A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis.

BACKGROUND: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. RESULTS: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. CONCLUSION: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.

A European inter-laboratory trial to evaluate the performance of three serological methods for diagnosis of Mycoplasma bovis infection in cattle using latent class analysis.

BACKGROUND: Mycoplasma bovis (M. bovis) is an emerging bovine pathogen, leading to significant economic losses in the livestock industry worldwide. Infection can result in a variety of clinical signs, such as arthritis, pneumonia, mastitis and keratoconjunctivitis, none of which are M. bovis-specific. Laboratory diagnosis is therefore important. Serological tests to detect M. bovis antibodies is considered an effective indicator of infection in a herd and often used as a herd test. Combined with clinical judgement, it can also be used to implement control strategies and/or to estimate the disease prevalence within a country. However, due to lack of harmonisation of approaches to testing, and serological tests used by different laboratories, comparisons of prevalence data between countries is often difficult. A network of researchers from six European countries designed and participated in an inter-laboratory trial, with the aim of evaluating the sensitivity (Se) and specificity (Sp) of two commercially available ELISA tests (ID Screen® ELISA (IDvet) and BIO K302 ELISA (BIO-X Diagnostics)) for diagnosis of M. bovis infection. Each laboratory received a blinded panel of bovine sera and tested independently, according to manufacturer's instructions. Western blot analyses (WB) performed by one of the participating laboratories was used as a third diagnostic test in the statistical evaluation of Se and Sp values using latent class analysis. RESULTS: The Se of WB, the ID Screen® ELISA and the BIO K302 ELISA were determined to be 91.8, 93.5 and 49.1% respectively, and corresponding Sp of the three tests were 99.6, 98.6 and 89.6%, respectively. CONCLUSIONS: The present study is, to our knowledge, the first to present an inter-laboratory comparison of the BIO K302 ELISA and the ID Screen® ELISA. Based on our results, the ID Screen® ELISA showed high consistency with WB and performed with higher precision and accuracy than the BIO K302 ELISA.

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid.

BACKGROUND: Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. RESULTS: The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10-1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. CONCLUSION: It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

Mycoplasma detection by triplex real-time PCR in bronchoalveolar lavage fluid from bovine respiratory disease complex cases.

BACKGROUND: In this study we evaluated the RespoCheck Mycoplasma triplex real-time PCR for the detection in bronchoalveolar lavage fluid (BALF) of Mycoplasma (M.) dispar, M. bovis and M. bovirhinis, all three associated with bovine respiratory disease (BRD). Primers and probes of the RespoCheck Mycoplasma triplex real-time PCR are based on the V3/V4 region of the 16S rRNA gene of the three Mycoplasma species. RESULTS: The analytical sensitivity of the RespoCheck triplex real-time PCR was, as determined by spiking experiments of the Mycoplasma strains in Phosphate Buffered Saline, 300 colony forming units (cfu)/mL for M. dispar, and 30 cfu/mL for M. bovis or M. bovirhinis. The analytical sensitivity of the RespoCheck Mycoplasma triplex real-time PCRwas, as determined on purified DNA, 10 fg DNA per assay for M. dispar and 100 fg fo rM. bovis and M. bovirhinis. The analytical specificity of the RespoCheck Mycoplasma triplex real-time PCR was, as determined by testing Mycoplasmas strains (n = 17) and other bacterial strains (n = 107), 100, 98.2 and 99.1% for M. bovis, M. dispar and M. bovirhinis respectively. The RespoCheck Mycoplasma triplex real-time PCR was compared with the PCR/DGGE analysis for M. bovis, M. dispar and M. bovirhinis respectively by testing 44 BALF samples from calves. CONCLUSION: In conclusion, the RespoCheck PCR assay can be a valuable tool for timely and accurate detection of three Mycoplasma species associated with in bovine respiratory disease.

Added Value of Sensor-Based Behavioural Monitoring in an Infectious Disease Study with Sheep Infected with Toxoplasma gondii.

