Study on inactivation of porcine epidemic diarrhoea virus, porcine sapelovirus 1 and adenovirus in the production and storage of laboratory spray-dried porcine plasma.

AIM: Evaluation of the thermal and physical conditions for inactivation of adenovirus (AdV), porcine sapelovirus 1 (PSV1) and the economically important viruses porcine epidemic diarrhoea virus (PEDV) and porcine circovirus 2 (PCV2) in the production of spray-dried porcine plasma (SDPP). METHODS AND RESULTS: Citrate-treated porcine plasma of pH 7·5, 9·8 and 10·2 (8·5% dry-matter) was spiked with PEDV, PSV1, PCV2 and AdV and incubated at 3°C for maximum 24 h, and at 44 or 48°C for maximum 10 min (Experiment 1). Spiked citrate-treated concentrated plasma of pH 7·5 and 9·8 (24% dry-matter) was spray dried in a laboratory scale apparatus (Experiment 2). Aliquots of SDPP were stored over a period of 0-10 weeks at 11 and 20°C (Experiment 3). Reverse transcription(RT)-quantitative PCR detected no notable reduction in viral genomes in treated plasma and SDPP samples. No infectious PSV1 was re-isolated from plasma and SDPP samples in cell culture. At pH 10·2 and 3°C, infectivity of PEDV in plasma was reduced with a reduction factor of 4·2 log 10 (LRF) at 10 h contact time, whereas heating to 44°C for at least 1 min at alkali pH was needed to achieve a LRF of 4·2 for AdV. Spray drying at an outlet temperature of 80°C reduced AdV infectivity effectively (LRF = 5·2) and PEDV infectivity for 95% (LRF = 1·4). After storage at 20°C for 2 weeks no infectious PEDV was re-isolated from SDPP anymore (LRF ≥4·0). Due to growth of antibiotic-resistant bacteria from plasma in cell cultures used for PCV2 isolation, no data regarding inactivation of PCV2 were obtained. CONCLUSIONS: Five percent of PEDV stayed infectious after our spray drying conditions. Spray drying in combination with storage for ≥2 weeks at 20°C eliminated infectivity of PEDV effectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The conditions for inactivation of virus in plasma and SDPP determined are important for producers to inactivate PEDV during production of SDPP.

ESBL carriage in pig slaughterhouse workers is associated with occupational exposure.

We investigated the prevalence of extended-spectrum ß-lactamase (ESBL) carriage in slaughterhouse workers and the association with occupational exposure to slaughter animals and products. Stool samples from 334 employees in a Dutch pig slaughterhouse were obtained. Presence of ESBL was determined by selective plating, microarray analysis, and gene sequencing. Questionnaires were used to collect personal and occupational information. The overall prevalence of ESBL carriage was 4·8% (16/334). All ESBL-producing isolates were Escherichia coli. The ESBL genes detected were bla CTX-M-1 (n = 8), bla CTX-M-15 (n = 3), bla CTX-M-27 (n = 2), bla CTX-M-24 (n = 1), bla CTX-M-55 (n = 1), and bla SHV-12 (n = 1). A higher prevalence of ESBL was seen in workers in jobs with as tasks 'removal of lungs, heart, liver, tongue' (33%), and 'removal of head and spinal cord' (25%). For further analysis, participants were divided in two groups based on potential exposure to ESBL as related to their job title. One group with an assumed higher exposure to ESBL (e.g. stable work, stabbing, dehairing, removal of organs) and another group with an assumed lower exposure to ESBL (e.g. refrigeration, packaging and expedition). In the 'higher exposure' group, ten out of 95 (10·5%) were carrying ESBL vs. six out of 233 (2·6%) in the 'lower exposure' group. Human ESBL carriage was significantly associated with job exposure in the slaughterhouse (OR 4·5, CI 1·6-12·6). Results suggest that ESBL carriage in slaughterhouse workers overall is comparable with the Dutch population. Within the slaughterhouse population a difference in carriage exists depending on their position along the slaughter line and tasks involved.

Prevalence of methicillin-resistant Staphylococcus aureus carrying mecA or mecC in dairy cattle.

