Liza Rosenbaum Nielsen

Evaluation of an indirect serum ELISA and a bacteriological faecal culture test for diagnosis of Salmonella serotype Dublin in cattle using latent class models

Aims:  To evaluate a conventional bacteriological test based on faecal culture and an indirect serum ELISA for detection of S. Dublin infected cattle. To compare the predictive values of the two tests in relation to the prevalence.

Factors influencing Salmonella carcass prevalence in Danish pig abattoirs

The Danish Salmonella Surveillance-and-Control Programme in finisher pigs includes both herd and carcass surveillance. Herd surveillance consists of serological testing of meat-juice samples and classification of herds into three Salmonella seroprevalence levels. At the abattoirs, carcass swabs from five pigs are collected daily and analysed as a pooled sample to evaluate the Salmonella carcass prevalence. This study aimed to investigate factors associated with Salmonella carcass prevalence in Denmark. A total of 20,196 pooled carcass swabs collected in 23 Danish abattoirs were included in the analysis. A multilevel logistic regression model was used taking into account the two-level data structure (abattoir, carcass pool) and adjusting the parameter estimates to the random variation at the abattoir level. Study results indicated that carcass contamination was mainly influenced by the probability that at least one pig contributing to the pool was seropositive, the log-transformed number of seropositive pigs delivered to the abattoir on the same day and weekday. No other factors were found to be significant (P>0.05). Large reductions in the number of seropositive pigs delivered to slaughter are unlikely to result in large reductions of the Salmonella carcass prevalence, unless the number of seropositive pigs can be kept below approximately 200. On average, individual Salmonella carcass prevalence can be kept below 1% by keeping a Salmonella input to the abattoir below approximately 50 seropositive pigs. Variation between abattoirs suggested that improved hygiene practices in some of the abattoirs would reduce the Salmonella carcass prevalence further.

Review of pathogenesis and diagnostic methods of immediate relevance for epidemiology and control of Salmonella Dublin in cattle

Salmonella enterica subsp. enterica serovar Dublin (S. Dublin) receives increasing attention in cattle production. It is host-adapted to cattle, and leads to unacceptable levels of morbidity, mortality and production losses in both newly and persistently infected herds. Cattle health promoting institutions in several countries are currently constructing active surveillance programmes or voluntary certification programmes, and encourage control and eradication of S. Dublin infected cattle herds. There is a need to understand the underlying pathogenesis of the infection at both animal and herd level to design successful programmes. Furthermore, knowledge about and access to diagnostic tests for use in practice including information about test accuracy and interpretation of available diagnostic test methods are requested. The aim is to synthesise the abundant literature on elements of pathogenesis and diagnosis of immediate relevance for epidemiology and control of S. Dublin at animal and herd level. Relatively few in vivo studies on S. Dublin pathogenesis in cattle included more than a few animals and often showed varying result. It makes it difficult to draw conclusions about mechanisms that affect dissemination in cattle and that might be targets for control methods directed towards improving resistance against the bacteria, e.g. new vaccines. It is recommended to perform larger studies to elucidate dose-response relationships and age- and genetic effects of immunity. Furthermore, it is recommended to attempt to develop faster and more sensitive methods for detection of S. Dublin for diagnosis of infectious animals.

A Blueprint to Evaluate One Health

One Health (OH) positions health professionals as agents for change and provides a platform to manage determinants of health that are often not comprehensively captured in medicine or public health alone. However, due to the organization of societies and disciplines, and the sectoral allocation of resources, the development of transdisciplinary approaches requires effort and perseverance. Therefore, there is a need to provide evidence on the added value of OH for governments, researchers, funding bodies, and stakeholders. This paper outlines a conceptual framework of what OH approaches can encompass and the added values they can provide. The framework was developed during a workshop conducted by the “Network for Evaluation of One Health,” an Action funded by the European Cooperation in Science and Technology. By systematically describing the various aspects of OH, we provide the basis for measuring and monitoring the integration of disciplines, sectors, and stakeholders in health initiatives. The framework identifies the social, economic, and environmental drivers leading to integrated approaches to health and illustrates how these evoke characteristic OH operations, i.e., thinking, planning, and working, and require supporting infrastructures to allow learning, sharing, and systemic organization. It also describes the OH outcomes (i.e., sustainability, health and welfare, interspecies equity and stewardship, effectiveness, and efficiency), which are not possible to obtain through sectoral approaches alone, and their alignment with aspects of sustainable development based on society, environment, and economy.

Epidemiologic and economic evaluation of risk-based meat inspection for bovine cysticercosis in Danish cattle.