Sensor technologies are increasingly used to monitor laboratory animal behaviour. The aim of this study was to investigate the added value of using accelerometers and video to monitor the activity and drinking behaviour of three rams from 5 days before to 22 days after inoculation with Toxoplasma gondii. We computed the activity from accelerometer data as the vectorial dynamic body acceleration (VDBA). In addition, we assessed individual drinking behaviour from video, using frame differencing above the drinker to identify drinking bouts, and Aruco markers for individual identification. Four days after inoculation, rams developed fever and activity decreased. The daytime VDBA from days 4 to 10 was 60-80% of that before inoculation. Animal caretakers scored rams as lethargic on days 5 and 6 and, for one ram, also on the morning of day 7. Video analysis showed that each ram decreased its number of visits to the drinker, as well as its time spent at the drinker, by up to 50%. The fever and corresponding sickness behaviours lasted until day 10. Overall, while we recognize the limited conclusiveness due to the small number of animals, the sensor technologies provided continuous, individual, detailed, and objective data and offered additional insights as compared to routine observations. We recommend the wider implementation of such technologies in animal disease trials to refine experiments and guarantee the quality of experimental results.

In vitro assay to determine inactivation of Toxoplasma gondii in meat samples.

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 µL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

Environmental Sampling Methods for Detection of Salmonella Infections in Laying Hens: A Systematic Review and Meta-Analysis.

Salmonellosis is the second most commonly reported foodborne gastrointestinal infection in humans in the European Union (EU). Most outbreaks are caused by Salmonella Enteritidis, present in contaminated food products, particularly in egg and egg products. In recent years, an increase in the prevalence of Salmonella in laying hen flocks in the EU has been observed. For the effective control of infection, adequate detection is key. In laying hen flocks, the occurrence of Salmonella in the EU is monitored by the culture of environmental samples (dust, faeces, and boot swabs). The performance of sampling procedures described in the literature for the detection of Salmonella in laying hens was reviewed. In total, 924 abstracts were screened, resulting in the selection of 87 abstracts and 18 publications for qualitative and quantitative analyses, respectively. Sample sizes and sampling locations of faecal material and dust were variable and poorly described. Microbiological culture methods used to detect Salmonella were variably described in the literature and were often incomplete. Overall, the available literature indicates higher sensitivity of environmental versus individual hen matrices and points to differences in sensitivity between environmental matrices. For non-cage housing systems, boot swabs are the preferred samples, while for cage housing systems dust might be a more reliable sample.

Systematic Review and Modelling of Age-Dependent Prevalence of Toxoplasma gondii in Livestock, Wildlife and Felids in Europe.

Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8-7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0-79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection.

Respiratory pathogens in veal calves: Inventory of circulating pathogens.

In the veal industry in The Netherlands, each year around 1.2 million "white" veal calves are produced on around 1100 farms. Bovine respiratory disease (BRD) causes serious health issues in these calves, also resulting in high usage of antimicrobials. To reduce antimicrobial usage, a more targeted treatment regime is needed, for which it is necessary to identify the causative agent. This study aimed at determining associations between pathogens and clinical disease, between prevalence of pathogens and BRD outbreaks, and BRD and performance. A cohort study was conducted involving ten veal farms, in which calf respiratory health was evaluated for the first 12 weeks. Whenever there was an outbreak of BRD, as determined by the farm veterinary surgeon, samples were taken from diseased and control calves through broncho-alveolar lavage. From these samples a broad spectrum of micro-organisms were isolated. Performance data were also collected. A total of 23 outbreaks happened during the 12 week study period, mostly in the first six weeks. BRD associated pathogens found were: BHV1, BPI3V, BRSV, BVDV, Pasteurella multocida, Mannheimia haemolytica, Trueperella pyogenes, Histophilus somni, Mycoplasma bovis, Mycoplasma bovirhinis and Mycoplasma dispar. For most BRD associated pathogens, there was no clear association between presence or prevalence of the micro-organisms and clinical issues. Only T. pyogenes (7.4% in healthy, 14.6% in diseased calves, p 0.013), M. bovis (37.6% and 63.2% respectively, p 0.001) and BVDV (9.9% and 16.9% respectively, p 0.03) were found more often in diseased animals. BPI3V was found in a few early outbreaks, which might suggest involvement in early outbreaks. It appears to be difficult to associate specific pathogens to outbreaks at the species level. BRD is the major reason for treatment with antimicrobials. More specific knowledge about the association between pathogens and health/disease could help to reduce antimicrobial use.