In the Netherlands, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been found in pigs, veal calves, horses and poultry. However, little is known about its prevalence in healthy dairy cattle. Recently, a new mec gene, called mecC, has been found in methicillin-resistant S. aureus (MRSA) isolates from humans and animals in several countries. The objective of our pilot study was to investigate the prevalence of MRSA (mecA and mecC) in dairy cows at a large slaughterhouse. Samples from the skin between the udder and hind leg were taken from 411 cows. The samples were incubated in Mueller-Hinton enrichment broth with 6.5% NaCl, followed by selective enrichment and plated onto Columbia agar with 5% sheep blood, Brilliance MRSA 2 agar and Baird-Parker agar. Suspected colonies were tested by PCR for a S. aureus specific DNA fragment, the mecA and mecC genes and the Panton-Valentine leucotoxin (PVL) genes. All MRSA isolates and methicillin-susceptible S. aureus (MSSA) isolates were typed by spa typing and MLVA typing. Sixteen of 411 (3.9%) cows, all originating from different farms, were found to be MRSA positive and this prevalence is lower than in Dutch pigs, veal calves and broilers. All MRSA isolates belonging to livestock-associated MLVA complex 398, were PVL-negative and spa type t011 predominated. MSSA isolates (n=39) were of many different MLVA types and spa type t543 was found most often. Four MSSA isolates belonging to MLVA clonal complex 398 and spa types t011 (n=2), t108 and t034 were isolated from different MRSA-negative animals. In conclusion, the prevalence of MRSA in dairy cows was low and isolates carrying the mecC gene were not found, indicating that it is absent or has a low prevalence (<0.73%) in Dutch dairy cows.

Quantifying the sources of Salmonella on dressed carcasses of pigs based on serovar distribution.

Salmonella serotyping data, qualitatively described by van Hoek et al. (2012), were used to quantify potential sources of Salmonella in a Dutch pig slaughterhouse. Statistical tests to compare per-day Salmonella prevalence and serotyping data from multiple points in the chain were used to find transmission pathways. A statistical model based on serotyping data was developed to attribute Salmonella on dressed carcasses to the most likely source. Approximately two-third of dressed carcasses carrying Salmonella on the medial surface had been contaminated by house flora. For carcasses carrying Salmonella on the distal surface, transient Salmonella from incoming pigs was a more important source. The relevance of the different sources of Salmonella varied within and between sampling days. Results were compared to those of another modeling approach, in which Salmonella concentration data from the same samples were used (Smid et al., 2012). They mostly agreed. The approach chosen by an individual slaughterhouse depends on the data that are collected.

Farm and management characteristics associated with boar taint.

Pig farms in the Netherlands producing boars have different levels of boar taint prevalence, as assessed by sensory evaluation with the human nose at the slaughter line. With a questionnaire to 152 Dutch pig producers (response rate 59%), farm and management characteristics were identified that are potentially associated with farm-level boar taint prevalence. Lower farm-level boar taint prevalence was associated with a smaller group size, a smaller pen surface per boar, newer housing equipment, not practicing restricted feeding in the last period before delivery, a longer fasting period before slaughter, a higher stocking weight and a lower fraction of boars from purebred dam line sows or from Pietrain terminal boars. These characteristics can be used to develop farm-level intervention strategies to control boar taint. More research effort is needed to establish causal relationships.

Evaluation of different heating methods for the detection of boar taint by means of the human nose.

No automated detection system for boar taint detection is currently available, thus boar taint at the slaughterline can currently only be assessed using the singeing method (olfactory scoring). This study compares several heating methods (microwave, soldering iron and pyropen) and evaluates the effect of habituation, cleaning the soldering iron, singeing the fat twice in the same place, and variations in the technical procedures. All methods seem to be suitable for detecting boar taint but the choice of heating method for sensory scoring of boar taint depends on habituation of the trained assessor and specific conditions applied. The pyropen seems to be most suitable because it does not contact the fat and is easy to handle (wireless). Finally, the intensity score may also be influenced by: contamination from not cleaning the soldering iron, singeing the fat twice in the same place, and the effect of habituation.

A human nose scoring system for boar taint and its relationship with androstenone and skatole.

A system for sensory evaluation of boar taint was used to evaluate boar taint in fat samples from 6574 entire males. The term "human nose scoring" has been used to describe this system. The samples from each boar were heated with a hot iron and three panelists assigned scores of 0 to 4. The reproducibility of HNS ranged from 0.19 to 0.32 reflecting natural variation in the ability of human beings to detect different odors. The correlations of HNS with androstenone ranged from 0.22 to 0.52, while those with skatole ranged from 0.31 to 0.89, suggesting that skatole is a better predictor of boar taint. Considering (1) the relationship of HNS with the boar taint compounds, (2) the ability of HNS to capture variation not accounted for by the boar taint compounds, (3) low estimation costs and (4) low time requirements, HNS can be used in large scale evaluations of boar taint.

A biotracing model of Salmonella in the pork production chain.

