequently a lower mortality. Mortality data have a potential use in disease surveillance. However, detected clusters might not be due to disease, but the result of changes such as herd management practices. Further analysis to explore other spatio-temporal monitoring methods is needed before mortality data can be incorporated into a Danish disease monitoring system.

BACKGROUND: Paratuberculosis (PTB) is a chronic disease which may lead to reduced milk yield, lower animal welfare and death in cattle. The causative agent is Mycobacterium avium subsp. paratuberculosis (MAP). The economic consequences are particularly important incentives in the control and eradication of the infection. One strategy to control PTB in a herd is to purchase animals from farms with a low risk of MAP infection. We wanted to investigate the epidemiological and economic consequences of buying livestock from different supplier farms of low, medium or high risk, as well as farms with unknown status. We also wanted to estimate the probability of spontaneous fadeout if the farmer of an initially MAP-free herd bought a specified number of infected animals in a single year, or continually bought infected animals. This was achieved through simulation modeling, and the effects of consistently introducing one, five or ten infected animals annually into an initially infection-free herd was also modeled.RESULTS: Our findings show that once infected, a farm can relatively safely purchase animals from other low and medium-risk farms without experiencing an increase in the prevalence, highlighting the importance of certification programmes. Furthermore, farms free of MAP are highly susceptible and cannot purchase more than a small number of animals per year without having a high risk of being infected. The probability of spontaneous fadeout after 10 years was 82% when introducing a single infected animal into an initially MAP-free herd. When purchasing ten infected animals, this probability was 46%. The continual purchase of infected animals resulted in very low probabilities of spontaneous fadeout.CONCLUSIONS: We demonstrated that MAP-free farms can purchase a small number of animals, preferably from certified farms, each year and still remain free of MAP. Already infected farms have little risk of increasing the prevalence on a farm when purchasing animals from other farms.

Precise estimates of disease transmission rates are critical for epidemiological simulation models. Most often these rates must be estimated from longitudinal field data, which are costly and time-consuming to conduct. Consequently, measures to reduce cost like increased sampling intervals or subsampling of the population are implemented. To assess the impact of such measures we implement two different SIS models to simulate disease transmission: A simple closed population model and a realistic dairy herd including population dynamics. We analyze the accuracy of different methods for estimating the transmission rate. We use data from the two simulation models and vary the sampling intervals and the size of the population sampled. We devise two new methods to determine transmission rate, and compare these to the frequently used Poisson regression method in both epidemic and endemic situations. For most tested scenarios these new methods perform similar or better than Poisson regression, especially in the case of long sampling intervals. We conclude that transmission rate estimates are easily biased, which is important to take into account when using these rates in simulation models.

Testing large quantities of samples in order to detect one or more test-positive sample(s) is expensive and time-consuming. It is possible to optimize this process by pooling samples. Two frameworks to produce different hierarchical and non-hierarchical pooling schemes were tested and compared to standard pooling. Their efficiency and the potential savings were determined as a function of prevalence and the number of pooled samples.The potential benefit of pooling samples is dependent upon the changes in the analytical sensitivity and specificity of the test used when diluting test-positive samples by pooling. To illustrate this, the sensitivity of antibody ELISA on pooled samples of bovine milk for Salmonella Dublin, Mycobacterium avium spp. paratuberculosis, and bovine virus diarrhea was tested. For these milk assays, the analytical sensitivity decreased rapidly with increasing pool sizes.The efficiency of pooling is usually only measured by the number of tests performed, yet real savings depend on all the costs involved in the pooling process. These may differ between laboratories depending on the available equipment and the salaries of the technicians, among other factors. Therefore, several cost parameters were introduced to describe the total cost and thereby calculate the total savings. In terms of overall savings, both tested schemes were potentially optimal depending on the prevalence, possible pool size, and the cost of retesting. For the pool sizes of interest in this study, the three-stage hierarchical pooling scheme was often marginally more efficient in terms of the total number of tests. However, if the price of re-pooling was high, the two-stage scheme performed better in terms of total savings. In addition, for low prevalences and the possibility of pooling a large number of samples, the two-stage non-hierarchical test may be more efficient, both in terms of number of tests and overall cost. In order to apply these results in different laboratory settings, a free Shiny WebApp was developed, to compare several pooling schemes with different cost parameters.