Potential risk factors for the presence of anti-Toxoplasma gondii antibodies in finishing pigs on conventional farms in the Netherlands.

BACKGROUND: The parasite Toxoplasma gondii (T. gondii) causes a substantial human disease burden worldwide. Ingesting improperly cooked pork containing T. gondii is considered one of the major sources of human infection in Europe and North America. Consequently, control of T. gondii infections in pigs is warranted. The European Food Safety Authority advised to perform serological monitoring of pigs and to conduct farm audits for the presence of risk factors. Serological monitoring was implemented in several Dutch slaughterhouses, one to six blood samples (a total of 5134 samples) were taken from each delivery of finishing pigs and samples were tested for the presence of anti-T. gondii antibodies. Using these test results, a cross-sectional study was initiated to assess the association between the within-herd T. gondii seroprevalence and the presence of risk factors for T. gondii infections at 69 conventional finishing pig farms in the Netherlands. RESULTS: A multivariable model showed significant (P ≤ 0.05) association with twelve potential risk factors: type of farm, presence of dogs, presence of ruminants, use of boots, use of shower and farm clothing, mode of rodent control, bedding accessibility for rodents, presence of cats, type of drinking water, heating of the feed, use of goat whey and shielding of birds. CONCLUSIONS: Serological monitoring of finishing pigs for T. gondii in slaughterhouses can be used to identify the presence of T. gondii risk factors on Dutch conventional finishing pig farms and seems a valuable tool to guide and monitor the control of T. gondii in pork production.

Genetic diversity of Streptococcus suis isolates as determined by comparative genome hybridization.

BACKGROUND: Streptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes. RESULTS: In this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH). Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF). Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP) and EF (MRP⁻EF⁻), suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7. CONCLUSIONS: In conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.

Qualitative Evaluation of Causes for Routine Salmonella Monitoring False-Positive Test Results in Dutch Poultry Breeding Flocks.

The Salmonella monitoring program, as outlined in the EU Commission regulation 200/2010, asks for repeated sampling in order to ascertain progress in achievement of the EU target. According to Article 2.2.2.2.c of this regulation, the competent authority may decide to do a resample and retest when it has reasons to question the results of initial testing. In the Netherlands, the competent authorities have been resampling and retesting all initial positive samplings for several years because of doubts about false positive initial test results. An analysis of population data in the period 2015-2019 indicates that 48% of initial samplings at the farm were classified as false positive after resampling and retesting by the competent authorities. A qualitative analysis, assessing factors that could be associated with the occurrence of false positives, indicates that cross-contamination during the sampling process by the poultry farmer is probably the most likely source. Cross-contamination of samples during transport from the farm to the laboratory and/or cross-contamination at the laboratory are also considered possible sources. Given the slightly non-optimal system-specificity of the Salmonella monitoring program, there is good reason to make, or consider, standard resampling and retesting of initial positive results by the competent veterinary authorities possible within the EU.

Intestinal Viral Loads and Inactivation Kinetics of Livestock Viruses Relevant for Natural Casing Production: a Systematic Review and Meta-Analysis.

Animal intestines are the source of edible sausage casings, which are traded worldwide and may come from areas where notifiable infectious animal diseases are prevalent. To estimate the risks of virus contamination, knowledge about the quantity of virus and decimal reduction values of the standard preservation method by salting is of great importance. A literature search, based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, was performed in search engine CAB Abstracts to determine the viral load of 14 relevant animal viruses in natural casings or intestines. Only a very limited number of scientific publications per virus were found and viral loads in the intestines varied from high for ASFV (five publications), BVDV (3), CSFV (6), PPRV (3), RPV(2) and TGEV (3) to moderate for PEDV (2) and SVDV (3), low for HEV (2) and FMDV (5), very low for VESV (1) and negative for PrV (2) and VSV (1). PRRSV was found in intestines, however, viral titers were not published. Three viruses (BVDV, CSFV and PPRV) with high viral loads were selected to search for their inactivation kinetics. For casings, no inactivation data were found, however, thermal inactivation data of these viruses were available, but differed in quantity, quality and matrices. In conclusion, important data gaps still exist when it comes to the quantitative inactivation of viruses in sausage casings or livestock intestines.