Under the current EU meat inspection regulation, every single carcase from all bovines above 6 weeks of age has to be examined for bovine cysticercosis (BC). This is time-consuming, costly, and is of limited value in countries with low prevalence. The aim of this study was to develop a stochastic simulation model for analysis of tentative risk-based meat inspection systems for BC in Danish cattle with regard to system sensitivity (SSSe), specificity and potential monetary benefits compared to the current system, which has an estimated SSSe of 15%. The relevant risk factors used to construct three alternative scenario trees were identified from previous Danish risk factor studies (1) gender, (2) grazing and (3) access to risky water sources. Thus, females, animals that had been grazing or animals with access to risky water sources were considered high-risk and would be subjected to invasive inspection at meat inspection. All animals in the low-risk groups (i.e. males, non-grazing or no access to risky water sources, respectively) would be subjected to visual inspection only. It was assumed that half of the cattle were slaughtered in abattoirs that would be able to reorganise the work at the slaughterline, allowing them to do with one meat inspector less. All abattoirs would gain on the price of sold uncut beef from the masseter muscles from visually inspected cattle. Under these assumptions, using gender and grazing were preferable due to them having SSSe only slightly lower than the current system, and highest effectiveness ratios, but they had a lower net economic effect (NEE) than the scenario using risky water sources. Using gender to differentiate high and low-risk groups was judged preferable over grazing due to feasibility, because the information is readily available at the slaughter line. The exact total NEE for the cattle sector depends on how many and which of the abattoirs that would be able to reorganise the work at the slaughter line to save money on inspection of the head of carcases. Overall, the SSSe was low in all scenarios leading to undetected BC-positive cattle both in the current meat inspection and under the investigated risk-based meat inspection systems. Therefore, improving the sensitivity of the methods used for inspection of high-risk cattle would be beneficial.

Occurrence and factors associated with bovine cysticercosis recorded in cattle at meat inspection in Denmark in 2004-2011.

Current EU regulation requires that every bovine carcass is examined for bovine cysticercosis (BC) at meat inspection. This is costly and might be superfluous at low BC prevalence. However, from a consumer view-point it may be important to identify and manage infected carcasses to avoid human infection. If relevant data could be effectively used to classify animals with respect to their risk of being infected, then the current meat inspection could be replaced by a more cost-effective system targeting high-risk animals. This study aimed to (1) describe the distribution of BC cases in the Danish cattle population, (2) estimate the animal level prevalence (3) provide descriptive statistics of potential risk factors for BC, and (4) determine attributable risks and fractions of selected risk factors potentially useful for a future risk-based meat inspection system. In total, 348 cases of BC were recorded among all cattle slaughtered (n=4,090,661) in Denmark between 2004 and 2011. The true animal level prevalence of BC was estimated to be 0.06%. The herd of origin of the cases were defined as the herd in which the animals spent most of their lifetimes. The detected cases were found to originate from 328 herds, with a maximum of two cases per herd indicating sporadic occurrence. Even though organic farming was associated with a higher risk (RR=1.9 in univariable analysis) of BC-positive animals being detected at slaughter, the population attributable fraction showed that only 5% of the animals with BC could be attributed to organic farming practices at the level of organic farming practiced in Denmark in the study period. Thus, organic farming status was not a suitable risk factor to use to target future risk-based meat inspection. However, 54% of the animals with BC in the cattle population were attributed to female gender. Increasing age at slaughter was also associated with high risk of BC. There may be overlaps between these effects in animals with multiple risk factors. Other underlying factors such as grazing patterns might explain the risk factors and attribution results found in this study. However, grazing practices are currently not recorded in the Danish cattle database. Therefore, animal level risk factors such as age and gender together with other risk factors such as grazing practices might be included as food chain information, required to be provided by the farmer prior to slaughter. The challenges and opportunities of this approach should be investigated further.