A cross-sectional study design is relatively inexpensive, fast and easy to conduct when compared to other study designs. Careful planning is essential to obtaining a representative sample of the population, and the recommended approach is to use simple random sampling from an exhaustive list of units in the target population. This approach is rarely feasible in practice, and other sampling procedures must often be adopted. For example, when slaughter pigs are the target population, sampling the pigs on the slaughter line may be an alternative to on-site sampling at a list of farms. However, it is difficult to sample a large number of farms from an exact predefined list, due to the logistics and workflow of an abattoir. Therefore, it is necessary to have a systematic sampling procedure and to evaluate the obtained sample with respect to the study objective. We propose a method for 1) planning, 2) conducting, and 3) evaluating the representativeness and reproducibility of a cross-sectional study when simple random sampling is not possible. We used an example of a cross-sectional study with the aim of quantifying the association of antimicrobial resistance and antimicrobial consumption in Danish slaughter pigs. It was not possible to visit farms within the designated timeframe. Therefore, it was decided to use convenience sampling at the abattoir. Our approach was carried out in three steps: 1) planning: using data from meat inspection to plan at which abattoirs and how many farms to sample; 2) conducting: sampling was carried out at five abattoirs; 3) evaluation: representativeness was evaluated by comparing sampled and non-sampled farms, and the reproducibility of the study was assessed through simulated sampling based on meat inspection data from the period where the actual data collection was carried out. In the cross-sectional study samples were taken from 681 Danish pig farms, during five weeks from February to March 2015. The evaluation showed that the sampling procedure was reproducible with results comparable to the collected sample. However, the sampling procedure favoured sampling of large farms. Furthermore, both under-sampled and over-sampled areas were found using scan statistics. In conclusion, sampling conducted at abattoirs can provide a spatially representative sample. Hence it is a possible cost-effective alternative to simple random sampling. However, it is important to assess the properties of the resulting sample so that any potential selection bias can be addressed when reporting the findings.

Before an efficient control strategy for livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) in pigs can be decided upon, it is necessary to obtain a betterunderstanding of how LA-MRSA spreads and persists within a pig herd, once it is introduced.We here present a mechanistic stochastic discrete-event simulation model forspread of LA-MRSA within a farrow-to-finish sow herd to aid in this. The model was individual-based and included three different disease compartments: susceptible, intermittent or persistent shedder of MRSA. The model was used for studying transmission dynamics and within-farm prevalence after different introductions of LA-MRSA into a farm. The spread of LA-MRSA throughout the farm mainly followed the movement of pigs. After spread of LA-MRSA had reached equilibrium, the prevalence of LA-MRSA shedders was predicted to be highest in the farrowing unit, independent of how LA-MRSA was introduced. LA-MRSA took longer to spread to the whole herd if introduced in the finisher stable, rather than by gilts in the mating stable. The more LA-MRSA positive animals introduced, the shorter time before the prevalence in the herd stabilised. Introduction of a low number of intermittently shedding pigs was predicted to frequently result in LA-MRSA fading out. The model is a potential decision support tool for assessments of short and long term consequences of proposed intervention strategies or surveillance options for LA-MRSA within pig herds.