Quarter of pig farmers readily viewed information on Toxoplasma gondii infection in pigs provided in a data exchange system of a Dutch slaughter company.

Toxoplasma gondii (T. gondii) is a food safety hazard causing a substantial human disease burden. Because infected pig meat is estimated to attribute 12 % to this disease burden, it is important to control T. gondii infection in pigs. Providing pig farmers with information on T. gondii infection in general, and more specific on the status on their farm, could motivate them to take actions. In this study, we analysed the strategy pig farmers used to view specific T. gondii information provided for the first time on webpages in an existing data exchange system of a Dutch pig slaughter company. The available information for farmers comprised a webpage displaying the farm-level T. gondii seroprevalence and a webpage with information on risk sources and control measures for T. gondii infection in pigs and on human health consequences of a T. gondii infection. A total of 1404 owners of pig farms logged in the data exchange system. Of these, a quarter viewed the webpage with information on T. gondii seroprevalence, and about of third of them also viewed the webpage with the information on risk sources and control measures. T. gondii seroprevalence exceeded 2.0 % at only 0.6 % of these 1404 farms. The seroprevalence level on a particular farm neither influenced the likelihood of the farmer viewing the webpage with the T. gondii seroprevalence, nor the likelihood of them continuing to the webpage with the additional information. In the days when the pop-up message was included, the number of views registered on the seroprevalence and the additional information webpages rose nine and two times, respectively. Since the majority of views was in the period with a pop-up message pointing to this information we conclude that a targeted pop-up might help to transfer needed information to farmers with higher T. gondii seroprevalence at farm-level. More general, our study provides valuable insight into pig farmers' viewing strategies of new information on food safety hazards provided in a slaughter data exchange system.

Determination of Intestinal Viral Loads and Distribution of Bovine Viral Diarrhea Virus, Classical Swine Fever Virus, and Peste Des Petits Ruminants Virus: A Pilot Study.

The aim of this pilot study was to determine viral loads and distribution over the total length, at short distances, and in the separate layers of the intestine of virus-infected animals for future inactivation studies. Two calves, two pigs, and two goats were infected with bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV), and peste des petits ruminants virus (PPRV), respectively. Homogenously distributed maximum BVDV viral loads were detected in the ileum of both calves, with a mean titer of 6.0 log10 TCID50-eq/g. The viral loads in colon and caecum were not distributed homogenously. In one pig, evenly distributed CSFV mean viral loads of 4.5 and 4.2 log10 TCID50-eq/g were found in the small and large intestines, respectively. Mucosa, submucosa, and muscular layer/serosa showed mean viral loads of 5.3, 3.4, and 4.0 log10 TCID50-eq/g, respectively. Homogenous distribution of PPRV was shown in the ileum of both goats, with a mean viral load of 4.6 log10 TCID50-eq/g. Mean mucosa, submucosa, and muscular layer/serosa viral loads were 3.5, 2.8, and 1.7 log10 TCID50-eq/g, respectively. This pilot study provides essential data for setting up inactivation experiments with intestines derived from experimentally infected animals, in which the level and the homogeneous distribution of intestinal viral loads are required.

Effectiveness and costs of interventions to reduce the within-farm Toxoplasma gondii seroprevalence on pig farms in the Netherlands.