In biotracing systems, downstream chain information and model-based approaches are used to trace the sources of microbial contamination in a food chain. This article includes the results of a biotracing model for Salmonella in the pork slaughter process chain. A Bayesian belief network model was used in which information on the Salmonella level at different locations in the slaughterhouse were used in combination with prior knowledge about the dynamics of Salmonella throughout the slaughter line. Data collected in a Dutch slaughterhouse were used to specify prior beliefs about the model inputs and to iteratively refine the distributions of the parameters in the model to obtain an optimal description of that specific slaughterhouse. The primary purpose of the model is to trace the sources of contamination for individual Salmonella-positive carcasses at the end of the slaughter line. The model results indicated that house flora on or in the carcass splitter was the source of contamination for many carcasses, especially for those that carried contamination on the cutting side. The results also indicated that the parameter values of the model may be subject to temporal variation and can be used as a tool to provide estimates of such trends. This model illustrates the concept of biotracing, gives insight into the dynamics of Salmonella in the slaughter line, and indicates the sites in the line where data collection is most effective for biotracing. This biotracing model was implemented as an interactive computer application, which is a step in the process toward an operational biotracing system by which a stakeholder can initiate immediate responses to Salmonella contamination and other hazards in the pork slaughterhouse.

Tracing and analytical results of the dioxin contamination incident in 2008 originating from the Republic of Ireland.

High levels of dioxins (PCDD/Fs) in pork were discovered in France and the Netherlands at the end of 2008. The contamination was rapidly traced back to a feed stock in the Republic of Ireland (RoI). Burning oil, used for the drying of bakery waste, appeared to be contaminated with PCBs. Consequently, very high levels up to 500 pg TEQ g⁻¹ fat were found in pork. The congener pattern clearly pointed to PCB-oil as a source, but the ratio between the non-dioxin-like indicator PCBs (PCBs 28, 52, 101, 138, 152 and 180) and PCDD/Fs was much lower than observed during the Belgian incident, thereby limiting the suitability of indicator PCBs as a marker for the presence of dioxins and dioxin-like PCBs. This paper describes the tracking and tracing of the incident, the public-private cooperation, the surveillance activities and its results. A major lesson to be learned from this incident is the importance of good private food safety systems. In this incident, it was the private surveillance systems that identified the origin of contamination within 10 days after the first signal of increased dioxin levels in a product. On the other hand, retrospective analyses showed that signals were missed that could have led to an earlier detection of the incident and the source. Above all, the incident would not have occurred when food safety assurance systems had been effectively implemented in the involved feed chain. It is discussed that besides primary responsibility for effective private food safety systems, the competent authorities have to supervise whether the food safety procedures are capable of coping with these kinds of complex food safety issues, while private food companies need to implement the law, and public authorities should supervise and enforce them. Finally, it is discussed whether the health risks derived from consumption of the contaminated batches of meat may have been underestimated during the incident due to the unusually high intake of dioxins.

[BSE in the Netherlands: an update]. / Boviene spongiforme encephalopathie (BSE) in Nederland: een update.

Up to 2006, there have been 82 cases of BSE in cattle born in the Netherlands. This article reviews the current situation regarding BSE in the Netherlands and summarizes the clinical symptoms of the disease. Data from the Netherlands show that a passive surveillance system, by which farmers and veterinarians have to report suspect clinical cases, has a low sensitivity. The epidemiology of, and risk factors for, BSE are discussed. All the Dutch cases of BSE can be attributed to cross-contamination of feed with meat-and-bone meal. On the basis of information about the epidemic and the cases reported to date, it is anticipated that the number of cases of BSE will continue to decline in the Netherlands and Europe. The European Commission has presented a road map that describes how the European BSE policy can be changed in the short and long term if the current favourable trend in BSE cases continues. It is time for a new phase in the management of BSE but with continued protection of the public's health and eradication of BSE.

Immunomodulation by probiotic lactobacilli in layer- and meat-type chickens.

1. The aim of the experiments was to evaluate whether selected probiotic lactobacillus strains have different immunomodulating effects in layer- and meat-type strain chickens. 2. Humoral and cellular specific and non-specific immune responses were studied by experiments on cellular proliferation, entry and survival of Salmonella bacteria in gut and spleen leukocytes, immunoglobulin isotypes and specific immunoglobulin titres. 3. The effects of two different feeding regimes (short and continuous feeding) and doses for administration of lactobacilli were studied. 4. The lactobacillus strains that were evaluated showed modulating effects on the immune system of layer- and meat-type chickens. 5. In meat-type strain chickens the lactobacilli had a stimulating effect when the chickens were young (up to 3 weeks) and the dose was relatively high, whereas in layer-type chickens a lower effective dose and discontinuous administration was also effective. 6. Immunoprobiotic lactobacilli can have a positive effect on humoral and cellular immune responses in layer- and meat-type strain chickens, but the lactobacillus strain to be used, the age of the animals and effective dose of lactobacilli to be administered need to be optimised.