Paratuberculosis is a chronic infection that in dairy cattle causes reduced milk yield, weight loss, and ultimately fatal diarrhea. Subclinical animals can excrete bacteria (Mycobacterium avium ssp. paratuberculosis, MAP) in feces and infect other animals. Farmers identify the infectious animals through a variety of test-strategies, but are challenged by the lack of perfect tests. Frequent testing increases the sensitivity but the costs of testing are a cause of concern for farmers. Here, we used a herd simulation model using milk ELISA tests to evaluate the epidemiological and economic consequences of continuously adapting the sampling interval in response to the estimated true prevalence in the herd. The key results were that the true prevalence was greatly affected by the hygiene level and to some extent by the test-frequency. Furthermore, the choice of prevalence that will be tolerated in a control scenario had a major impact on the true prevalence in the normal hygiene setting, but less so when the hygiene was poor. The net revenue is not greatly affected by the test-strategy, because of the general variation in net revenues between farms. An exception to this is the low hygiene herd, where frequent testing results in lower revenue. When we look at the probability of eradication, then it is correlated with the testing frequency and the target prevalence during the control phase. The probability of eradication is low in the low hygiene herd, and a test-and-cull strategy should probably not be the primary strategy in this herd. Based on this study we suggest that, in order to control MAP, the standard Danish dairy farm should use an adaptive strategy where a short sampling interval of three months is used when the estimated true prevalence is above 1%, and otherwise use a long sampling interval of one year.

Background This study evaluated how dosing regimen for intramuscularly-administered ampicillin, composition of Escherichia coli strains with regard to ampicillin susceptibility, and excretion of bacteria from the intestine affected the level of resistance among Escherichia coli strains in the intestine of nursery pigs. It also examined the dynamics of the composition of bacterial strains during and after the treatment. The growth responses of strains to ampicillin concentrations were determined using in vitro growth curves. Using these results as input data, growth predictions were generated using a mathematical model to simulate the competitive growth of E. coli strains in a pig intestine under specified plasma concentration profiles of ampicillin. Results In vitro growth results demonstrated that the resistant strains did not carry a fitness cost for their resistance, and that the most susceptible strains were more affected by increasing concentrations of antibiotics that the rest of the strains. The modeling revealed that short treatment duration resulted in lower levels of resistance and that dosing frequency did not substantially influence the growth of resistant strains. Resistance levels were found to be sensitive to the number of competing strains, and this effect was enhanced by longer duration of treatment. High excretion of bacteria from the intestine favored resistant strains over sensitive strains, but at the same time it resulted in a faster return to pre-treatment levels after the treatment ended. When the duration of high excretion was set to be limited to the treatment time (i.e. the treatment was assumed to result in a cure of diarrhea) resistant strains required longer time to reach the previous level. Conclusion No fitness cost was found to be associated with ampicillin resistance in E. coli. Besides dosing factors, epidemiological factors (such as number of competing strains and bacterial excretion) influenced resistance development and need to be considered further in relation to optimal treatment strategies. The modeling approach used in the study is generic, and could be used for prediction of the effect of treatment with other drugs and other administration routes for effect on resistance development in the intestine of pigs. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0823-3) contains supplementary material, which is available to authorized users.

Background Transparent calculation methods are crucial when investigating trends in antimicrobial consumption over time and between populations. Until 2011, one single standardized method was applied when quantifying the Danish pig antimicrobial consumption with the unit “Animal Daily Dose” (ADD). However, two new methods for assigning values for ADDs have recently emerged, one implemented by DANMAP, responsible for publishing annual reports on antimicrobial consumption, and one by the Danish Veterinary and Food Administration (DVFA), responsible for the Yellow Card initiative. In addition to new ADD assignment methods, Denmark has also experienced a shift in the production pattern, towards a larger export of live pigs. The aims of this paper were to (1) describe previous and current ADD assignment methods used by the major Danish institutions and (2) to illustrate how ADD assignment method and choice of population and population measurement affect the calculated national antimicrobial consumption in pigs (2007–2013). Results The old VetStat ADD-values were based on SPCs in contrast to the new ADD-values, which were based on active compound, concentration and administration route. The new ADD-values stated by both DANMAP and DVFA were only identical for 48 % of antimicrobial products approved for use in pigs. From 2007 to 2013, the total number of ADDs per year increased by 9 % when using the new DVFA ADD-values, but decreased by 2 and 7 % when using the new DANMAP ADD-values or the old VetStat ADD-values, respectively. Through 2007 to 2013, the production of pigs increased from 26.1 million pigs per year with 18 % exported live to 28.7 million with 34 % exported live. In the same time span, the annual pig antimicrobial consumption increased by 22.2 %, when calculated using the new DVFA ADD-values and pigs slaughtered per year as population measurement (13.0 ADDs/pig/year to 15.9 ADDs/pig/year). However, when based on the old VetStat ADD values and pigs produced per year (including live export), a 10.9 % decrease was seen (10.6 ADDs/pig/year to 9.4 ADDs/pig/year). Conclusion The findings of this paper clearly highlight that calculated national antimicrobial consumption is highly affected by chosen population measurement and the applied ADD-values.