BACKGROUND: The parasite Toxoplasma gondii (T. gondii) is recognized as one of the major foodborne pathogens with a high human disease burden. To control T. gondii infections in pigs, European Food Safety Agency (EFSA) advises serological testing of pigs and audits of pig farms to identify risk factors for T. gondii infection. In line with this approach, the aim of the current study was to assess the effectiveness and costs of intervention measures implemented to reduce the T. gondii seroprevalence on finishing pig farms in the Netherlands. A crossover clinical trial was conducted at five case farms were their own control and the cross-over moment was the implementation of interventions to reduce risk factors. Each of the case farms had a farm-specific intervention strategy with one principal intervention measure (neutering of cats, professional rodent control or covering food storage). RESULTS: All finishing pig farms (n = 5) showed a reduction in T. gondii seroprevalence within one year of implementing the intervention strategy. Cat neutering (n = 3) and feed coverage (n = 1) showed statistically significant reductions in seroprevalence. Rodent control (n = 1) did not show a statistically significant reduction. The estimated reduction in seroprevalence in response to the neutering of cats and feed coverage were 67 and 96 %, respectively. CONCLUSIONS: Our work demonstrates that it is possible to reduce the within-farm T. gondii seroprevalence within one year after interventions were implemented to reduce T. gondii risk factors. This information is essential and encouraging for policy makers, food business operators, and farmers to implement in their risk assessment and to apply to food safety control systems.

Behavioural factors of Dutch pig producers related to control of toxoplasma gondii infections in pigs.

Toxoplasma gondii (T. gondii) is a food safety hazard which causes a substantial human disease burden. Infected pig meat is a common risk source of toxoplasmosis. Therefore, it is important to control T. gondii infections in pigs. Improving farm management to control the introduction risk likely contributes to that aim. A pig producer only implements control measures when he or she is aware of the underlying problem, wants to solve it, and is able to solve it. If a pig producer is not implementing appropriate control measures, behavioural change interventions can be introduced to overcome constraining behavioural factors. To aid in designing behaviour change interventions, this study analysed behavioural factors of Dutch pig producers in terms of capability, opportunity and motivation to control T. gondii infections in pigs. Key risk sources analysed focused on the life cycle of T. gondii, with cats as primary host, rodents as intermediate host, and uncovered feed as an important risk source. A survey was conducted among Dutch pig producers. Responses were analysed using descriptive and cluster analysis. Results showed that around 80% of the 67 responding pig producers was aware of key risk sources of T. gondii infections in pigs. Respondents also rated risk sources that are not known to increase the risk of T. gondii infections in pigs as somewhat important. Many respondents did not know about potential consequences of a T. gondii infection in pigs on human health. Two third expected some impact on pig performance, which is incorrect because T. gondii generally does not make pigs ill. Most respondents indicated to have the motivation and opportunity to control the risk sources cats, rodents and uncovered feed. Three pig producer clusters were identified: one with higher capability to control rodents, one with lower motivation to control rodents and cats and to cover feed storages, and one with lower scores on the importance of rodent control for pigs, human health and farm profit. We conclude that, although many pig producers have knowledge about risk sources for and consequences of T. gondii infections in pigs, the public health impact and risks of T. gondii infections in pigs are not yet common knowledge among all Dutch pig producers. Furthermore, Dutch pig producers differ in opportunity and motivation to control T. gondii infections. Targeted interventions to address these specific constraining behavioural factors can help to improve the control of T. gondii infections in pigs.

Mycoplasma bovis in Nordic European Countries: Emergence and Dominance of a New Clone.

Mycoplasma (M.) bovis is an important pathogen of cattle implicated in a broad range of clinical manifestations that adversely impacts livestock production worldwide. In the absence of a safe, effective commercial vaccine in Europe, the reported reduced susceptibility to antimicrobials for this organism has contributed to difficulties in controlling infection. Despite global presence, some countries have only recently experienced outbreaks of this pathogen. In the present study, M. bovis isolates collected in Denmark between 1981 and 2016 were characterized to determine (i) genetic diversity and phylogenetic relationships using whole genome sequencing and various sequence-based typing methods and (ii) patterns of antimicrobial resistance compared to other European isolates. The M. bovis population in Denmark was found to be highly homogeneous genomically and with respect to the antimicrobial resistance profile. Previously dominated by an old genotype shared by many other countries (ST17 in the PubMLST legacy scheme), a new predominant type represented by ST94-adh1 has emerged. The same clone is also found in Sweden and Finland, where M. bovis introduction is more recent. Although retrieved from the Netherlands, it appears absent from France, two countries with a long history of M. bovis infection where the M. bovis population is more diverse.