Effect of acidified feed on susceptibility of broiler chickens to intestinal infection by Campylobacter and Salmonella.

Consumption of poultry meat is associated with human Campylobacter and Salmonella infections. One way to control the presence of these bacteria in broiler flocks is to make chickens less susceptible for colonisation. Acidification of feed may be a tool to reduce the Campylobacter and Salmonella carriage in broiler chickens. In the present experiments an acidified feed with high levels of organic acid, 5.7% lactic acid and 0.7% acetic acid, was applied. In an in vitro experiment the reduction or growth of Campylobacter and Salmonella was measured after addition of 10(7)cfu of these bacteria into a conventional broiler feed, acidified feed and fermented feed, whereas the numbers of Salmonella increased in non-acidified feed. The number of Campylobacter decreased 2-3 (10)log cfu. In the acidified and fermented feed a complete reduction of Campylobacter was observed within 20 min, and a total Salmonella reduction started after 1h, and was complete after 2h. Subsequently, an in vivo experiment with 100 individually housed broiler chickens showed that chickens fed acidified feed were less susceptible to an infection with Campylobacter than were chickens fed conventional feed. The size of reduction was however limited. The susceptibility for Salmonella colonisation was not affected by acidified feed. It is concluded that the role for acidified feed in the control of Campylobacter and Salmonella is limited.

Transmission of Salmonella between broiler chickens fed with fermented liquid feed.

In the light of food safety and the control of Salmonella at chicken farms, fermented liquid feed (FLF) was studied. This moistened feed reduced the susceptibility of chickens for Salmonella. To assess the effect of the fermented feed on the transmission of Salmonella between chickens, a transmission experiment was performed. Salmonella shedding was followed within groups of two susceptible chickens together with two previously inoculated chickens. The between-chicken transmission was quantified by calculating a reproduction ratio (R0) and a transmission rate parameter (beta). R0 and beta in the FLF-treated groups were reduced, but a typical infectious chicken fed with FLF, could on average still infect more than one new infectious case. FLF can therefore reduce the transmission of Salmonella in chicken flocks, but it will not prevent the occurrence of major outbreaks.

Fermented liquid feed reduces susceptibility of broilers for Salmonella enteritidis.

The presence of Salmonella in chickens is a problem because poultry meat is recognized as a source of human salmonellosis. Fermented feed has characteristics like a high number of lactobacilli and high concentration of lactic acid, which could make chickens less susceptible for infection with Salmonella. Fermented feed might therefore prevent the colonization of chickens with Salmonella. Two studies were performed to quantify the effect of fermented liquid feed on the susceptibility of broilers for Salmonella. The fermented feed was prepared by fermenting a dry broiler feed supplemented with 1.4 parts of water. Lactobacillus plantarum was used for fermentation. The fermented liquid feed (FLF) contained 10(9) to 10(10) cfu lactobacilli per gram, and the pH was 4. Individually housed control chickens and FLF-fed chickens were inoculated with 10(2) to 10(7) cfu Salmonella enteritidis (SE). Colonization was estimated by cloacal swabs and quantitative caecal culture. The proportion of SE-shedding chickens was decreased in FLF-fed chickens. FLF-fed chickens required a longer time after inoculation or a higher inoculation dose to get the same proportion of infected chickens in comparison with dry feed-fed chickens. The level of cecal colonization with Salmonella in the ceca was not different at the end of the experimental period. The results indicate that FLF can hamper the introduction of Salmonella in broiler flocks because the chickens are less susceptible for infection. Fermented liquid feed might therefore be a new hurdle in the strategy to control Salmonella in chicken flocks.

Validation of a new method of visual oestrus detection on the farm.

Since visual observation is the most commonly used way of detecting oestrus and is supposed to be as effective as detection with technical devices, we evaluated a recently developed oestrus detection scoring system in daily dairy practice. In this scoring system nine signs of oestrus are scored with points, ranging 3 to 100. Twenty-one dairy farmers used the scoring system during a period of 3 weeks. All cows that were more than 30 days post partum and not confirmed pregnant were monitored, using the scoring system, by the herd owners. Oestrus was confirmed by measuring progesterone concentrations in milk. With the scoring system a detection rate of 47% was achieved. This was lower than expected, because in an earlier control period, the detection rate was 64%. We concluded that this new method might be too complicated to introduce to normal herd management, because in daily practice it is too demanding to watch cows twice a day for 30 minutes, especially if the cows show only vague and infrequent symptoms of oestrus. It also appeared to be too complicated to watch the herd at the most appropriate time. However, if the scoring system is included in the daily routine, meaning that farmers are trained to watch for other symptoms than standing heat only and are able to recognize their different values, it can be a valuable aid to oestrus detection.