Background Mycoplasma bovis is an important pathogen causing severe disease outbreaks in cattle farms. Since 2011, there has been an apparent increase in M. bovis outbreaks among Danish dairy cattle herds. The dairy cattle industry performed cross-sectional antibody screening for M. bovis on four occasions, using the indirect BIO K 302 M. bovis enzyme-linked immunosorbent assay (ELISA) (Bio-X, Belgium) in bulk tank milk from all dairy herds between June 2013 and July 2014. The objective of this study was to investigate the evolution of the spatial distribution of M. bovis in the Danish dairy herd population throughout the study period. Repeated bulk tank milk samples were used as a proxy for the herd-level diagnosis. Descriptive and spatial analyses were performed for the four screening rounds. Based on a previous diagnostic test evaluation study, the M. bovis status for each herd was determined as test-positive or test-negative using a cut-off of 50 optical density coefficient %. The spatial global clustering was evaluated through a modified K-function method, and local clusters were identified by scan statistics. Results The results showed that M. bovis test-positive herds had a dynamic pattern in space. The global clustering analysis showed that M. bovis test-positive herds were spatially correlated in rounds one, three and four. These findings were supported to some extent by the local clustering analysis, which found significant high- and low-risk spatial clusters in rounds one and three in the north and south of the mainland. Conclusion The clusters with a high risk of observing test-positive herds did not remain between sampling rounds, indicating that M. bovis did not tend to persist upon emergence in dairy herds. In contrast, the clusters with a low risk of observing test-positive herds persisted in the same area throughout the study period.

To study the spread of African swine fever (ASF) within a pig unit and the impact of unit size on ASF spread, a simulation model was created. In the model, an animal can be in one of the following stages: susceptible, latent, subclinical, clinical, or recovered. Animals can be infectious during the subclinical stage and are fully infectious during the clinical stage. ASF virus (ASFV) infection through residues of dead animals in the slurries was also modeled in an exponentially fading-out pattern. Low and high transmission rates for ASFV were tested in the model. Robustness analysis was carried out in order to study the impact of uncertain parameters on model predictions. The results showed that the disease may fade out within the pig unit without a major outbreak. Furthermore, they showed that spread of ASFV is dependent on the infectiousness of subclinical animals and the residues of dead animals, the transmission rate of the virus, and importantly the unit size. Moreover, increasing the duration of the latent or the subclinical stages resulted in longer time to disease fade out. The proposed model is a simple and robust tool simulating the spread of ASFV within a pig house taking into account dynamics of ASFV spread and the unit size. The tool can be implemented in simulation models of ASFV spread between herds.