Methods to assess the effect of meat processing on viability of Toxoplasma gondii: towards replacement of mouse bioassay by in vitro testing.

Consumption of meat containing viable tissue cysts is considered one of the main sources of human infection with Toxoplasma gondii. In contrast to fresh meat, raw meat products usually undergo processing, including salting and mixing with other additives such as sodium acetate and sodium lactate, which affects the viability of T. gondii. However, the experiments described in the literature are not always performed in line with the current processing methods applied in industry. It was our goal to study the effect of salting and additives according to the recipes used by industrial producers. Mouse or cat bioassay is the 'gold standard' to demonstrate the presence of viable T. gondii. However, it is costly, time consuming and for ethical reasons not preferred for large-scale studies.Therefore, we first aimed to develop an alternative for mouse bioassay that can be used to determine the effect of processing on the viability of T. gondii tissue cysts. The assays studied were (i) a cell culture method to determine the parasite's ability to multiply, and (ii) a propidium monoazide (PMA) dye-based assay to selectively detect DNA from intact parasites. Processing experiments were performed with minced meat incubated for 20 h with low concentrations of NaCl, sodium lactate and sodium acetate. NaCl appeared to be the most effective ingredient with only one or two out of eight mice infected after inoculation with pepsin-digest of portions processed with 1.0, 1.2 and 1.6% NaCl. Results of preliminary experiments with the PMA-based method were inconsistent and did not sufficiently discriminate between live and dead parasites. In contrast, the cell culture method showed promising results, but further optimization is needed before it can replace or reduce the number of mouse bioassays needed. In future, standardised in vitro methods are necessary to allow more extensive testing of product-specific processing methods, thereby providing a better indication of the risk of T. gondii infection for consumers.

Large-scale serological screening of slaughter pigs for Toxoplasma gondii infections in The Netherlands during five years (2012-2016): Trends in seroprevalence over years, seasons, regions and farming systems.

Toxoplasma gondii is the causative agent of the parasitic disease toxoplasmosis, which is an important foodborne zoonosis. Eating undercooked meat of infected animals, including pigs, has been considered the major transmission route of T. gondii to humans. Therefore, it is urgent to develop and implement intervention measures in the pork meat chain to reduce risks of acquiring a T. gondii infection. Proposed measures for control of T. gondii in pigs include serological testing of pigs and audits of pig farms on risk factors for T. gondii infection. So far, these ideas have not been tested in practice. In order to generate knowledge about the epidemiology and seroprevalence of T. gondii, as a basis for developing a surveillance system, we studied the long term seroprevalence over years, farming systems and regions, and seasonal patterns of T. gondii seroprevalence in Dutch slaughter pigs. During a five year study period from 2012 to 2016, serum samples were routinely collected in five Dutch pig slaughterhouses. The sera were tested in an ELISA for the presence of antibodies against Toxoplasma. In total 226,340 serum samples were collected and tested during the study period. The observed seroprevalence varied over years, with the highest overall seroprevalence in 2014 (2.8%) and the lowest in 2016 (1.4%). A higher seroprevalence was observed in pigs from organic farms compared to pigs from conventional farms. The overall risk of infection was on average 2.63 times significantly (p < 0.001) higher for organically raised pigs than for conventionally raised pigs. A seasonal pattern in seroprevalence was observed: the results showed a dominant annual periodicity with a seroprevalence peak in winter around week 1 and a minimum seroprevalence in summer around week 27. To our knowledge, this is the first large scale study on the seroprevalence of T. gondii in slaughter pigs. In comparison to other European serological studies, the observed seroprevalence seems to be relatively low. However, care is needed when comparing the results with other studies because of differences in test setup, the number of samples and time period of sampling. The results can be used as a starting point for developing a surveillance system for T. gondii, and for implementation of intervention measures.