Factors affecting persistence of high Salmonella serology in Danish pig herds

In 1995, Denmark implemented a National Surveillance-and-Control Programme for Salmonella in finisher pigs. It was based on serological surveillance that enabled assignment of pig herds into three prevalence levels. Herds in levels 2 and 3 (corresponding to moderate and high Salmonella seroprevalence) were subjected to control measures, compulsory pen faecal sampling for bacteriological examination and serotyping. The initial dataset contained all herds that had mandatory bacteriological samples taken (N=3691 samples), out of which 44.4% were negative. Data from January 2005 to November 2007 obtained from the Danish programme were used to investigate the persistence of high serology (Salmonella index > or =40, corresponding to levels 2 or 3) for herds infected with one of the four most common Salmonella serotypes found in pig herds, S. Typhimurium, S. Derby, S. Infantis or S. Livingstone, using survival analysis. The effects of other potential factors, such as season, year, herd size and herd health status were also evaluated. An event was defined as return to low serology measured by a Salmonella index <40, for at least 6 consecutive months. A total of 1242 pig herds positive for one of the four serotypes of interest and 1433 events were included in the preliminary analysis using the Kaplan-Meyer method. The median high serology time (survival time) was 8 months and all explanatory variables were found to be significant (p<0.05), except the herd health status (p=0.49). Cox proportional hazards regression was used for herds infected with S. Typhimurium (N=1066) or S. Derby (N=314). Herds infected with S. Typhimurium presented a lower probability for returning to low serology (HR=0.69, p<0.001), compared to S. Derby, indicating longer high serology periods. Herds that started having high seroprevalence in Summer and Fall had a higher probability of returning to low serology compared to those starting in Winter and Spring (p<0.001). Compared to 2005, periods with high Salmonella serology were fewer and shorter in 2006 and 2007 (p<0.001). A positive impact of herd size was found, with larger herds being associated with shorter high serology periods, compared to smaller herds (p=0.012). This might indicate effective implementation of biosecurity measures in large herds. The health status of the herds was not found to be a significant factor (p=0.87), which might reflect that biosecurity measures in place in SPF herds are not effective for clearing Salmonella infections.

Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure.

Aims:  To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces.

Latent class analysis of bulk tank milk PCR and ELISA testing for herd level diagnosis of Mycoplasma bovis.

The bacterium Mycoplasma bovis causes disease in cattle of all ages. An apparent increase in the occurrence of M. bovis associated outbreaks among Danish dairy cattle herds since 2011 has prompted a need for knowledge regarding herd-level diagnostic performance. Therefore, the objective of this study was to evaluate the herd-level diagnostic performance of an indirect ELISA test by comparison to a real-time PCR test when diagnosing M. bovis in cattle herds of bulk tank milk. Bulk tank milk samples from Danish dairy herds (N=3437) were analysed with both the antibody detecting BIO K 302 M. bovis ELISA kit and the antigen detecting PathoProof Mastitis Major-3 kit. As none of these are considered a gold standard test for herd-level diagnostics we applied a series of Bayesian latent class analyses for a range of ELISA cut-off values. The negative and positive predictive values were calculated for hypothetical true national prevalences (1, 5, 10, 15 and 20%) of infected herds. We estimated that the ELISA test had a median sensitivity and specificity of 60.4 [37.5-96.2 95% Posterior Credibility Interval] and 97.3 [94.0-99.8 95% PCI] at the currently recommended cut-off (37% Optical density Coefficient). These changed to 43.5 [21.1-92.5 95% PCI] and 99.6 [98.8-100 95% PCI] if the cut-off was increased to 50 ODC%. In addition, herd-level diagnosis by ELISA would result in fewer false positives at a cut-off value of 50 ODC% compared to 37 ODC% without compromising the negative predictive value.

Exposure assessment of extended-spectrum beta-lactamases/AmpC beta-lactamases-producing Escherichia coli in meat in Denmark

Introduction Extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases (AmpC) are of concern for veterinary and public health because of their ability to cause treatment failure due to antimicrobial resistance in Enterobacteriaceae. The main objective was to assess the relative contribution (RC) of different types of meat to the exposure of consumers to ESBL/AmpC and their potential importance for human infections in Denmark. Material and methods The prevalence of each genotype of ESBL/AmpC-producing E. coli in imported and nationally produced broiler meat, pork and beef was weighted by the meat consumption patterns. Data originated from the Danish surveillance program for antibiotic use and antibiotic resistance (DANMAP) from 2009 to 2011. DANMAP also provided data about human ESBL/AmpC cases in 2011, which were used to assess a possible genotype overlap. Uncertainty about the occurrence of ESBL/AmpC-producing E. coli in meat was assessed by inspecting beta distributions given the available data of the genotypes in each type of meat. Results and discussion Broiler meat represented the largest part (83.8%) of the estimated ESBL/AmpC-contaminated pool of meat compared to pork (12.5%) and beef (3.7%). CMY-2 was the genotype with the highest RC to human exposure (58.3%). However, this genotype is rarely found in human infections in Denmark. Conclusion The overlap between ESBL/AmpC genotypes in meat and human E. coli infections was limited. This suggests that meat might constitute a less important source of ESBL/AmpC exposure to humans in Denmark than previously thought – maybe because the use of cephalosporins is restricted in cattle and banned in poultry and pigs. Nonetheless, more detailed surveillance data are required to determine the contribution of meat compared to other sources, such as travelling, pets, water resources, community and hospitals in the pursuit of a full source attribution model.