An epidemic of foot-and-mouth disease (FMD) in a FMD-free country with large exports of livestock and livestock products would result in profound economic damage. This could be reduced by rapid and efficient control of the disease spread. The objectives of this study were to estimate the economic impact of a hypothetical FMD outbreak in Denmark based on changes to the economic assumptions of the model, and to investigate whether the control of an FMD epidemic can be improved by combining the enlargement of protection or surveillance zones with pre-emptive depopulation or emergency vaccination. The stochastic spatial simulation model DTU-DADS was used to simulate the spread of FMD in Denmark. The control strategies were: the basic EU and Danish strategy; pre-emptive depopulation; suppressive or protective vaccination; enlarging protection or surveillance zones, and a combination of pre-emptive depopulation or emergency vaccination with enlarged protection or surveillance zones. Herds are detected either based on basic detection through the appearance of clinical signs, or as a result of surveillance in the control zones. The economic analyses consisted of direct costs and export losses. Sensitivity analysis was performed on uncertain and potentially influential input parameters. Enlarging the surveillance zones from 10km to 15km, combined with pre-emptive depopulation over a 1km radius around detected herds resulted in the lowest total costs. This was still the case even when the different input parameters were changed in the sensitivity analysis. Changing the resources for clinical surveillance did not affect the epidemic consequences. In conclusion, an FMD epidemic in Denmark would have a larger economic impact on the agricultural sector than previously anticipated. Furthermore, the control of a potential FMD outbreak in Denmark may be improved by combining pre-emptive depopulation with an enlarged protection or surveillance zone.

The complex relationship between drug concentrations and bacterial growth rates require not only the minimum inhibitory concentration but also other parameters to capture the dynamic nature of the relationship. To analyse this relationship between tetracycline concentration and growth of Escherichia coli representative of those found in the Danish pig population, we compared the growth of 50 randomly selected strains. The observed net growth rates were used to describe the in vitro pharmacodynamic relationship between drug concentration and net growth rate based on E max model with three parameters: maximum net growth rate (α max ); concentration for a half-maximal response (E max ); and the Hill coefficient (γ). The net growth rate in the absence of antibiotic did not differ between susceptible and resistant isolates (P = 0.97). The net growth rate decreased with increasing tetracycline concentrations, and this decline was greater in susceptible strains than resistant strains. The lag phase, defined as the time needed for the strain to reach an OD600 value of 0.01, increased exponentially with increasing tetracycline concentration. The pharmacodynamic parameters confirmed that the $$ \alpha_{max} $$ between susceptible and resistant strains in the absence of a drug was not different. EC 50 increased linearly with MIC on a log–log scale, and γ was different between susceptible and resistant strains. The in vitro model parameters described the inhibition effect of tetracycline on E. coli when strains were exposed to a wide range of tetracycline concentrations. These parameters, along with in vivo pharmacokinetic data, may be useful in mathematical models to predict in vivo competitive growth of many different strains and for development of optimal dosing regimens for preventing selection of resistance.

BACKGROUND: Canine cognitive dysfunction (CCD) is an age-dependent neurodegenerative condition dominated by changes in behavioral patterns. Cohort studies investigating cognitive status in dogs are lacking.OBJECTIVES: To investigate cognitive function, progression of age-related behavioral changes, survival, and possible biomarkers of CCD in aged dogs.ANIMALS: Fifty-one dogs >8 years old; 21 with no cognitive deficits, 17 with mild cognitive impairments (MCI) and 13 with CCD.METHODS: Longitudinal study. Recruitment period of 12 months and an observational period of 24 months including a baseline and 3 planned subsequent assessments. Cognitive status was determined using validated questionnaires. Plasma Aβ-peptides were quantified using commercial ELISA assays and cytokines by a validated immunoassay.RESULTS: Signs characterizing dogs with CCD were aimless wandering, staring into space, avoid getting patted, difficulty finding dropped food and anxiety. Thirty-three percent of dogs with a normal cognitive status progressed to MCI and 22% classified as MCI progressed to CCD during the study period. For 6 dogs diagnosed with CCD, signs of cognitive dysfunction increased with time. A diagnosis of CCD did not affect survival. The level of plasma Aβ42 was significantly increased (P < .05) in the CCD group (92.8 ± 24.0 pg/mL) compared to the MCI (77.0 ± 12.3 pg/mL) and normal group (74.9 ± 10.0 pg/mL), but no significant differences in concentrations of systemic inflammatory markers were detected.CONCLUSIONS: Canine cognitive dysfunction is a progressive disorder with an individual variability in the rate of cognitive decline and clinical signs. Plasma Aβ42 seems to be an interesting plasma biomarker of CCD.